Establishment of HSV1 Latency in Immunodeficient Mice Facilitates Efficient In Vivo Reactivation

The establishment of latent infections in sensory neurons is a remarkably effective immune evasion strategy that accounts for the widespread dissemination of life long Herpes Simplex Virus type 1 (HSV1) infections in humans. Periodic reactivation of latent virus results in asymptomatic shedding and transmission of HSV1 or recurrent disease that is usually mild but can be severe. An in-depth understanding of the mechanisms regulating the maintenance of latency and reactivation are essential for developing new approaches to block reactivation. However, the lack of a reliable mouse model that supports efficient in vivo reactivation (IVR) resulting in production of infectious HSV1 and/or disease has hampered progress. Since HSV1 reactivation is enhanced in immunosuppressed hosts, we exploited the antiviral and immunomodulatory activities of IVIG (intravenous immunoglobulins) to promote survival of latently infected immunodeficient Rag mice. Latently infected Rag mice derived by high dose (HD), but not low dose (LD), HSV1 inoculation exhibited spontaneous reactivation. Following hyperthermia stress (HS), the majority of HD inoculated mice developed HSV1 encephalitis (HSE) rapidly and synchronously, whereas for LD inoculated mice reactivated HSV1 persisted only transiently in trigeminal ganglia (Tg). T cells, but not B cells, were required to suppress spontaneous reactivation in HD inoculated latently infected mice. Transfer of HSV1 memory but not OVA specific or naïve T cells prior to HS blocked IVR, revealing the utility of this powerful Rag latency model for studying immune mechanisms involved in control of reactivation. Crossing Rag mice to various knockout strains and infecting them with wild type or mutant HSV1 strains is expected to provide novel insights into the role of specific cellular and viral genes in reactivation, thereby facilitating identification of new targets with the potential to block reactivation.     

Analysis of Single Locus Trajectories for Extracting In Vivo Chromatin Tethering Interactions

Is it possible to extract tethering forces applied on chromatin from the statistics of a single locus trajectories imaged in vivo? Chromatin fragments interact with many partners such as the nuclear membrane, other chromosomes or nuclear bodies, but the resulting forces cannot be directly measured in vivo. However, they impact chromatin dynamics and should be reflected in particular in the motion of a single locus. We present here a method based on polymer models and statistics of single trajectories to extract the force characteristics and in particular when they are generated by the gradient of a quadratic potential well. Using numerical simulations of a Rouse polymer and live cell imaging of the MAT-locus located on the yeast Saccharomyces cerevisiae chromosome III, we recover the amplitude and the distance between the observed and the interacting monomer. To conclude, the confined trajectories we observed in vivo reflect local interaction on chromatin.     

MicroRNA Profiling in Human Neutrophils during Bone Marrow Granulopoiesis and In Vivo Exudation

The purpose of this study was to describe the microRNA (miRNA) expression profiles of neutrophils and their precursors from the initiation of granulopoiesis in the bone marrow to extravasation and accumulation in skin windows. We analyzed three different cell populations from human bone marrow, polymorphonuclear neutrophil (PMNs) from peripheral blood, and extravasated PMNs from skin windows using the Affymetrix 2.0 platform. Our data reveal 135 miRNAs differentially regulated during bone marrow granulopoiesis. The majority is differentially regulated between the myeloblast/promyelocyte (MB/PM) and myelocyte/metamyelocyte (MC/MM) stages of development. These 135 miRNAs were divided into six clusters according to the pattern of their expression. Several miRNAs demonstrate a pronounced increase or reduction at the transition between MB/PM and MC/MM, which is associated with cell cycle arrest and the initiation of terminal differentiation. Seven miRNAs are differentially up-regulated between peripheral blood PMNs and extravasated PMNs and only one of these (miR-132) is also differentially regulated during granulopoiesis. The study indicates that several different miRNAs participate in the regulation of normal granulopoiesis and that miRNAs might also regulate activities of extravasated neutrophils. The data present the miRNA profiles during the development and activation of the neutrophil granulocyte in healthy humans and thus serves as a reference for further research of normal and malignant granulocytic development.     

Stem Cell Selection In Vivo Using Foamy Vectors Cures Canine Pyruvate Kinase Deficiency

Background

Hematopoietic stem cell (HSC) gene therapy has cured immunodeficiencies including X-linked severe combined immunodeficiency (SCID-X1) and adenine deaminase deficiency (ADA). For these immunodeficiencies corrected cells have a selective advantage in vivo, and low numbers of gene-modified cells are sufficient to provide therapeutic benefit. Strategies to efficiently transduce and/or expand long-term repopulating cells in vivo are needed for treatment of diseases that require higher levels of corrected cells, such as hemoglobinopathies. Here we expanded corrected stem cells in vivo in a canine model of a severe erythroid disease, pyruvate kinase deficiency.

Methodology/Principal Findings

We used a foamy virus (FV) vector expressing the P140K mutant of methylguanine methyltransferase (MGMTP140K) for in vivo expansion of corrected hematopoietic repopulating cells. FV vectors are attractive gene transfer vectors for hematopoietic stem cell gene therapy since they efficiently transduce repopulating cells and may be safer than more commonly used gammaretroviral vectors. Following transplantation with HSCs transduced ex vivo using a tri-cistronic FV vector that expressed EGFP, R-type pyruvate kinase, and MGMTP140K, we were able to increase marking from approximately 3.5% to 33% in myeloid long-term repopulating cells resulting in a functional cure.

Conclusions/Significance

Here we describe in one affected dog a functional cure for a severe erythroid disease using stem cell selection in vivo. In addition to providing a potential cure for patients with pyruvate kinase deficiency, in vivo selection using foamy vectors with MGMTP140K has broad potential for several hematopoietic diseases including hemoglobinopathies.     

In Vivo and In Vitro Morphological Analysis of Melanocytes Homozygous for the misp Allele at the Murine Microphthalmia Locus

The mi^8p allele (microphthalmia-spotted), a mutant allele at the murine microphthalmia (mi) locus, when homozygous, results in a normal phenotype in which there is no apparent alteration in pelage pigmentation or ocular development. However, when heterozygous with other mi locus alleles, specifically Mi^wh (microphthalmia-white) the mi^8p allele exerts an affect on the phenotype. We examined the ultrastructure of melanocytes in the anagen hair bulb and the choroid plus the retinal pigmented epithelium of C57BL/6J-mi^8p/mi^8p mice, C57BL/6J-Mi^wh/Mi^wh mice, C57BL/6J-Mi^wh/mi^8p mice, and C57BL/6J-⁺/⁺ control mice. Melanocytes of the mi^8p/mi^8p mice appeared normal in situ. However, melanocyte cultures derived from neonatal skins of mi^8p/mi^8p mice exhibited small primary colonies that did not dramatically expand in size. Occasionally, abnormalities in the structure of the Golgi apparatus were observed in primary cultures of mi^8p/mi^8p melanocytes. These results demonstrate that while the mi^8p allele has no obvious effect on the phenotype of the mouse, it does dramatically suppress the survival of melanocytes in normal culture conditions.     

KSHV Latency Locus Cooperates with Myc to Drive Lymphoma in Mice

Kaposi sarcoma-associated herpesvirus (KSHV) has been linked to Kaposi sarcoma and B-cell malignancies. Mechanisms of KSHV-induced oncogenesis remain elusive, however, in part due to lack of reliable in vivo models. Recently, we showed that transgenic mice expressing the KSHV latent genes, including all viral microRNAs, developed splenic B cell hyperplasia with 100% penetrance, but only a fraction converted to B cell lymphomas, suggesting that cooperative oncogenic events were missing. Myc was chosen as a possible candidate, because Myc is deregulated in many B cell lymphomas. We crossed KSHV latency locus transgenic (latency) mice to Cα Myc transgenic (Myc) mice. By itself these Myc transgenic mice develop lymphomas only rarely. In the double transgenic mice (Myc/latency) we observed plasmacytosis, severe extramedullary hematopoiesis in spleen and liver, and increased proliferation of splenocytes. Myc/latency mice developed frank lymphoma at a higher rate than single transgenic latency or Myc mice. These data indicate that the KSHV latency locus cooperates with the deregulated Myc pathways to further lymphoma progression.     

The M33 Chemokine Receptor Homolog of Murine Cytomegalovirus Exhibits a Differential Tissue-Specific Role during In Vivo Replication and Latency

M33, encoded by murine cytomegalovirus (MCMV), is a member of the UL33 homolog G-protein-coupled receptor (GPCR) family and is conserved across all the betaherpesviruses. Infection of mice with recombinant viruses lacking M33 or containing specific signaling domain mutations in M33 results in significantly diminished MCMV infection of the salivary glands. To determine the role of M33 in viral dissemination and/or infection in other tissues, viral infection with wild-type K181 virus and an M33 mutant virus, ΔM33B_T2, was characterized using two different routes of inoculation. Following both intraperitoneal (i.p.) and intranasal (i.n.) inoculation, M33 was attenuated for infection of the spleen and pancreas as early as 7 days after infection. Following i.p. inoculation, ΔM33B_T2 exhibited a severe defect in latency as measured by a diminished capacity to reactivate from spleens and lungs in reactivation assays (P < 0.001). Subsequent PCR analysis revealed markedly reduced ΔM33B_T2 viral DNA levels in the latently infected spleens, lungs, and bone marrow. Following i.n. inoculation, latent ΔM33B_T2 viral DNA was significantly reduced in the spleen and, in agreement with results from i.p. inoculation, did not reactivate from the spleen (P < 0.001). Furthermore, in vivo complementation of ΔM33B_T2 virus replication and/or dissemination to the salivary glands and pancreas was achieved by coinfection with wild-type virus. Overall, our data suggest a critical tissue-specific role for M33 during infection in the salivary glands, spleen, and pancreas but not the lungs. Our data suggest that M33 contributes to the efficient establishment or maintenance of long-term latent MCMV infection.Since the discovery of the G protein-coupled receptors (GPCRs) encoded by the betaherpesviruses, there has been intense speculation on the biological role these viral proteins play during infection (15, 16, 22). Human cytomegalovirus (HCMV), a betaherpesvirus, is a ubiquitous pathogen that asymptomatically infects humans and establishes a long-term persistent infection. HCMV is life-threatening, however, to immunocompromised individuals, such as neonates, AIDS patients, and transplant recipients. HCMV, similar to a number of herpesviruses, encodes viral genes that are predicted to impact virus-host interactions that may promote efficient long-term infection of the host. The CMVs encode genes for proteins that potentially enhance viral dissemination and replication and promote immune evasion by mimicry of host functions that influence the conditions of primary infection, the virus-specific immune response, and even long-term host control of persistent or latent infection (reviewed in references 1, 44, and 68).HCMV encodes four GPCRs (UL33, UL78, US28, and US27) which share homology to host chemokine receptors (16). This suggests that these virally encoded chemokine receptors may function similarly to their cellular receptor counterparts. Chemokines are chemoattractant cytokines that bind and activate chemokine receptors that are on the surfaces of cells. Host chemokine receptors then mediate the activation of cellular signaling pathways and cell migration to sites of inflammation by transmitting signals through G proteins (56, 70). In humans, approximately 50 chemokines and 20 chemokine receptors have been identified, many of which have close homologs in mice and other species (39). Chemokines are divided into two classes, lymphoid chemokines, which are constitutively expressed and involved in lymphoid tissue organization, and inflammatory chemokines, which are induced following infection and part of the inflammatory response (21, 39, 51). Growing evidence indicates that chemokines play a critical role in the host response to infection and inflammation during both the innate and adaptive immune responses (26), thus suggesting that the betaherpesviruses have “hijacked” the chemokine receptors from the host genome to subvert or alter these responses during infection. Besides chemokine receptors, HCMV also encodes a CXC chemokine (UL146) that induces the migration of neutrophils (48); a second CXC chemokine homolog (UL147) whose function is not yet known; a viral CC chemokine (UL131) that is critical for infection of macrophages, endothelial cells, and epithelial cells (25, 57, 73); and a RANTES decoy protein (72). A CC chemokine (vMCK or m131/129) is also encoded by murine CMV (MCMV), and a homolog in rat CMV ([RCMV] r131) that promotes monocyte-associated viremia (20, 37, 59, 60). The MCMV m131/129 chemokine was shown to recruit myelomonocytic progenitors from the bone marrow, perhaps to facilitate cell-type-specific viremia (46). Clearly, the CMVs have invested a great deal of effort into manipulating or subverting the host chemokine system, thus making it reasonable to speculate that these viral members of the chemokine system play an important role during CMV pathogenesis.Of the HCMV-encoded GPCRs, US28 has been well characterized in vitro and functions as a bona fide chemokine receptor, whereas much less is known about the receptor activity of US27, UL33, and UL78. US28 binds and sequesters CC chemokines, induces smooth-muscle cell migration, and constitutively activates signaling pathways (5, 7-9, 42, 52, 64, 67, 71). US28 and US27 are found only in primate CMVs, whereas both UL33 and UL78 are highly conserved across all betaherpesvirus genomes, suggesting an important evolutionary function for UL33 and UL78 during CMV infection. Two other betaherpesviruses, human herpesviruses 6 and 7 (HHV6 and HHV7), encode homologs to the UL33 and UL78 receptors, U12 and U51, respectively. The U12 receptors of HHV6 and HHV7 (34, 45, 66) and the HHV6-encoded U51 receptor (22) exhibit chemokine binding activity. UL33, along with its rodent CMV homologs, M33 (MCMV) and R33 (RCMV), constitutively activates signaling pathways (13, 23, 71). M33 induces smooth-muscle cell migration (39), similar to US28-mediated smooth-muscle cell migration (64). Thus, members of the UL33 family potentially function during viral infection by modulating or influencing the composition of leukocytes at sites of infection, the migration of infected cells or infiltrating leukocytes, or modulation of intracellular signaling pathways.Due to the species specificity of CMV, the in vivo role of the HCMV-encoded GPCRs cannot be addressed. However, the importance of UL33 and UL78 for viral dissemination and virulence in vivo has been indicated by disruption of the viral homologs in MCMV and RCMV (6, 19, 36, 47). Disruption of the UL33, M33, and R33 genes demonstrated that they are dispensable for replication in vitro, indicating that the UL33 family members are not required for replication or cell entry in at least some cell types (6, 19, 40). Infection of mice with M33-deficient MCMV or infection of rats with R33-deficient RCMV results in highly attenuated viruses and diminished infection of the salivary glands. The RCMV R33 protein also appears to play a role in virulence since rats infected with an R33 deletion virus had a lower mortality rate (6). More recently, constitutive M33-mediated activation of signaling pathways was shown to be essential for MCMV infection of salivary glands (14). Significantly, the UL33 protein partially rescued the defect in salivary gland infection attributed to disruption of M33, indicating the evolutionary conservation of function between the HCMV (UL33) and MCMV (M33) chemokine receptor homologs.In this paper, the role of M33 is further investigated using two routes of infection to assess viral dissemination and viral replication kinetics at different tissue sites, the numbers of infected cells following infection, and the possibility that M33 plays a role during latent infection. In addition to the critical role that M33 plays in salivary gland infection, this study reveals that M33 is important for MCMV infection of the spleen and the pancreas but not the lungs. Significantly, our studies provide preliminary evidence that disruption of M33 leads to reduced latent viral load in the spleen, lungs, and bone marrow, perhaps due to defects in the establishment and/or maintenance of latent infection. Lastly, we demonstrate that the tissue defects observed during acute infection with an M33 mutant virus (ΔM33B_T2) can be complemented in vivo when mice are coinfected with ΔM33B_T2 virus and wild-type MCMV. Taken together, our findings indicate that M33 plays a critical tissue-specific role during acute MCMV infection and, importantly, contributes to the efficient establishment or maintenance of latent MCMV infection.     

Repair of the UL21 Locus in Pseudorabies Virus Bartha Enhances the Kinetics of Retrograde, Transneuronal Infection In Vitro and In Vivo

The attenuated pseudorabies virus (PRV) strain Bartha contains several characterized mutations that affect its virulence and ability to spread through neural circuits. This strain contains a small genomic deletion that abrogates anterograde spread and is widely used as a retrograde-restricted neural circuit tracer. Previous studies showed that the retrograde-directed spread of PRV Bartha is slower than that of wild-type PRV. We used compartmented neuronal cultures to characterize the retrograde defect and identify the genetic basis of the phenotype. PRV Bartha is not impaired in retrograde axonal transport, but transneuronal spread among neurons is diminished. Repair of the U_L21 locus with wild-type sequence restored efficient transneuronal spread both in vitro and in vivo. It is likely that mutations in the Bartha U_L21 gene confer defects that affect infectious particle production, causing a delay in spread to presynaptic neurons and amplification of infection. These events manifest as slower kinetics of retrograde viral spread in a neural circuit.     

A Neuron-Specific Host MicroRNA Targets Herpes Simplex Virus-1 ICP0 Expression and Promotes Latency

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Kaposi's Sarcoma-Associated Herpesvirus Latency Locus Compensates for Interleukin-6 in Initial B Cell Activation

Interleukin 6 (IL-6) is considered a proliferation and survival factor for B cells. To assess the role of IL-6 in Kaposi sarcoma-associated herpesvirus (KSHV) latency, KSHV latency locus-transgenic mice (referred to as latency mice) lacking IL-6 were evaluated. IL-6^−/− latency mice had the same phenotypes as the latency mice, i.e., increased frequency of marginal zone B cells, hyperplasia, and hyperglobulinemia, indicating that the KSHV latency locus, which includes all viral microRNAs (miRNAs), can compensate for lack of IL-6 in premalignant B cell activation.     

MicroRNA miR-155 Affects Antiviral Effector and Effector Memory CD8 T Cell Differentiation

MicroRNAs are key regulators of the immune response, but their role in CD8 T cell differentiation in vivo is not known. We show that miR-155 is important in both effector and memory antiviral CD8 T cell responses. Without miR-155, there was a weaker effector response and a skewing toward memory precursor cells. At the memory stage, miR-155-deficient CD8 T cells preferentially differentiated into central memory cells and were capable of mounting a potent secondary response.     

MicroRNA Let-7a Inhibits Proliferation of Human Prostate Cancer Cells In Vitro and In Vivo by Targeting E2F2 and CCND2

Background

Previous work has shown reduced expression levels of let-7 in lung tumors. But little is known about the expression or mechanisms of let-7a in prostate cancer. In this study, we used in vitro and in vivo approaches to investigate whether E2F2 and CCND2 are direct targets of let-7a, and if let-7a acts as a tumor suppressor in prostate cancer by down-regulating E2F2 and CCND2.

Methodology/Principal

Findings Real-time RT-PCR demonstrated that decreased levels of let-7a are present in resected prostate cancer samples and prostate cancer cell lines. Cellular proliferation was inhibited in PC3 cells and LNCaP cells after transfection with let-7a. Cell cycle analysis showed that let-7a induced cell cycle arrest at the G1/S phase. A dual-luciferase reporter assay demonstrated that the 3′UTR of E2F2 and CCND2 were directly bound to let-7a and western blotting analysis further indicated that let-7a down-regulated the expression of E2F2 and CCND2. Our xenograft models of prostate cancer confirmed the capability of let-7a to inhibit prostate tumor development in vivo.

Conclusions/Significance

These findings help to unravel the anti-proliferative mechanisms of let-7a in prostate cancer. Let-7a may also be novel therapeutic candidate for prostate cancer given its ability to induce cell-cycle arrest and inhibit cell growth, especially in hormone-refractory prostate cancer.     

Differential In Vivo Regulation of mRNA Encoding the Norepinephrine Transporter and Tyrosine Hydroxylase in Rat Adrenal Medulla and Locus Ceruleus

Abstract: To investigate the regulation of norepinephrine transporter mRNA in vivo, we analyzed the effects of reserpine on its expression in the rat adrenal medulla and locus ceruleus. First, PCR was used to clone a 0.5-kb rat cDNA fragment that exhibits 87% nucleotide identity to the corresponding human norepinephrine transporter cDNA sequence. In situ, the cDNA hybridizes specifically within norepinephrine-secreting cells, but in neither dopamine nor serotonin neurons, suggesting strongly it is a partial rat norepinephrine transporter cDNA. Reserpine, 10 mg/kg administered 24 h premortem, decreased steady-state levels of norepinephrine transporter mRNA in the adrenal medulla by ∼65% and in the locus ceruleus by ∼25%, as determined by quantitative in situ hybridization. Northern analysis confirmed the results of the in situ hybridization analysis in the adrenal medulla but did not detect the smaller changes observed in the locus ceruleus. Both analyses showed that reserpine increased tyrosine hydroxylase expression in the adrenal medulla and locus ceruleus. These results suggest that noradrenergic neurons and adrenal chromaffin cells can coordinate opposing changes in systems mediating catecholamine uptake and synthesis, to compensate for catecholamine depletion.     

In Vivo Microdialysis Study of the Extracellular 3,4-Dihydroxyphenylacetic Acid in the Rat Locus Ceruleus: Topographical and Pharmacological Aspects

Abstract: In vivo microdialysis coupled with HPLC and electrochemical detection was used to monitor extracellular levels of 3, 4-dihydroxyphenylacetic acid (DOPAC) of the locus ceruleus (LC) in halothane-anesthetized rats. The identity of DOPAC was confirmed by experiments showing that the chromatographic peak was totally suppressed after inhibition of monoamine oxidase by pargyline. Histological examinations allowed to relate the quantity of DOPAC measured in the dialysates with the localization of the probe implantation site. We found that the DOPAC concentration was inversely proportional to the distance between the probe and the lateral border of the LC. Regardless of the caudorostral level of the nucleus, concentrations were maximal when the axis of the probe was 100 μ M from the lateral border of the LC and decreased by 53% when this distance reached 300 μ M . Activation of LC noradrenergic neurons by systemic administration of the α₂-antagonist piperoxane increased by 100% DOPAC concentrations in LC dialysates. These results suggest that the DOPAC measured by microdialysis could be considered an indicator of the functional state of LC noradrenergic neurons.     

In Vivo Muscle Electroporation Threshold Determination: Realistic Numerical Models and In Vivo Experiments

In vivo electroporation is used as an effective technique for delivery of therapeutic agents such as chemotherapeutic drugs or DNA into target tissue cells for different biomedical purposes. In order to successfully electroporate a target tissue, it is essential to know the local electric field distribution produced by an application of electroporation voltage pulses. In this study three-dimensional finite element models were built in order to analyze local electric field distribution and corresponding tissue conductivity changes in rat muscle electroporated either transcutaneously or directly (i.e., two-plate electrodes were placed either on the skin or directly on the skeletal muscle after removing the skin). Numerical calculations of electroporation thresholds and conductivity changes in skin and muscle were validated with in vivo measurements. Our model of muscle with skin also confirms the in vivo findings of previous studies that electroporation “breaks” the skin barrier when the applied voltage is above 50?V.