CMV Chemokines and Co-infection: A Dissemination Plot that Peptides Can Foil

Human Cytomegalovirus (HCMV) is the leading cause of both non-hereditary mental retardation and hearing loss, and CMV infection/reactivation causes serious complications in transplant and immune compromised patients. Due to these issues, development of a CMV vaccine and/or therapeutics is required. To achieve this goal, it is necessary to gain a better understanding of CMV pathogenesis. Because of its coevolution with humans, HCMV has evolved genes with homology to human immune modulatory genes. Several of these genes help CMV establish a successful and lifelong infection within the host. An example is the viral CXC chemokine homolog UL146 gene (vCXCL-1). UL146 varies between clinical isolates and has been associated with clinical outcomes of HCMV infection. In this dissertation we characterized the vCXCL-1 protein from different clinical isolates in vitro (chapter 1). We hypothesized that, variability in vCXCL-1 leads to differential activation of neutrophils, which in turn leads to the observed differences in HCMV pathogenesis. In this study we identified the similarities and differences in the functional activity of vCXCL-1 from different HCMV isolates and suggest how the variability can affect neutrophil function and CMV pathogenesis. In order to understand the contribution of vCXCL-1 in the pathogenesis of CMV infection in vivo (chapter 2), we tested the hypothesis that vCXCL-1 from chimpanzee CMV (vCXCL-1CCMV) is a functional CXC chemokine and contributes to viral dissemination, similar to MCMV CC chemokine. However, contrary to this hypothesis, we found that overexpression of the chemokine is detrimental to the dissemination of MCMV by recruiting more inflammatory monocytes and NK cells to the site of infection. In an effort to develop a novel anti-CMV treatment, we tested the hypothesis that heparan sulfate binding peptides can act as potential antivirals (chapter 3). Peptides of different lengths and net charge were generated and tested for their ability to prevent MCMV infections. Of those tested the cationic peptides reduced MCMV infection in vitro by ~ 90%. In summary my research suggests that over expression of chemokines can attenuate CMV dissemination making it a potential vaccine candidate and that a peptide that binds to heparan sulfate can be a potential CMV therapeutic

PER PREFIX FLEXIBLE METRIC FOR INTENT BASED ROUTING AND TRAFFIC ENGINEERING USING EIGRP

Routing protocols are typically designed to use a single-cost formula for all of a protocol\u27s routes. In some instances, a network administrator can adjust the cost formula of the Enhanced Interior Gateway Routing Protocol (EIGRP), but the protocol applies the formula uniformly to all prefixes, which can cause all applications to suffer from lower performance. In order to address such issues, techniques are presented herein through which a flexible metric can be applied that is isolated to prefixes. for example, the techniques presented herein provide for the reduced use of Virtual Private Network (VPN) routing & forwarding (VRF) instances on routers and ease network configuration as configurations provided in accordance with the techniques presented herein are only needed at prefix originators

Tissue Determinants of Human NK Cell Development, Function, and Residence.

Immune responses in diverse tissue sites are critical for protective immunity and homeostasis. Here, we investigate how tissue localization regulates the development and function of human natural killer (NK) cells, innate lymphocytes important for anti-viral and tumor immunity. Integrating high-dimensional analysis of NK cells from blood, lymphoid organs, and mucosal tissue sites from 60 individuals, we identify tissue-specific patterns of NK cell subset distribution, maturation, and function maintained across age and between individuals. Mature and terminally differentiated NK cells with enhanced effector function predominate in blood, bone marrow, spleen, and lungs and exhibit shared transcriptional programs across sites. By contrast, precursor and immature NK cells with reduced effector capacity populate lymph nodes and intestines and exhibit tissue-resident signatures and site-specific adaptations. Together, our results reveal anatomic control of NK cell development and maintenance as tissue-resident populations, whereas mature, terminally differentiated subsets mediate immunosurveillance through diverse peripheral sites. VIDEO ABSTRACT

Novel Heparan Sulfate-Binding Peptides for Blocking Herpesvirus Entry

Human cytomegalovirus (HCMV) infection can lead to congenital hearing loss and mental retardation. Upon immune suppression, reactivation of latent HCMV or primary infection increases morbidity in cancer, transplantation, and late stage AIDS patients. Current treatments include nucleoside analogues, which have significant toxicities limiting their usefulness. In this study we screened a panel of synthetic heparin-binding peptides for their ability to prevent CMV infection in vitro. A peptide designated, p5+14 exhibited ~ 90% reduction in murine CMV (MCMV) infection. Because negatively charged, cell-surface heparan sulfate proteoglycans (HSPGs), serve as the attachment receptor during the adsorption phase of the CMV infection cycle, we hypothesized that p5+14 effectively competes for CMV adsorption to the cell surface resulting in the reduction in infection. Positively charged Lys residues were required for peptide binding to cell-surface HSPGs and reducing viral infection. We show that this inhibition was not due to a direct neutralizing effect on the virus itself and that the peptide blocked adsorption of the virus. The peptide also inhibited infection of other herpesviruses: HCMV and herpes simplex virus 1 and 2 in vitro, demonstrating it has broad-spectrum antiviral activity. Therefore, this peptide may offer an adjunct therapy for the treatment of herpes viral infections and other viruses that use HSPGs for entry

Anticytomegalovirus Peptides Point to New Insights for CMV Entry Mechanisms and the Limitations of In Vitro Screenings

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that can cause severe disease following in utero exposure, during primary infection, or la- tent virus reactivation in immunocompromised populations. These complications lead to a 1- to 2-billion-dollar economic burden, making vaccine development and/or alternative treatments a high priority. Current treatments for HCMV include nucleoside analogues such as ganciclovir (GCV), foscarnet, and cidofovir. Recently, letermovir, a terminase complex inhibitor, was approved for prophylaxis after stem cell transplantation. These treatments have unwanted side effects, and HCMV is be- coming resistant to them. Therefore, we sought to develop an alternative treatment that targets a different stage in viral infection. Currently, small antiviral peptides are being investigated as anti-influenza and anti-HIV treatments. We have developed heparan sulfate-binding peptides as tools for preventing CMV infections. These pep- tides are highly effective at stopping infection of fibroblasts with in vitro-derived HCMV and murine cytomegalovirus (MCMV). However, they do not prevent MCMV infection in vivo. Interestingly, these peptides inhibit infectivity of in vivo-derived CMVs, albeit not as well as tissue culture-grown CMVs. We further demonstrate that this class of heparan sulfate-binding peptides is incapable of inhibiting MCMV cell- to-cell spread, which is independent of heparan sulfate usage. These data indicate that inhibition of CMV infection can be achieved using synthetic polybasic peptides, but cell-to-cell spread and in vivo-grown CMVs require further investigation to de- sign appropriate anti-CMV peptides

The Human Cytomegalovirus Chemokine vCXCL-1 Modulates Normal Dissemination Kinetics of Murine Cytomegalovirus In Vivo

Human cytomegalovirus (HCMV) is a betaherpesvirus that is a significant pathogen within newborn and immunocompromised populations. Morbidity associated with HCMV infection is the consequence of viral dissemination. HCMV has evolved to manipulate the host immune system to enhance viral dissemination and ensure long-term survival within the host. The immunomodulatory protein vCXCL-1, a viral chemokine functioning primarily through the CXCR2 chemokine receptor, is hypothesized to attract CXCR2+ neutrophils to infection sites, aiding viral dissemination. Neutrophils harbor HCMV in vivo; however, the interaction between vCXCL-1 and the neutrophil has not been evaluated in vivo. Using the mouse model and mouse cytomegalovirus (MCMV) infection, we show that murine neutrophils harbor and transfer infectious MCMV and that virus replication initiates within this cell type. Utilizing recombinant MCMVs expressing vCXCL-1 from the HCMV strain (Toledo), we demonstrated that vCXCL-1 significantly enhances MCMV dissemination kinetics. Through cellular depletion experiments, we observe that neutrophils impact dissemination but that overall dissemination is largely neutrophil independent. This work adds neutrophils to the list of innate cells (i.e., dendritic and macrophages/monocytes) that contribute to MCMV dissemination but refutes the hypothesis that neutrophils are the primary cell responding to vCXCL-1

Novel Human Cytomegalovirus Viral Chemokines, vCXCL-1s, Display Functional Selectivity for Neutrophil Signaling and Function

Human CMV (HCMV) uses members of the hematopoietic system including neutrophils for dissemination throughout the body. HCMV encodes a viral chemokine, vCXCL-1, that is postulated to attract neutrophils for dissemination within the host. The gene encoding vCXCL-1, UL146, is one of the most variable genes in the HCMV genome. Why HCMV has evolved this hypervariability and how this affects the virus' dissemination and pathogenesis is unknown. Because the vCXCL-1 hypervariability maps to important binding and activation domains, we hypothesized that vCXCL-1s differentially activate neutrophils, which could contribute to HCMV dissemination, pathogenesis, or both. To test whether these viral chemokines affect neutrophil function, we generated vCXCL-1 proteins from 11 different clades from clinical isolates from infants infected congenitally with HCMV. All vCXCL-1s were able to induce calcium flux at a concentration of 100 nM and integrin expression on human peripheral blood neutrophils, despite differences in affinity for the CXCR1 and CXCR2 receptors. In fact, their affinity for CXCR1 or CXCR2 did not correlate directly with chemotaxis, G protein-dependent and independent (β-arrestin-2) activation, or secondary chemokine (CCL22) expression. Our data suggest that vCXCL-1 polymorphisms affect the binding affinity, receptor usage, and differential peripheral blood neutrophil activation that could contribute to HCMV dissemination and pathogenesis

Capacity building through operational research training in tobacco control: Experiences and lesson learned

Background: Several competing priorities with health and development sector currently deter research, and as a result of which evidence does not drive policy- or decision-making. There is limited operational research (OR) within the India's National Tobacco Control Programme, as it is in other middle- and low-income countries, primarily due to limited capacity and skills in undertaking OR and lack of dedicated funding. Few models of OR have been developed to meet the needs of different settings; however, they were found to be costly and time-consuming. Objective: To elucidate a cost-effective and less resource arduous training model for building capacity in OR focused on tobacco control. Materials and Methods: This 5½-day partly funded course enrolled 15 participants across the country and nine facilitators. The facilitator-participants interactions were initiated 2 weeks before the course, which enabled them to develop possible research questions and a plan for data analysis. Results: This article presents the new OR model along with experiences of the participants which will provide useful insights on lessons learned for planning similar courses in the future. While we faced several challenges in the process and the outputs were modest, several lessons were learned which will be instrumental in the future courses that we are planning to conduct. Conclusion: This low cost and less time intensive model can be applied in similar settings across range of public health issues