A Viral Immunoevasin Controls Innate Immunity by Targeting the Prototypical Natural Killer Cell Receptor Family.

Natural killer (NK) cells play a key role in innate immunity by detecting alterations in self and non-self ligands via paired NK cell receptors (NKRs). Despite identification of numerous NKR-ligand interactions, physiological ligands for the prototypical NK1.1 orphan receptor remain elusive. Here, we identify a viral ligand for the inhibitory and activating NKR-P1 (NK1.1) receptors. This murine cytomegalovirus (MCMV)-encoded protein, m12, restrains NK cell effector function by directly engaging the inhibitory NKR-P1B receptor. However, m12 also interacts with the activating NKR-P1A/C receptors to counterbalance m12 decoy function. Structural analyses reveal that m12 sequesters a large NKR-P1 surface area via a "polar claw" mechanism. Polymorphisms in, and ablation of, the viral m12 protein and host NKR-P1B/C alleles impact NK cell responses in vivo. Thus, we identify the long-sought foreign ligand for this key immunoregulatory NKR family and reveal how it controls the evolutionary balance of immune recognition during host-pathogen interplay.

MicroRNA-142 Is Critical for the Homeostasis and Function of Type 1 Innate Lymphoid Cells.

Natural killer (NK) cells are cytotoxic type 1 innate lymphoid cells (ILCs) that defend against viruses and mediate anti-tumor responses, yet mechanisms controlling their development and function remain incompletely understood. We hypothesized that the abundantly expressed microRNA-142 (miR-142) is a critical regulator of type 1 ILC biology. Interleukin-15 (IL-15) signaling induced miR-142 expression, whereas global and ILC-specific miR-142-deficient mice exhibited a cell-intrinsic loss of NK cells. Death of NK cells resulted from diminished IL-15 receptor signaling within miR-142-deficient mice, likely via reduced suppressor of cytokine signaling-1 (Socs1) regulation by miR-142-5p. ILCs persisting in Mir142-/- mice demonstrated increased expression of the miR-142-3p target αV integrin, which supported their survival. Global miR-142-deficient mice exhibited an expansion of ILC1-like cells concurrent with increased transforming growth factor-ß (TGF-ß) signaling. Further, miR-142-deficient mice had reduced NK-cell-dependent function and increased susceptibility to murine cytomegalovirus (MCMV) infection. Thus, miR-142 critically integrates environmental cues for proper type 1 ILC homeostasis and defense against viral infection.

Cytomegalovirus infection induces Alzheimer's disease-associated alterations in tau.

Alzheimer's disease (AD) manifests with loss of neurons correlated with intercellular deposition of amyloid (amyloid plaques) and intracellular neurofibrillary tangles of hyperphosphorylated tau. However, targeting AD hallmarks has not as yet led to development of an effective treatment despite numerous clinical trials. A better understanding of the early stages of neurodegeneration may lead to development of more effective treatments. One underexplored area is the clinical correlation between infection with herpesviruses and increased risk of AD. We hypothesized that similar to work performed with herpes simplex virus 1 (HSV1), infection with the cytomegalovirus (CMV) herpesvirus increases levels and phosphorylation of tau, similar to AD tauopathy. We used murine CMV (MCMV) to infect mouse fibroblasts and rat neuronal cells to test our hypothesis. MCMV infection increased steady-state levels of primarily high molecular weight forms of tau and altered the patterns of tau phosphorylation. Both changes required viral late gene products. Glycogen synthase kinase 3 beta (GSK3ß) was upregulated in the HSVI model, but inhibition with lithium chloride suggested that this enzyme is unlikely to be involved in MCMV infection mediated tau phosphorylation. Thus, we confirm that MCMV, a beta herpes virus, like alpha herpes viruses (e.g., HSV1), can promote tau pathology. This suggests that CMV infection can be useful as another model system to study mechanisms leading to neurodegeneration. Since MCMV infects both mice and rats as permissive hosts, our findings from tissue culture can likely be applied to a variety of AD models to study development of abnormal tau pathology.

Murine cytomegalovirus localization and uveitic cell infiltration might both contribute to trabecular meshwork impairment in Posner-Schlossman syndrome: Evidence from an open-angle rat model.

As a special type of glaucoma, Posner-Schlossman syndrome (PSS) is characterized by elevated intraocular pressure (IOP) and anterior uveitis. Cytomegalovirus (CMV) anterior chamber infection has now been considered the leading cause of PSS. We used murine CMV (MCMV) intracameral injection to establish a rat model manifested in IOP elevation and mild anterior uveitis, much like PSS; viral localization and gene expression at various time points and inflammatory cell infiltration derived from innate and adaptive immunity were investigated, as well as pathogenetic changes of the trabecular meshwork (TM). The IOP and uveitic manifestations peaked at 24 h post-infection (p.i.) and returned to normal after 96 h; the iridocorneal angle remained open consistently. At 24 h p.i., leucocytes gathered at the chamber angle. Maximum transcription of MCMV immediate early 1 (IE1) was reached at 24 h in the cornea and 48 h in the iris and ciliary body. MCMV localized in aqueous humor outflow facilities and the iris from 24 h to 28 d p.i. and was detected by in situ hybridization, though it did not transcribe after 7 d p.i. TM and iris pigment epithelial cells harboring viral inclusion bodies and autophagosomes were present at 28 d p.i. These findings shed light on how and where innate and adaptive immunity reacted after MCMV was found and transcribed in a highly ordered cascade, as well as pathogenetic changes in TM as a result of virus and uveitis behaviors.

Selective Inhibition of Murine Cytomegalovirus Viral Gene Expression by the Antiviral Peptide TAT-I24.

The effect of the antiviral peptide TAT-I24 on viral gene expression in cells infected with murine cytomegalovirus (MCMV) was investigated. The expression of immediate-early, early and late genes was highly induced upon infection with MCMV. In the presence of the peptide, the expression of all tested genes was sustainably reduced to a similar extent, independent of whether they were immediate-early, early or late genes. In contrast, the expression of host genes, such as NF-κB inhibitor alpha (Nfkbia), interferon-induced protein with tetratricopeptide repeats 1 (Ifit1), chemokine (C-X-C motif) ligand 10 (Cxcl10), chemokine (C-C motif) ligand 7 (Ccl7) and chemokine (C-C motif) ligand 5 (Ccl5), which are induced early upon virus infection, was only transiently suppressed in peptide-treated cells. The expression of other host genes which are affected by MCMV infection and play a role in endoplasmic reticulum stress or DNA-damage repair was not inhibited by the peptide. A combination of TAT-I24 with the nucleoside analogue cidofovir showed enhancement of the antiviral effect, demonstrating that viral replication can be more efficiently inhibited with a combination of drugs acting at different stages of the viral life-cycle.

Inhibition of autophagy by 3-methyladenine restricts murine cytomegalovirus replication.

Cytomegalovirus (CMV) induced autophagy affects virus replication and survival of the infected cells. The purpose of this study was to investigate the role of autophagy inhibition by 3-methyladenine (3-MA) on murine cytomegalovirus (MCMV) replication and whether it is associated with caspase-3 dependent apoptosis. The eyecup isolated from adult C57BL/6J mice (6-8 weeks old) and mouse embryo fibroblast cells (MEFs) were infected with MCMV K181 strain, followed by the treatment of 3-methyladenine (3-MA), chloroquine, or rapamycin to block or stimulate autophagy. In cultured MEFs, the ratio of LC3I/II was reduced at 24 hours post infection (hpi), but was increased at 48 hpi In the eyecup culture, LC3I/II ratio was also decreased at 4 and 7 days post infection (dpi). In addition, caspase-3 cleavage was increased at 48 hpi in MEFs and also elevated in MCMV infected eyecups at 4, 7, 10, and 14 dpi. 3-MA treatment significantly inhibited the virus replication in MEFs and eyecups. The expression of early antigen (EA) of MCMV was also decreased in MEFs and eyecups. Meanwhile, cleaved caspase-3 dependent cell death was promoted with the presence of 3-MA in MCMV infected MEFs and eyecups, while RIPK1/RIPK3/MLKL pathway was inhibited by 3-MA in eyecups. Inhibition of autophagy by 3-MA restricts virus replication and promotes caspase-3 dependent apoptosis in the eyecup and MEFs with MCMV infection. It can be explained that during the early period of MCMV infection, the suppressed autophagy process directly reduced virus release, but later caspase-3 dependent apoptosis dominated and resulted in decreased virus replication.

Atypical cytomegalovirus retinal disease in pyroptosis-deficient mice with murine acquired immunodeficiency syndrome.

Pyroptosis is a caspase-dependent programmed cell death pathway that initiates and sustains inflammation through release of pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 following formation of gasdermin D (GSDMD)-mediated membrane pores. To determine the possible pathogenic contributions of pyroptosis toward development of full-thickness retinal necrosis during AIDS-related human cytomegalovirus retinitis, we performed a series of studies using an established model of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS). Initial investigations demonstrated significant transcription and translation of key pyroptosis-associated genes within the ocular compartments of MCMV-infected eyes of mice with MAIDS. Subsequent investigations compared MCMV-infected eyes of groups of wildtype MAIDS mice with MCMV-infected eyes of groups of caspase-1-/- MAIDS mice, GSDMD-/- MAIDS mice, or IL-18-/- MAIDS mice to explore a possible contribution of pyroptosis towards the pathogenesis of MAIDS-related MCMV retinitis. Histopathologic analysis revealed typical full-thickness retinal necrosis in 100% of MCMV-infected eyes of wildtype MAIDS mice. In sharp contrast, none (0%) of MCMV-infected eyes of MAIDS mice that were deficient in either caspase-1, GSDMD, or IL-18 developed full-thickness retinal necrosis but instead exhibited an atypical pattern of retinal disease characterized by thickening and proliferation of the retinal pigmented epithelium layer with relative sparing of the neurosensory retina. Surprisingly, MCMV-infected eyes of all groups of deficient MAIDS mice harbored equivalent intraocular amounts of infectious virus as seen in MCMV-infected eyes of groups of wildtype MAIDS mice despite failure to develop full-thickness retinal necrosis. We conclude that pyroptosis plays a significant role in the development of full-thickness retinal necrosis during the pathogenesis of MAIDS-related MCMV retinitis. This observation may extend to the pathogenesis of AIDS-related HCMV retinitis and other AIDS-related opportunistic virus infections.

The Role of Caspase-12 in Retinal Bystander Cell Death and Innate Immune Responses against MCMV Retinitis.

(1) Background: caspase-12 is activated during cytomegalovirus retinitis, although its role is presently unclear. (2) Methods: caspase-12-/- (KO) or caspase-12+/+ (WT) mice were immunosup eyes were analyzed by plaque assay, TUNEL assay, immunohistochemical staining, western blotting, and real-time PCR. (3) Results: increased retinitis and a more extensive virus spread were detected in the retina of infected eyes of KO mice compared to WT mice at day 14 p.i. Compared to MCMV injected WT eyes, mRNA levels of interferons α, ß and γ were significantly reduced in the neural retina of MCMV-infected KO eyes at day 14 p.i. Although similar numbers of MCMV infected cells, similar virus titers and similar numbers of TUNEL-staining cells were detected in injected eyes of both KO and WT mice at days 7 and 10 p.i., significantly lower amounts of cleaved caspase-3 and p53 protein were detected in infected eyes of KO mice at both time points. (4) Conclusions: caspase-12 contributes to caspase-3-dependent and independent retinal bystander cell death during MCMV retinitis and may also play an important role in innate immunity against virus infection of the retina.

Viral Induced Protein Aggregation: A Mechanism of Immune Evasion.

Various intrinsic and extrinsic factors can interfere with the process of protein folding, resulting in protein aggregates. Usually, cells prevent the formation of aggregates or degrade them to prevent the cytotoxic effects they may cause. However, during viral infection, the formation of aggregates may serve as a cellular defense mechanism. On the other hand, some viruses are able to exploit the process of aggregate formation and removal to promote their replication or evade the immune response. This review article summarizes the process of cellular protein aggregation and gives examples of how different viruses exploit it. Particular emphasis is placed on the ribonucleotide reductases of herpesviruses and how their additional non-canonical functions in viral immune evasion are closely linked to protein aggregation.

Parthanatos-associated proteins are stimulated intraocularly during development of experimental murine cytomegalovirus retinitis in mice with retrovirus-induced immunosuppression.

The mechanisms that contribute to retinal tissue destruction during the onset and progression of AIDS-related human cytomegalovirus (HCMV) retinitis remain unclear. Evidence for the stimulation of multiple cell death pathways including apoptosis, necroptosis, and pyroptosis during the pathogenesis of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS) has been reported. Parthanatos is a caspase-independent cell death pathway mediated by rapid overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) and distinct from other cell death pathways. Using the MAIDS model of MCMV retinitis, studies were performed to test the hypothesis that intraocular MCMV infection of mice with MAIDS stimulates parthanatos-associated messenger RNAs (mRNAs) and proteins within the eye during the development of retinal necrosis that takes place by 10 days after MCMV infection. MCMV-infected eyes of MAIDS mice exhibited significant stimulation of PARP-1 mRNA and proteins at 3 days after infection but declined thereafter at 6 and 10 days after infection. Additional studies showed the intraocular stimulation of mRNAs or proteins before MCMV retinitis development for two additional participants in parthanatos, polymer of ADP-ribose and poly (ADP-ribose) glycohydrolase. These results provide new evidence for a role for parthanatos during MAIDS-related MCMV retinitis that may also extend to AIDS-related HCMV retinitis.

Mast cells within cellular networks.

Mast cells are highly versatile in terms of their mode of activation by a host of stimuli and their ability to flexibly release a plethora of biologically highly active mediators. Within the immune system, mast cells can best be designated as an active nexus interlinking innate and adaptive immunity. Here we try to draw an arc from initiation of acute inflammatory reactions to microbial pathogens to development of adaptive immunity and allergies. This multifaceted nature of mast cells is made possible by interaction with multiple cell types of immunologic and nonimmunologic origin. Examples for the former include neutrophils, eosinophils, T cells, and professional antigen-presenting cells. These interactions allow mast cells to orchestrate inflammatory innate reactions and complex adaptive immunity, including the pathogenesis of allergies. Important partners of nonimmunologic origin include cells of the sensory neuronal system. The intimate association between mast cells and sensory nerve fibers allows bidirectional communication, leading to neurogenic inflammation. Evidence is accumulating that this mast cell/nerve crosstalk is of pathophysiologic relevance in patients with allergic diseases, such as asthma.

Suppressor of Cytokine Signaling 1 (SOCS1) and SOCS3 Are Stimulated within the Eye during Experimental Murine Cytomegalovirus Retinitis in Mice with Retrovirus-Induced Immunosuppression.

AIDS-related human cytomegalovirus retinitis remains the leading cause of blindness among untreated HIV/AIDS patients worldwide. To study mechanisms of this disease, we used a clinically relevant animal model of murine cytomegalovirus (MCMV) retinitis with retrovirus-induced murine AIDS (MAIDS) that mimics the progression of AIDS in humans. We found in this model that MCMV infection significantly stimulates ocular suppressor of cytokine signaling 1 (SOCS1) and SOCS3, host proteins which hinder immune-related signaling by cytokines, including antiviral type I and type II interferons. The present study demonstrates that in the absence of retinal disease, systemic MCMV infection of mice without MAIDS, but not in mice with MAIDS, leads to mild stimulation of splenic SOCS1 mRNA. In sharp contrast, when MCMV is directly inoculated into the eyes of retinitis-susceptible MAIDS mice, high levels of intraocular SOCS1 and SOCS3 mRNA and protein are produced which are associated with significant intraocular upregulation of gamma interferon (IFN-γ) and interleukin-6 (IL-6) mRNA expression. We also show that infiltrating macrophages, granulocytes, and resident retinal cells are sources of intraocular SOCS1 and SOCS3 protein production during development of MAIDS-related MCMV retinitis, and SOCS1 and SOCS3 mRNA transcripts are detected in retinal areas histologically characteristic of MCMV retinitis. Furthermore, SOCS1 and SOCS3 are found in both MCMV-infected cells and uninfected cells, suggesting that these SOCS proteins are stimulated via a bystander mechanism during MCMV retinitis. Taken together, our findings suggest a role for MCMV-related stimulation of SOCS1 and SOCS3 in the progression of retinal disease during ocular, but not systemic, MCMV infection.IMPORTANCE Cytomegalovirus infection frequently causes blindness in untreated HIV/AIDS patients. This virus manipulates host cells to dysregulate immune functions and drive disease. Here, we use an animal model of this disease to demonstrate that cytomegalovirus infection within eyes during retinitis causes massive upregulation of immunosuppressive host proteins called SOCS. As viral overexpression of SOCS proteins exacerbates infection with other viruses, they may also enhance cytomegalovirus infection. Alternatively, the immunosuppressive effect of SOCS proteins may be protective against immunopathology during cytomegalovirus retinitis, and in such a case SOCS mimetics or overexpression treatment strategies might be used to combat this disease. The results of this work therefore provide crucial basic knowledge that contributes to our understanding of the mechanisms of AIDS-related cytomegalovirus retinitis and, together with future studies, may contribute to the development of novel therapeutic targets that could improve the treatment or management of this sight-threatening disease.

Immunometabolic phenotype of BV-2 microglia cells upon murine cytomegalovirus infection.

Microglia are resident brain macrophages with key roles in development and brain homeostasis. Cytomegalovirus (CMV) readily infects microglia cells, even as a possible primary target of infection in development. Effects of CMV infection on a cellular level in microglia are still unclear; therefore, the aim of this research was to assess the immunometabolic changes of BV-2 microglia cells following the murine cytomegalovirus (MCMV) infection. In light of that aim, we established an in vitro model of ramified BV-2 microglia (BV-2∅FCS, inducible nitric oxide synthase (iNOSlow), arginase-1 (Arg-1high), mannose receptor CD206high, and hypoxia-inducible factor 1α (HIF-1αlow)) to better replicate the in vivo conditions by removing FCS from the cultivation media, while the cells cultivated in 10% FCS DMEM displayed an ameboid morphology (BV-2FCS high, iNOShigh, Arg-1low, CD206low, and HIF-1αhigh). Experiments were performed using both ramified and ameboid microglia, and both of them were permissive to productive viral infection. Our results indicate that MCMV significantly alters the immunometabolic phenotypic properties of BV-2 microglia cells through the manipulation of iNOS and Arg-1 expression patterns, along with an induction of a glycolytic shift in the infected cell cultures.

Murine cytomegalovirus IE3-dependent transcription is required for DAI/ZBP1-mediated necroptosis.

DNA-dependent activator of interferon regulatory factors/Z-DNA binding protein 1 (DAI/ZBP1) is a crucial sensor of necroptotic cell death induced by murine cytomegalovirus (MCMV) in its natural host. Here, we show that viral capsid transport to the nucleus and subsequent viral IE3-dependent early transcription are required for necroptosis. Necroptosis induction does not depend on input virion DNA or newly synthesized viral DNA A putative RNA-binding domain of DAI/ZBP1, Zα2, is required to sense virus and trigger necroptosis. Thus, MCMV IE3-dependent transcription from the viral genome plays a crucial role in activating DAI/ZBP1-dependent necroptosis. This implicates RNA transcripts generated by a large double-stranded DNA virus as a biologically relevant ligand for DAI/ZBP1 during natural viral infection.

Autophagy protects against retinal cell death in mouse model of cytomegalovirus retinitis.

BACKGROUND: Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis (CMV). Our previous results have shown that there is a functional relationship between autophagy and apoptosis during MCMV infection of retinal pigment epithelium (RPE). The purpose of this study was to determine whether autophagy plays a significant role in the death of retinal cells during MCMV retinitis. METHODS: The retinas of adult BALB/c mice were infected with MCMV via supraciliary injection. Rapamycin, a mTOR inhibitor, was injected to MCMV-infected BALB/c mice intraperitoneally. Immunohistochemistry and western blot were performed to observe the spread pattern of virus in retinas and the levels of targeted proteins. Plaque assay was performed to determine the virus titer in different groups. Since Atg5 is a key gene regulating autophagy, we bred Atg5flox/flox; Nestin-Cre mice to deeply elucidate the role of autophagy during MCMV retinitis. Atg5flox/flox; Nestin-Cre mice were genotyped and infected with MCMV. Immunohistochemistry was performed to observe the type of virus-infected cells and apoptosis in retinas during MCMV retinitis. RESULTS: In MCMV mouse model, MCMV infection in outer nuclear layer (ONL) and inner nuclear layer (INL) in the retinas caused cleaved caspase 3 positive apoptosis, which is not co-localized with early antigen (EA) positive virus infected cells in rapamycin treated group. Rapamycin treatment increased the levels of LC3B-II by inhibiting mTOR and decreased the levels of cleaved caspase-3 during MCMV retinitis. However, virus propagation was not affected by rapamycin. In Atg5flox/flox; Nestin-Cre mice, RPE and glial cells were the main targets of viral infection, and number of EA positive retinal cells and TUNEL positive retinal cells was significantly increased compared to Atg5flox/+; Nestin-Cre mice though there was no difference of virus propagation between Atg5flox/flox; Nestin-Cre mice and Atg5flox/+; Nestin-Cre mice. CONCLUSIONS: Autophagy protects retinal cells from MCMV infection induced apoptosis through mTOR-mediated signaling pathway.

The murine cytomegalovirus immunoevasin gp40 binds MHC class I molecules to retain them in the early secretory pathway.

In the presence of the murine cytomegalovirus (mCMV) gp40 (m152) protein, murine major histocompatibility complex (MHC) class I molecules do not reach the cell surface but are retained in an early compartment of the secretory pathway. We find that gp40 does not impair the folding or high-affinity peptide binding of the class I molecules but binds to them, leading to their retention in the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, most likely by retrieval from the cis-Golgi to the ER. We identify a sequence in gp40 that is required for both its own retention in the early secretory pathway and for that of class I molecules.

Heterologous Immunity and Persistent Murine Cytomegalovirus Infection.

One's history of infections can affect the immune response to unrelated pathogens and influence disease outcome through the process of heterologous immunity. This can occur after acute viral infections, such as infections with lymphocytic choriomeningitis virus (LCMV) and vaccinia virus, where the pathogens are cleared, but it becomes a more complex issue in the context of persistent infections. In this study, murine cytomegalovirus (MCMV) was used as a persistent infection model to study heterologous immunity with LCMV. If mice were previously immune to LCMV and then infected with MCMV (LCMV+MCMV), they had more severe immunopathology, enhanced viral burden in multiple organs, and suppression of MCMV-specific T cell memory inflation. MCMV infection initially reduced the numbers of LCMV-specific memory T cells, but continued MCMV persistence did not further erode memory T cells specific to LCMV. When MCMV infection was given first (MCMV+LCMV), the magnitude of the acute T cell response to LCMV declined with age though this age-dependent decline was not dependent on MCMV. However, some of these MCMV persistently infected mice with acute LCMV infection (7 of 36) developed a robust immunodominant CD8 T cell response apparently cross-reactive between a newly defined putative MCMV epitope sequence, M57727-734, and the normally subdominant LCMV epitope L2062-2069, indicating a profound private specificity effect in heterologous immunity between these two viruses. These results further illustrate how a history of an acute or a persistent virus infection can substantially influence the immune responses and immune pathology associated with acute or persistent infections with an unrelated virus. IMPORTANCE: This study extends our understanding of heterologous immunity in the context of persistent viral infection. The phenomenon has been studied mostly with viruses such as LCMV that are cleared, but the situation can be more complex with a persistent virus such as MCMV. We found that the history of LCMV infection intensifies MCMV immunopathology, enhances MCMV burden in multiple organs, and suppresses MCMV-specific T cell memory inflation. In the reverse infection sequence, we show that some of the long-term MCMV-immune mice mount a robust CD8 T cell cross-reactive response between a newly defined putative MCMV epitope sequence and a normally subdominant LCMV epitope. These results further illustrate how a history of infection can substantially influence the immune responses and immune pathology associated with infections with an unrelated virus.

Electrochemical detection of a single cytomegalovirus at an ultramicroelectrode and its antibody anchoring.

We report observations of stochastic collisions of murine cytomegalovirus (MCMV) on ultramicroelectrodes (UMEs), extending the observation of discrete collision events on UMEs to biologically relevant analytes. Adsorption of an antibody specific for a virion surface glycoprotein allowed differentiation of MCMV from MCMV bound by antibody from the collision frequency decrease and current magnitudes in the electrochemical collision experiments, which shows the efficacy of the method to size viral samples. To add selectivity to the technique, interactions between MCMV, a glycoprotein-specific primary antibody to MCMV, and polystyrene bead "anchors," which were functionalized with a secondary antibody specific to the Fc region of the primary antibody, were used to affect virus mobility. Bead aggregation was observed, and the extent of aggregation was measured using the electrochemical collision technique. Scanning electron microscopy and optical microscopy further supported aggregate shape and extent of aggregation with and without MCMV. This work extends the field of collisions to biologically relevant antigens and provides a novel foundation upon which qualitative sensor technology might be built for selective detection of viruses and other biologically relevant analytes.

Reduced frequency of murine cytomegalovirus retinitis in C57BL/6 mice correlates with low levels of suppressor of cytokine signaling (SOCS)1 and SOCS3 expression within the eye during corticosteroid-induced immunosuppression.

AIDS-related human cytomegalovirus retinitis remains a leading cause of blindness worldwide. We compared two C57BL/6 mouse models of experimental murine cytomegalovirus (MCMV) retinitis for intraocular expression of suppressors of cytokine signaling (SOCS)1 and SOCS3, host proteins that are inducible negative feedback regulators of cytokine signaling. These mouse models differed in method of immune suppression, one by retrovirus-induced immune suppression (MAIDS) and the other by corticosteroid-induced immune suppression. Following subretinal injection of MCMV to induce retinitis, intraocular SOCS1 and SOCS3 were only mildly stimulated, and often without significance, within MCMV-infected eyes during the progression of MCMV retinitis in corticosteroid-immunosuppressed mice, contrary to MCMV-infected eyes of mice with MAIDS that showed significant high stimulation of SOCS1 and SOCS3 expression in agreement with previous findings. Frequency and severity of retinitis as well as amounts of intraocular infectious MCMV in corticosteroid-immunosuppressed mice were also unexpectedly lower than values previously reported for MAIDS animals during MCMV retinitis. These data reveal a major difference between two mouse models of experimental MCMV retinitis and suggest a possible link between the amplitude of SOCS1 and SOCS3 stimulation and severity of disease in these models.

Expression pattern of CD11c on lung immune cells after disseminated murine cytomegalovirus infection.

BACKGROUND: Cytomegalovirus (CMV) infection occurs frequently and is widespread globally. Numerous studies have shown that various types of immune cells play roles in mediating the response to CMV infection. CD11c, a commonly used dendritic cell (DC) marker, is expressed by other immune cells as well, such as T cells. This study analyzed the immune cells that express CD11c and monitored the expression level of their specific cell surface markers in the lung following a disseminated murine (M)CMV infection. METHODS: Mouse models of disseminated MCMV infection were used; uninfected and lipopolysaccharide (LPS)-treated mice were used as controls. At 1, 3 and 7 days following infection, single cell suspensions prepared from freshly digested lung tissue were stained for CD11c, CD86 and MHC II. Stained cells were analyzed using flow cytometry. Peripheral blood and single cell suspensions from spleen were sorted as well. Then these cells were subjected to analyze the CD11c expression pattern on natural killer (NK) cells and T cells. RESULTS: This assay showed that after MCMV infection, the expression of CD86 on pulmonary CD11chiMHC-IIhi cells (encompassing conventional DCs) was higher at 3 days post-infection than at 1 or 7 days post-infection, accompanied by a downregulation of MHC II. In addition, expression of CD11c was greatly increased in the MCMV infection group at 7 days post infection. This study also detected a large population of cells displaying an intermediate level of expression of CD11c (CD11cint); these cells were in the MCMV groups exclusively, and were subsequently identified as CD8+ T cells. In lung, spleen and blood, different proportions of CD11cint cells among the NK cell and T cell populations were observed between the BALB/c and C57BL/6 mice with or without MCMV infection. The expression level of NKp46 in NK cells dropped to a lower level after MCMV infection. CONCLUSIONS: The findings collectively indicate that CD11cintCD8+ T cells might play a key role in anti-MCMV adaptive immune response in lungs, as well as in spleen and blood. B220+CD11cint NK cells might be a more effective type of NK cell, participating in anti-MCMV infection. The downregulation of NKp46, in particular, might be linked with the immune evasion of MCMV.