The Role of Cytomegalovirus Viral Chemokines in Neutrophil Activation and Viral Dissemination

CMV is the leading cause of both non-hereditary mental retardation and hearing loss and CMV infection following transplantation carries a serious risk for complications. The development of a CMV vaccine or better therapeutic treatment is desired but to develop these a more complete understanding of CMV pathogenesis is necessary. Sequence comparisons between attenuated and virulent strains of HCMV map major differences to a 15kb region (ULb’) containing the chemokine homolog, vCXCL-1. The vCXCL-1 protein of the Toledo (Tol) strain was previously shown to function in vitro as a CXC chemokine. Murine CMV (MCMV) also encodes a viral chemokine, MCK2. Prior research has shown that infection of mice with a recombinant MCMV, RM4511, which lacks a functional MCK2, resulted in decreased inflammation at the site of infection and decreased dissemination to the salivary gland. Analysis of the role of vCXCL-1 in the context of HCMV infection is limited by the species specificity of the CMVs. One possible model system for analyzing the function of this chemokine is the chimpanzee model of CMV infection. Chimpanzee CMV (CCMV) has at least one gene with similarity to the vCXCL-1 gene of the Toledo strain of HCMV. The hypothesis of this study was that vCXCL-1CCMV is a functional CXC chemokine that contributes to viral dissemination. To address this we initially compared the functional and biochemical characteristics in vitro of vCXCL-1 from Toledo HCMV (vCXCL-1Tol) and CCMV (vCXCL-1ccmv) by analyzing receptor binding, activation, chemotaxis, signaling, and anti apoptotic suppression in neutrophils. The CCMV chemokine had a ~70-fold lower affinity for human CXCR2 compared to (vCXCL-1Tol. Nevertheless, the homologs functioned very similarly and we only observed differences in integrin induction and the ability of the two chemokines to reduce neutrophil apoptosis. We evaluated the in vivo function of vCXCL-1ccmv using MCMV infection in mice. Due to the strict species specificity of CMV we used MCMV RM4511 to produce recombinant MCMVs expressing vCXCL-1ccmv or a control host chemokine, mCXCL1, under the control of the HCMV IE promoter. RMvCXCL1ccmv and RMmCXCL1 grew with similar kinetics to RM4511 in cell culture and expressed vCXCL-1ccmv and mCXCL1, respectively. Primary dissemination of RMvCXCL1ccmv and RMmCXCL1 was similar to RM4511 but neither recombinant was recovered from the salivary gland at any time point. Maintenance of this phenotype in SCID mice showed the recombinant viruses were not cleared from the salivary gland by the adaptive immune response. RMvCXCL1ccmv and RMmCXCL1 were also not recovered in neutrophil-depleted mice, although RM4511 titers were reduced in these mice indicating neutrophils may play a role in dissemination of MCMV to the salivary gland. This study is the first to characterize the CCMV viral chemokine, vCXCL-1, and show it is functionally similar to vCXCL-1Tol This provides evidence that the study of vCXCL-1ccmv may contribute to a better understanding of its HCMV homolog. Furthermore, this research is the first to evaluate the role of a CMV CXC chemokine in vivo Future work aimed at evaluating the role of other viral and host chemokines in vivo using the mouse model will likely advance our understanding of viral chemokines and their role in virus-host interactions

Feeling manipulated: cytomegalovirus immune manipulation

No one likes to feel like they have been manipulated, but in the case of cytomegalovirus (CMV) immune manipulation, we do not really have much choice. Whether you call it CMV immune modulation, manipulation, or evasion, the bottom line is that CMV alters the immune response in such a way to allow the establishment of latency with lifelong shedding. With millions of years of coevolution within their hosts, CMVs, like other herpesviruses, encode numerous proteins that can broadly influence the magnitude and quality of both innate and adaptive immune responses. These viral proteins include both homologues of host proteins, such as MHC class I or chemokine homologues, and proteins with little similarity to any other known proteins, such as the chemokine binding protein. Although a strong immune response is launched against CMV, these virally encoded proteins can interfere with the host's ability to efficiently recognize and clear virus, while others induce or alter specific immune responses to benefit viral replication or spread within the host. Modulation of host immunity allows survival of both the virus and the host. One way of describing it would be a kind of "mutually assured survival" (as opposed to MAD, Mutually Assured Destruction). Evaluation of this relationship provides important insights into the life cycle of CMV as well as a greater understanding of the complexity of the immune response to pathogens in general

An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ

Introduction: Human models of noninvasive breast tumors are limited, and the existing in vivo models do not mimic inter- and intratumoral heterogeneity. Ductal carcinoma in situ (DCIS) is the most common type (80%) of noninvasive breast lesions. The aim of this study was to develop an in vivo model whereby the natural progression of human DCIS might be reproduced and studied. To accomplish this goal, the intraductal human-in-mouse (HIM) transplantation model was developed. The resulting models, which mimicked some of the diversity of human noninvasive breast cancers in vivo, were used to show whether subtypes of human DCIS might contain distinct subpopulations of tumor-initiating cells.Methods The intraductal models were established by injection of human DCIS cell lines (MCF10DCIS.COM and SUM-225), as well as cells derived from a primary human DCIS (FSK-H7), directly into the primary mouse mammary ducts via cleaved nipple. Six to eight weeks after injections, whole-mount, hematoxylin and eosin, and immunofluorescence staining were performed to evaluate the type and extent of growth of the DCIS-like lesions. To identify tumor-initiating cells, putative human breast stem/progenitor subpopulations were sorted from MCF10DCIS.COM and SUM-225 with flow cytometry, and their in vivo growth fractions were compared with the Fisher's Exact test. Results: Human DCIS cells initially grew within the mammary ducts, followed by progression to invasion in some cases into the stroma. The lesions were histologically almost identical to those of clinical human DCIS. This method was successful for growing DCIS cell lines (MCF10DCIS.COM and SUM-225) as well as a primary human DCIS (FSK-H7). MCF10DCIS.COM represented a basal-like DCIS model, whereas SUM-225 and FSK-H7 cells were models for HER-2[super]+ DCIS. With this approach, we showed that various subtypes of human DCIS appeared to contain distinct subpopulations of tumor-initiating cells. Conclusions: The intraductal HIM transplantation model provides an invaluable tool that mimics human breast heterogeneity at the noninvasive stages and allows the study of the distinct molecular and cellular mechanisms of breast cancer progression

Progress in manufacturing the first 8.4 m off-axis segment for the Giant Magellan Telescope

ABSTRACT The first of the 8.4 m off-axis segments for the primary mirror of the Giant Magellan Telescope is being manufactured at the Steward Observatory Mirror Lab. In addition to the manufacture of the segment, this project includes the development of a complete facility to make and measure all seven segments. We have installed a new 28 m test tower and designed a set of measurements to guide the fabrication and qualify the finished segments. The first test, a lasertracker measurement of the ground surface, is operational. The principal optical test is a full-aperture interferometric test with a null corrector that includes a 3.75 m spherical mirror, a smaller sphere, and a computer-generated hologram. We have also designed a scanning pentaprism test to validate the measurement of low-order aberrations. The first segment has been cast and generated, and is in the process of loose-abrasive grinding

Human cytomegalovirus latency-associated proteins elicit immune-suppressive IL-10 producing CD4⁺ T cells.

Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4⁺ T cell mediated. These UL138-specific CD4⁺ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CDCD4⁺ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4⁺ T cell responses included CD4⁺ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4⁺ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4⁺ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4⁺ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo

Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ

Human cytomegalovirus (hCMV) is a highly prevalent pathogen that, upon primary infection, establishes life-long persistence in all infected individuals. Acute hCMV infections cause a variety of diseases in humans with developmental or acquired immune deficits. In addition, persistent hCMV infection may contribute to various chronic disease conditions even in immunologically normal people. The pathogenesis of hCMV disease has been frequently linked to inflammatory host immune responses triggered by virus-infected cells. Moreover, hCMV infection activates numerous host genes many of which encode pro-inflammatory proteins. However, little is known about the relative contributions of individual viral gene products to these changes in cellular transcription. We systematically analyzed the effects of the hCMV 72-kDa immediate-early 1 (IE1) protein, a major transcriptional activator and antagonist of type I interferon (IFN) signaling, on the human transcriptome. Following expression under conditions closely mimicking the situation during productive infection, IE1 elicits a global type II IFN-like host cell response. This response is dominated by the selective up-regulation of immune stimulatory genes normally controlled by IFN-γ and includes the synthesis and secretion of pro-inflammatory chemokines. IE1-mediated induction of IFN-stimulated genes strictly depends on tyrosine-phosphorylated signal transducer and activator of transcription 1 (STAT1) and correlates with the nuclear accumulation and sequence-specific binding of STAT1 to IFN-γ-responsive promoters. However, neither synthesis nor secretion of IFN-γ or other IFNs seems to be required for the IE1-dependent effects on cellular gene expression. Our results demonstrate that a single hCMV protein can trigger a pro-inflammatory host transcriptional response via an unexpected STAT1-dependent but IFN-independent mechanism and identify IE1 as a candidate determinant of hCMV pathogenicity

Human Cytomegalovirus Fcγ Binding Proteins gp34 and gp68 Antagonize Fcγ Receptors I, II and III

Human cytomegalovirus (HCMV) establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG). Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs). Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs) interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab) and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity.DFG grant He 2526/6-2.European Commission grants QLRT-2001-01112 and MRTN-CT-2005-019248.Helmholtz Association through VISTRIE VH-VI-242.UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí