Relationship between physical and psychological functioning and health-related quality of life in congenital Aniridia.

PURPOSE: Congenital aniridia is a serious eye disease characterized by absence of iris to various degrees. The aims of this study were to investigate health-related quality of life (HRQoL) in adults with aniridia and assess the relationships between HRQoL, psychological status, ocular health and obesity. METHODS: Twenty-nine adults with congenital aniridia (48% male, aged 18-79 years) participated. HRQoL was measured with SF-36 and the EQ visual analogue scale (VAS). The physical (PCS) and mental (MCS) component summaries of the SF-36 were calculated with higher scores indicating better HRQoL. Symptoms of anxiety and depression were measured using the Hospital Anxiety and Depression Scale (HADS). Obesity was assessed with the Patient-Reported Outcomes in Obesity (PROS). Sociodemographic characteristics, genetic variants and ocular and medical health variables were also analysed. RESULTS: The participants scored significantly lower in the general health domain of the SF-36 than the general population (65.2 vs. 75.3, p = 0.017). The EQ VAS score was also lower in the aniridia group (64.9 vs. 77.9, p = 0.021). Low PCS score was correlated with presence of ocular pain (p = 0.019), high HADS score (p = 0.017) and high PROS score (p = 0.009). Low MCS score was related to higher educational level (p = 0.038) and high HADS score (p < 0.001). High HADS and PROS scores were both related to low EQ VAS scores. CONCLUSION: Adults with congenital aniridia scored worse on certain measures of HRQoL than the general population. Poorer HRQoL was associated with increased symptoms of anxiety, depression and obesity and with presence of ocular pain.

Self-reported symptoms of everyday executive dysfunction, daytime sleepiness, and fatigue and health status among adults with congenital aniridia: a descriptive study.

Background: Congenital aniridia is a rare genetic disorder of the eye characterized by visual impairment and progressive vision loss. While prior research has focused on ocular manifestations in individuals with aniridia, there is a dearth of research on impacts on cognition and mental health. The aims of this study were to describe subjective symptoms of everyday executive functioning, fatigue and sleepiness in adults with aniridia and to compare self-reported health status with that of a normative reference group. Methods: Twenty-nine adults (aged 18-79 years) with congenital aniridia were included in this online survey, of whom 52% were females. Participants completed self-report measures of executive functioning (The Behavior Rating Inventory of Executive Function-Adult Version), sleepiness, fatigue, and health status (EQ-5D-5L). Results: Participants reported relatively few problems in everyday executive functioning, with only 14% experiencing impaired executive functioning. Scores on the five EQ-5D-5L domains (mobility, self-care, usual activities, pain, and anxiety/depression) did not differ from those of the normative reference group. The frequencies of excessive daytime sleepiness and severe fatigue were 17% and 38%, respectively. Ocular pain was experienced by 62% of participants. Conclusions: The findings show that cognitive problems are related to and reflect self-reported health status and extent of fatigue. Moreover, those who suffered from ocular pain reported more difficulties with executive functioning, sleepiness and fatigue. These findings are important for understanding this disorder and supporting patients.

Pregnancy enables antibody protection against intracellular infection.

Adaptive immune components are thought to exert non-overlapping roles in antimicrobial host defence, with antibodies targeting pathogens in the extracellular environment and T cells eliminating infection inside cells1,2. Reliance on antibodies for vertically transferred immunity from mothers to babies may explain neonatal susceptibility to intracellular infections3,4. Here we show that pregnancy-induced post-translational antibody modification enables protection against the prototypical intracellular pathogen Listeria monocytogenes. Infection susceptibility was reversed in neonatal mice born to preconceptually primed mothers possessing L. monocytogenes-specific IgG or after passive transfer of antibodies from primed pregnant, but not virgin, mice. Although maternal B cells were essential for producing IgGs that mediate vertically transferred protection, they were dispensable for antibody acquisition of protective function, which instead required sialic acid acetyl esterase5 to deacetylate terminal sialic acid residues on IgG variable-region N-linked glycans. Deacetylated L. monocytogenes-specific IgG protected neonates through the sialic acid receptor CD226,7, which suppressed IL-10 production by B cells leading to antibody-mediated protection. Consideration of the maternal-fetal dyad as a joined immunological unit reveals protective roles for antibodies against intracellular infection and fine-tuned adaptations to enhance host defence during pregnancy and early life.

Increased Dietary Manganese Impairs Neutrophil Extracellular Trap Formation Rendering Neutrophils Ineffective at Combating Staphylococcus aureus.

Dietary metals can modify the risk to infection. Previously, we demonstrated that heightened dietary manganese (Mn) during systemic Staphylococcus aureus infection increases S. aureus virulence. However, immune cells also operate in these same environments and the effect of dietary Mn on neutrophil function in vivo has not been assessed. This study reveals that increased concentrations of Mn impairs mitochondrial respiration and superoxide production in neutrophils responding to S. aureus. As a result, high Mn accelerates primary degranulation, while impairing suicidal neutrophil extracellular trap (NET) formation, which decreases bactericidal activity. In vivo, elevated dietary Mn accumulated extracellularly in the heart, indicating that excess Mn may be more bioavailable in the heart. Coinciding with this phenotype, neutrophil function in the heart was most impacted by a high Mn diet, as neutrophils produced lower levels of mitochondrial superoxide and underwent less suicidal NET formation. Consistent with an ineffective neutrophil response when mice are on a high Mn diet, S. aureus burdens were increased in the heart and mice were more susceptible to systemic infection. Therefore, elevated dietary Mn not only affects S. aureus but also renders neutrophils less capable of restricting staphylococcal infection.

Altered Mitochondrial Homeostasis during Systemic Lupus Erythematosus Impairs Neutrophil Extracellular Trap Formation Rendering Neutrophils Ineffective at Combating Staphylococcus aureus.

Inflammation involves a delicate balance between pathogen clearance and limiting host tissue damage, and perturbations in this equilibrium promote disease. Patients suffering from autoimmune diseases, such as systemic lupus erythematosus (SLE), have higher levels of serum S100A9 protein and increased risk for infection. S100A9 is highly abundant within neutrophils and modulates antimicrobial activity in response to bacterial pathogens. We reasoned that increased serum S100A9 in SLE patients reflects accumulation of S100A9 protein in neutrophils and may indicate altered neutrophil function. In this study, we demonstrate elevated S100A9 protein within neutrophils from SLE patients, and MRL/lpr mice associates with lower mitochondrial superoxide, decreased suicidal neutrophil extracellular trap formation, and increased susceptibility to Staphylococcus aureus infection. Furthermore, increasing mitochondrial superoxide production restored the antibacterial activity of MRL/lpr neutrophils in response to S. aureus These results demonstrate that accumulation of intracellular S100A9 associates with impaired mitochondrial homeostasis, thereby rendering SLE neutrophils inherently less bactericidal.

Mitochondrial Calcium Uniporter Affects Neutrophil Bactericidal Activity during Staphylococcus aureus Infection.

Neutrophils simultaneously restrict Staphylococcus aureus dissemination and facilitate bactericidal activity during infection through the formation of neutrophil extracellular traps (NETs). Neutrophils that produce higher levels of mitochondrial superoxide undergo enhanced terminal NET formation (suicidal NETosis) in response to S. aureus; however, mechanisms regulating mitochondrial homeostasis upstream of neutrophil antibacterial processes are not fully resolved. Here, we demonstrate that mitochondrial calcium uptake 1 (MICU1)-deficient (MICU1-/-) neutrophils accumulate higher levels of calcium and iron within the mitochondria in a mitochondrial calcium uniporter (MCU)-dependent manner. Corresponding with increased ion flux through the MCU, mitochondrial superoxide production is elevated, thereby increasing the propensity for MICU1-/- neutrophils to undergo suicidal NETosis rather than primary degranulation in response to S. aureus. Increased NET formation augments macrophage killing of bacterial pathogens. Similarly, MICU1-/- neutrophils alone are not more antibacterial toward S. aureus, but rather, enhanced suicidal NETosis by MICU1-/- neutrophils facilitates increased bactericidal activity in the presence of macrophages. Similarly, mice with a deficiency in MICU1 restricted to cells expressing LysM exhibit lower bacterial burdens in the heart with increased survival during systemic S. aureus infection. Coinciding with the decrease in S. aureus burdens, MICU1-/- neutrophils in the heart produce higher levels of mitochondrial superoxide and undergo enhanced suicidal NETosis. These results demonstrate that ion flux by the MCU affects the antibacterial function of neutrophils during S. aureus infection.

Adolescents and parents' perception of Young Person's Face IT: An online intervention for adolescents struggling with conditions affecting their appearance.

Objective: A visible difference in appearance caused by a congenital or acquired condition can negatively affect adolescents' psychosocial well-being. Young Person's Face IT (YPF) is an online intervention based on cognitive behavioural therapy and social skills training, developed to help adolescents who struggle with adjusting to a visible difference. The objective of the present study was to explore adolescents' and parents' perceptions of the intervention's relevance and usefulness in supporting young people with appearance-related psychosocial concerns. Methods: Participants were adolescents (N = 76, aged 11-18) and parents (N = 15), recruited in a larger randomised controlled trial aiming at evaluating YPF. This qualitative study with descriptive data includes adolescents' ratings on YPF's usefulness, and interview data from adolescents and parents on their experiences with YPF. The interviews were analysed using a thematic approach. Results: Results indicated that YPF was experienced as useful and relevant. Interviews showed that adolescents felt validated through the programme's content, discovered that other young people had similar experiences and felt that YPF could contribute to changing self-perceptions for the better. However, results could not confirm whether perceived usefulness led to the development and use of new social skills in real-life situations. Conclusion: This study offers new perspectives on the relevance and usefulness of YPF in supporting adolescents with appearance-related psychosocial concerns. Findings suggest that updates and modifications are required so that YPF stays relevant and useful for adolescents in need of support. Trial registration number: NCT03165331.

Neutrophil extracellular traps enhance macrophage killing of bacterial pathogens.

Neutrophils and macrophages are critical to the innate immune response, but cooperative mechanisms used by these cells to combat extracellular pathogens are not well understood. This study reveals that S100A9-deficient neutrophils produce higher levels of mitochondrial superoxide in response to Staphylococcus aureus and, as a result, form neutrophil extracellular traps (suicidal NETosis). Increased suicidal NETosis does not improve neutrophil killing of S. aureus in isolation but augments macrophage killing. NET formation enhances antibacterial activity by increasing phagocytosis by macrophages and by transferring neutrophil-specific antimicrobial peptides to them. Similar results were observed in response to other phylogenetically distinct bacterial pathogens including Streptococcus pneumoniae and Pseudomonas aeruginosa, implicating this as an immune defense mechanism that broadly enhances antibacterial activity. These results demonstrate that achieving maximal bactericidal activity through NET formation requires macrophages and that accelerated and more robust suicidal NETosis makes neutrophils adept at increasing antibacterial activity, especially when A9 deficient.

Seroprevalence of SARS-CoV-2 infection in Cincinnati Ohio USA from August to December 2020.

The world is currently in a pandemic of COVID-19 (Coronavirus disease-2019) caused by a novel positive-sense, single-stranded RNA ß-coronavirus referred to as SARS-CoV-2. Here we investigated rates of SARS-CoV-2 infection in the greater Cincinnati, Ohio, USA metropolitan area from August 13 to December 8, 2020, just prior to initiation of the national vaccination program. Examination of 9,550 adult blood donor volunteers for serum IgG antibody positivity against the SARS-CoV-2 Spike protein showed an overall prevalence of 8.40%, measured as 7.56% in the first 58 days and 9.24% in the last 58 days, and 12.86% in December 2020, which we extrapolated to ~20% as of March, 2021. Males and females showed similar rates of past infection, and rates among Hispanic or Latinos, African Americans and Whites were also investigated. Donors under 30 years of age had the highest rates of past infection, while those over 60 had the lowest. Geographic analysis showed higher rates of infectivity on the West side of Cincinnati compared with the East side (split by I-75) and the lowest rates in the adjoining region of Kentucky (across the Ohio river). These results in regional seroprevalence will help inform efforts to best achieve herd immunity in conjunction with the national vaccination campaign.

Speech and language difficulties in Huntington's disease: A qualitative study of patients' and professional caregivers' experiences.

BACKGROUND: Huntington's disease (HD) is a neurodegenerative disease characterized by a triad of motor, cognitive and psychological symptoms, leading to a gradual breakdown of communication skills. Few studies have investigated how people affected by HD and their professional caregivers, for example, medical doctors, physiotherapists and nurses, experience the patients' gradual loss of speech and language. AIMS: To examine communication-related experiences of patients and professional caregivers. Experiences with speech therapy and the use of augmentative and alternative communication aids (AAC) were also investigated. METHODS & PROCEDURES: Seven individuals with HD and seven professional caregivers were interviewed individually, using a semi-structured interview guide. Transcripts were analysed using a conventional content analysis, and the results presented in three main categories. OUTCOMES & RESULTS: Most individuals with HD were aware of having communication difficulties, struggling with understanding others as well as being understood. This was confirmed by professional caregivers, who also raised ethical issues encountered when patients struggled with communication. Both groups talked about external factors (such as noise or crowded social settings) as disrupting communication, and shared recommendations on how people in general, and speech and language therapists (SLTs) in particular, could optimize communication. Very few patients had received information about communication aids, and none was using AACs. Professional caregivers underlined the importance of interdisciplinary collaborations, including SLTs, in order to optimize care. CONCLUSIONS & IMPLICATIONS: Findings shed a light on everyday communication challenges faced by people with HD and their professional caregivers, and the lack of implementation of communication aids in this group. The dramatic impact of HD on patients' communication skills underscores the need to include SLTs in the follow-up of this patient group, ideally from the early stages of the disease, while the patient is still capable of voicing his/her own wishes and thoughts. Future research that explores how to optimize communication and implement the use of AACs for individuals with HD is needed. What this paper adds What is already known on this subject Although the ability to communicate gradually deteriorates in individuals affected by Huntington's disease (HD), there is little knowledge about how affected individuals experience the loss of speech and language skills. Interdisciplinary care is recognised as essential for this patient group. However, professional caregivers' thoughts and experiences of communicating with their patients have not been fully explored. Recent years have seen a rapid growth of available communication supporting technologies that could potentially be helpful for individuals with HD, but limited attention has been given to this subject. What this paper adds to existing knowledge What do we now know as a result of this study that we did not know before the results highlight that patients are aware of problems with speech and language even in early phases of the disease, and include patients' personal outlook on problems with communication. Professional caregivers raised ethical issues encountered when patients struggled with communication. Both groups described specific strategies that could facilitate communication. There was a significant lack of experience and knowledge about augmentative and alternative communication aids (AACs). What are the potential or actual clinical implications of this work? Clinical implications of this study. The introduction of communication aids in the follow-up of patients with HD needs to be discussed with the patients in the early phases of the disease, and implemented while the individual still has the capacity to learn and take advantage of alternative communication support. Results underline the importance of including speech and language therapists (SLTs) into multidisciplinary care of patients with HD. SLTs should be available also for professional caregivers who need advice on how to facilitate conversations and social interactions, in order to optimise care of patients with HD.

Clostridioides difficile Senses and Hijacks Host Heme for Incorporation into an Oxidative Stress Defense System.

Clostridioides difficile infection of the colon leads to severe inflammation and damage to the gastrointestinal epithelium due to the production of potent toxins. This inflammatory tissue damage causes the liberation of high concentrations of host heme at infection sites. Here, we identify the C. difficile heme-sensing membrane protein system (HsmRA) and show that this operon induces a protective response that repurposes heme to counteract antimicrobial oxidative stress responses. HsmR senses vertebrate heme, leading to increased expression of the hsmRA operon and subsequent deployment of HsmA to capture heme and reduce redox damage caused by inflammatory mediators of protection and antibiotic therapy. Strains with inactivated hsmR or hsmA have increased sensitivity to redox-active compounds and reduced colonization persistence in a murine model of relapse C. difficile infection. These results define a mechanism exploited by C. difficile to repurpose toxic heme within the inflamed gut as a shield against antimicrobial compounds.

Light pollution increases West Nile virus competence of a ubiquitous passerine reservoir species.

Among the many anthropogenic changes that impact humans and wildlife, one of the most pervasive but least understood is light pollution. Although detrimental physiological and behavioural effects resulting from exposure to light at night are widely appreciated, the impacts of light pollution on infectious disease risk have not been studied. Here, we demonstrate that artificial light at night (ALAN) extends the infectious-to-vector period of the house sparrow (Passer domesticus), an urban-dwelling avian reservoir host of West Nile virus (WNV). Sparrows exposed to ALAN maintained transmissible viral titres for 2 days longer than controls but did not experience greater WNV-induced mortality during this window. Transcriptionally, ALAN altered the expression of gene regulatory networks including key hubs (OASL, PLBD1 and TRAP1) and effector genes known to affect WNV dissemination (SOCS). Despite mounting anti-viral immune responses earlier, transcriptomic signatures indicated that ALAN-exposed individuals probably experienced pathogen-induced damage and immunopathology, potentially due to evasion of immune effectors. A simple mathematical modelling exercise indicated that ALAN-induced increases of host infectious-to-vector period could increase WNV outbreak potential by approximately 41%. ALAN probably affects other host and vector traits relevant to transmission, and additional research is needed to advise the management of zoonotic diseases in light-polluted areas.

Clustering, Spatial Distribution, and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen I.

Discoidin domain receptors (DDR1 and DDR2) are receptor tyrosine kinases that signal in response to collagen. We had previously shown that collagen binding leads to clustering of DDR1b, a process partly mediated by its extracellular domain (ECD). In this study, we investigated (i) the impact of the oligomeric state of DDR2 ECD on collagen binding and fibrillogenesis, (ii) the effect of collagen binding on DDR2 clustering, and (iii) the spatial distribution and phosphorylation status of DDR1b and DDR2 after collagen stimulation. Studies were conducted using purified recombinant DDR2 ECD proteins in monomeric, dimeric or oligomeric state, and MC3T3-E1 cells expressing full-length DDR2-GFP or DDR1b-YFP. We show that the oligomeric form of DDR2 ECD displayed enhanced binding to collagen and inhibition of fibrillogenesis. Using atomic force and fluorescence microscopy, we demonstrate that unlike DDR1b, DDR2 ECD and DDR2-GFP do not undergo collagen-induced receptor clustering. However, after prolonged collagen stimulation, both DDR1b-YFP and DDR2-GFP formed filamentous structures consistent with spatial re-distribution of DDRs in cells. Immunocytochemistry revealed that while DDR1b clusters co-localized with non-fibrillar collagen, DDR1b/DDR2 filamentous structures associated with collagen fibrils. Antibodies against a tyrosine phosphorylation site in the intracellular juxtamembrane region of DDR1b displayed positive signals in both DDR1b clusters and filamentous structures. However, only the filamentous structures of both DDR1b and DDR2 co-localized with antibodies directed against tyrosine phosphorylation sites within the receptor kinase domain. Our results uncover key differences and similarities in the clustering abilities and spatial distribution of DDR1b and DDR2 and their impact on receptor phosphorylation.

Immobilized fibrinogen activates human platelets through glycoprotein VI.

Glycoprotein VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.

Fibrin and D-dimer bind to monomeric GPVI.

Fibrin has recently been shown to activate platelets through the immunoglobulin receptor glycoprotein VI (GPVI). In the present study, we show that spreading of human platelets on fibrin is abolished in patients deficient in GPVI, confirming that fibrin activates human platelets through the immunoglobulin receptor. Using a series of proteolytic fragments, we show that D-dimer, but not the E fragment of fibrin, binds to GPVI and that immobilized D-dimer induces platelet spreading through activation of Src and Syk tyrosine kinases. In contrast, when platelets are activated in suspension, soluble D-dimer inhibits platelet aggregation induced by fibrin and collagen, but not by a collagen-related peptide composed of a repeat GPO sequence or by thrombin. Using surface plasmon resonance, we demonstrate that fibrin binds selectively to monomeric GPVI with a KD of 302 nM, in contrast to collagen, which binds primarily to dimeric GPVI. These results establish GPVI as the major signaling receptor for fibrin in human platelets and provide evidence that fibrin binds to a distinct configuration of GPVI. This indicates that it may be possible to develop agents that selectively block the interaction of fibrin but not collagen with the immunoglobulin receptor. Such agents are required to establish whether selective targeting of either interaction has the potential to lead to development of an antithrombotic agent with a reduced effect on bleeding relative to current antiplatelet drugs.

Structural basis for collagen recognition by the immune receptor OSCAR.

The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 µM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2.

Activation of glycoprotein VI (GPVI) and C-type lectin-like receptor-2 (CLEC-2) underlies platelet activation by diesel exhaust particles and other charged/hydrophobic ligands.

Platelets are activated by a range of stimuli that share little or no resemblance in structure to each other or to recognized ligands, including diesel exhaust particles (DEP), small peptides [4N1-1, Champs (computed helical anti-membrane proteins), LSARLAF (Leu-Ser-Ala-Arg-Leu-Ala-Phe)], proteins (histones) and large polysaccharides (fucoidan, dextran sulfate). This miscellaneous group stimulate aggregation of human and mouse platelets through the glycoprotein VI (GPVI)-FcR γ-chain complex and/or C-type lectin-like receptor-2 (CLEC-2) as shown using platelets from mice deficient in either or both of these receptors. In addition, all of these ligands stimulate tyrosine phosphorylation in GPVI/CLEC-2-double-deficient platelets, indicating that they bind to additional surface receptors, although only in the case of dextran sulfate does this lead to activation. DEP, fucoidan and dextran sulfate, but not the other agonists, activate GPVI and CLEC-2 in transfected cell lines as shown using a sensitive reporter assay confirming a direct interaction with the two receptors. We conclude that this miscellaneous group of ligands bind to multiple proteins on the cell surface including GPVI and/or CLEC-2, inducing activation. These results have pathophysiological significance in a variety of conditions that involve exposure to activating charged/hydrophobic agents.

US28 is a potent activator of phospholipase C during HCMV infection of clinically relevant target cells.

Members of the cytomegalovirus family each encode two or more genes with significant homology to G-protein coupled receptors (GPCRs). In rodent models of pathogenesis, these viral encoded GPCRs play functionally significant roles, as their deletion results in crippled viruses that cannot traffic properly and/or replicate in virally important target cells. Of the four HCMV encoded GPCRs, US28 has garnered the most attention due to the fact that it exhibits both agonist-independent and agonist-dependent signaling activity and has been demonstrated to promote cellular migration and proliferation. Thus, it appears that the CMV GPCRs play important roles in viral replication in vivo as well as promote the development of virus-associated pathology. In the current study we have utilized a series of HCMV/US28 recombinants to investigate the expression profile and signaling activities of US28 in a number of cell types relevant to HCMV infection including smooth muscle cells, endothelial cells and cells derived from glioblastoma multiforme (GBM) tumors. The results indicate that US28 is expressed and exhibits constitutive agonist-independent signaling activity through PLC-ß in all cell types tested. Moreover, while CCL5/RANTES and CX3CL1/Fractalkine both promote US28-dependent Ca(++) release in smooth muscle cells, this agonist-dependent effect appears to be cell-specific as we fail to detect US28 driven Ca(++) release in the GBM cells. We have also investigated the effects of US28 on signaling via endogenous GPCRs including those in the LPA receptor family. Our data indicate that US28 can enhance signaling via endogenous LPA receptors. Taken together, our results indicate that US28 induces a variety of signaling events in all cell types tested suggesting that US28 signaling likely plays a significant role during HCMV infection and dissemination in vivo.

The carboxy-terminal tail of human cytomegalovirus (HCMV) US28 regulates both chemokine-independent and chemokine-dependent signaling in HCMV-infected cells.

The human cytomegalovirus (HCMV)-encoded G-protein-coupled receptor (GPCR) US28 is a potent activator of a number of signaling pathways in HCMV-infected cells. The intracellular carboxy-terminal domain of US28 contains residues critical for the regulation of US28 signaling in heterologous expression systems; however, the role that this domain plays during HCMV infection remains unknown. For this study, we constructed an HCMV recombinant virus encoding a carboxy-terminal domain truncation mutant of US28, FLAG-US28/1-314, to investigate the role that this domain plays in US28 signaling. We demonstrate that US28/1-314 exhibits a more potent phospholipase C-beta (PLC-beta) signal than does wild-type US28, indicating that the carboxy-terminal domain plays an important role in regulating agonist-independent signaling in infected cells. Moreover, HMCV-infected cells expressing the US28/1-314 mutant exhibit a prolonged calcium signal in response to CCL5, indicating that the US28 carboxy-terminal domain also regulates agonist-dependent signaling. Finally, while the chemokine CX3CL1 behaves as an inverse agonist or inhibitor of constitutive US28 signaling to PLC-beta, we demonstrate that CX3CL1 functions as an agonist with regard to US28-stimulated calcium release. This study is the first to demonstrate that the carboxy terminus of US28 controls US28 signaling in the context of HCMV infection and indicates that chemokines such as CX3CL1 can decrease constitutive US28 signals and yet simultaneously promote nonconstitutive US28 signals.