Molecular docking remains an important tool for structure-based screening to find new ligands and chemical probes. As docking ambitions grow to include new scoring function terms, and to address ever more targets, the reliability and extendability of the orientation sampling, and the throughput of the method, become pressing. Here we explore sampling techniques that eliminate stochastic behavior in DOCK3.6, allowing us to optimize the method for regularly variable sampling of orientations. This also enabled a focused effort to optimize the code for efficiency, with a three-fold increase in the speed of the program. This, in turn, facilitated extensive testing of the method on the 102 targets, 22,805 ligands and 1,411,214 decoys of the Directory of Useful Decoys - Enhanced (DUD-E) benchmarking set, at multiple levels of sampling. Encouragingly, we observe that as sampling increases from 50 to 500 to 2000 to 5000 to 20000 molecular orientations in the binding site (and so from about 1×1010 to 4×1010 to 1×1011 to 2×1011 to 5×1011 mean atoms scored per target, since multiple conformations are sampled per orientation), the enrichment of ligands over decoys monotonically increases for most DUD-E targets. Meanwhile, including internal electrostatics in the evaluation ligand conformational energies, and restricting aromatic hydroxyls to low energy rotamers, further improved enrichment values. Several of the strategies used here to improve the efficiency of the code are broadly applicable in the field.
It is well known that flow patterns at the anastomosis of coronary artery bypass graft (CABG) are complex and may affect the long-term patency. Various attempts at optimal designs of anastomosis have not improved long-term patency. Here, we hypothesize that mild anastomotic stenosis (area stenosis of about 40–60%) may be adaptive to enhance the hemodynamic conditions, which may contribute to slower progression of atherosclerosis. We further hypothesize that proximal/distal sites to the stenosis have converse changes that may be a risk factor for the diffuse expansion of atherosclerosis from the site of stenosis. Twelve (12) patient-specific models with various stenotic degrees were extracted from computed tomography images using a validated segmentation software package. A 3-D finite element model was used to compute flow patterns including wall shear stress (WSS) and its spatial and temporal gradients (WSS gradient, WSSG, and oscillatory shear index, OSI). The flow simulations showed that mild anastomotic stenosis significantly increased WSS (>15 dynes⋅cm−2) and decreased OSI (<0.02) to result in a more uniform distribution of hemodynamic parameters inside anastomosis albeit proximal/distal sites to the stenosis have a decrease of WSS (<4 dynes⋅cm−2). These findings have significant implications for graft adaptation and long-term patency.
Genetic variants that contribute to asthma susceptibility may be present at varying frequencies in different populations, which is an important consideration and advantage for performing genetic association studies in admixed populations.
To identify asthma-associated loci in African Americans.
We compared local African and European ancestry estimated from dense single nucleotide polymorphism (SNP) genotype data in African American adults with asthma and non-asthmatic controls. Allelic tests of association were performed within the candidate regions identified, correcting for local European admixture.
We identified a significant ancestry association peak on chromosomes 6q. Allelic tests for association within this region identified a SNP (rs1361549) on 6q14.1 that was associated with asthma exclusively in African Americans with local European admixture (OR=2.2). The risk allele is common in Europe (42% in the HapMap CEU) but absent in West Africa (0% in the HapMap YRI), suggesting the allele is present in African Americans due to recent European admixture. We replicated our findings in Puerto Ricans and similarly found that the signal of association is largely specific to individuals who are heterozygous for African and non-African ancestry at 6q14.1. However, we found no evidence for association in European Americans or in Puerto Ricans in the absence of local African ancestry, suggesting that the association with asthma at rs1361549 is due to an environmental or genetic interaction.
We identified a novel asthma-associated locus that is relevant to admixed populations with African ancestry, and highlight the importance of considering local ancestry in genetic association studies of admixed populations.
asthma; population structure; genome-wide association study; admixture mapping; ancestry association testing; admixed populations; African Americans; Puerto Ricans
Cellular treatments for repairing diseased tissues represent a promising clinical strategy. Umbilical cord tissue-derived cells (UTC) are a unique source of cells with a low immunogenic profile and potential for tissue repair. By using UTC from miniature swine, we previously demonstrated that despite their low immunogenic phenotype, UTC could induce an immune response under certain inflammatory conditions and after multiple subcutaneous (SC) injections. Given that repeat dosing of cells may be necessary to achieve a lasting therapeutic benefit, in this study, we examined approaches to avoid an immune response to multiple SC injections of UTC.
By using in vitro and in vivo measures of sensitization to SC cellular injections, we assessed the effects of varying the location of administration site, prolongation of timing between injections, and use of immunosuppressive treatments on repeated cellular injections in Massachusetts General Hospital major histocompatibility complex-defined miniature swine.
Although under normal conditions, a single SC injection of major histocompatibility complex-mismatched UTC did not induce a detectable immune response, multiple SC injections of UTC demonstrated rapid humoral and cell-mediated immune responses. Avoidance of an immune response to repeat SC injection was achieved by concurrent immunosuppression with each dose of UTC.
UTC and other similar cell types believed to be nonimmunogenic have the potential to induce immune responses under certain conditions. These studies provide important considerations and guidelines for preclinical studies investigating allogeneic cellular therapies.
Umbilical cord tissue derived cells; Immunogenicity; Miniature swine; Cellular therapy
Although experiencing alcohol-related consequences has some influence on future drinking, this effect may be stronger based on the degree to which the consequence is viewed as positive versus negative, either by the individual or pre-defined by researchers. This study explored the relationship between experiencing positive and negative alcohol-related consequences and college students’ perceptions of how likely those consequences were to occur in the future (i.e. likelihood), and their view of how positive or negative experiencing those consequences would be if they did experience them as a result of drinking (i.e. valence). Data were collected from 491 college students (mean age = 19.26; 56.4% female; 55.0% Caucasian; 33.2% Asian/Pacific Islander) through a computerized survey. Results indicated that experiencing more positive consequences in the past year was associated with viewing those consequences as both more likely to occur and more positive, while experiencing more negative consequences was associated with viewing them as less negative and no more likely to occur, except for those who had experienced the highest levels of negative consequences. These findings suggest that finding ways to reduce both perceptions as well as consequences themselves may be effective intervention tools.
alcohol; consequences; college students; expectancies; likelihood
Aberrant T-cell activation underlies many autoimmune disorders, yet most attempts to induce T-cell tolerance have failed. Building on previous strategies for tolerance induction that exploited natural mechanisms for clearing apoptotic debris, we show that antigen-decorated microparticles (500-nm diameter) induce long-term T-cell tolerance in mice with relapsing experimental autoimmune encephalomyelitis. Specifically, intravenous infusion of either polystyrene or biodegradable poly(lactide-co-glycolide) microparticles bearing encephalitogenic peptides prevents the onset and modifies the course of the disease. These beneficial effects require microparticle uptake by marginal zone macrophages expressing the scavenger receptor MARCO and are mediated in part by the activity of regulatory T cells, abortive T-cell activation and T-cell anergy. Together these data highlight the potential for using microparticles to target natural apoptotic clearance pathways to inactivate pathogenic T cells and halt the disease process in autoimmunity.
In recent years, substantial advances in T-cell immunosuppressive strategies and their translation to routine clinical practice have revolutionized management and outcomes in autoimmune disease and solid organ transplantation. More than 80 diseases have been considered to have an autoimmune etiology, such that autoimmune-associated morbidity and mortality rank as third highest in developed countries, after cardiovascular diseases and cancer. Solid organ transplantation has become the therapy of choice for many end-stage organ diseases. Short-term outcomes such as patient and allograft survival at 1 year, acute rejection rates, as well as time course of disease progression and symptom control have steadily improved. However, despite the use of newer immunosuppressive drug combinations, improvements in long-term allograft survival and complete resolution of autoimmunity remain elusive. In addition, the chronic use of nonspecifically targeted immunosuppressive drugs is associated with significant adverse effects and increased morbidity and mortality. In this article, we discuss the current clinical tools for immune suppression and attempts to induce long-term T-cell tolerance induction as well as much-needed future approaches to produce more short-acting, antigen-specific agents, which may optimize outcomes in the clinic.
autoimmunity; chimerism; immune suppression; infection; maintenance suppression; T-cell tolerance; transplantation
Squamous cell carcinomas (SCC) are sun-induced skin cancers that are particularly numerous and aggressive in immunosuppressed individuals. SCC evade immune detection at least in part by down-regulating E-selectin on tumor vessels, thereby restricting entry of skin homing T cells into tumors. We find that nitric oxide potently suppresses E-selectin expression on human endothelial cells and that SCC are infiltrated by nitric oxide-producing iNOS+ CD11b+ CD33+ CD11c− HLA-DR− myeloid-derived suppressor cells (MDSC). MDSC from SCC produced NO, TGFβ and arginase and inhibited endothelial E-selectin expression in vitro. MDSC from SCC expressed the chemokine receptor CCR2 and tumors expressed the CCR2 ligand HBD3, suggesting CCR2-HBD3 interactions may contribute to MDSC recruitment to SCC. Treatment of SCC in vitro with the iNOS inhibitor L-NNA induced E-selectin expression at levels comparable to imiquimod-treated SCC undergoing immunologic destruction. Our results suggest that local production of NO in SCC may impair vascular E-selectin expression. We show that MDSC are critical producers of NO in SCC and that NO inhibition restores vascular E-selectin expression, potentially enhancing T cell recruitment. iNOS inhibitors and other therapies that reduce NO production may therefore be effective in the treatment of SCC and their premalignant precursor lesions actinic keratoses.
The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis.
Spontaneous antigen-specific T cell responses can be generated in hosts harboring a variety of solid malignancies, but are subverted by immune evasion mechanisms active within the tumor microenvironment. In contrast to solid tumors, the mechanisms that regulate T cell activation versus tolerance to hematological malignancies have been underexplored. A murine acute myeloid leukemia (AML) model was used to investigate antigen-specific T cell responses against AML cells inoculated i.v. versus s.c. Robust antigen-specific T cell responses were generated against AML cells after s.c., but not i.v., inoculation. In fact, i.v. AML cell inoculation prevented functional T cell activation in response to subsequent s.c. AML cell challenge. T cell dysfunction was antigen specific and did not depend on Tregs or myeloid-derived suppressor cells (MDSCs). Antigen-specific TCR-Tg CD8+ T cells proliferated, but failed to accumulate, and expressed low levels of effector cytokines in hosts after i.v. AML induction, consistent with abortive T cell activation and peripheral tolerance. Administration of agonistic anti-CD40 Ab to activate host APCs enhanced accumulation of functional T cells and prolonged survival. Our results suggest that antigen-specific T cell tolerance is a potent immune evasion mechanism in hosts with AML that can be reversed in vivo after CD40 engagement.
Although many adults with B cell acute lymphoblastic leukemia (B-ALL) are induced into remission, most will relapse, underscoring the dire need for novel therapies for this disease. We developed murine CD19-specific chimeric antigen receptors (CARs) and an immunocompetent mouse model of B-ALL that recapitulates the disease at genetic, cellular, and pathologic levels. Mouse T cells transduced with an all-murine CD3ζ/CD28-based CAR that is equivalent to the one being used in our clinical trials, eradicate B-ALL in mice and mediate long-term B cell aplasias. In this model, we find that increasing conditioning chemotherapy increases tumor eradication, B cell aplasia, and CAR-modified T cell persistence. Quantification of recipient B lineage cells allowed us to estimate an in vivo effector to endogenous target ratio for B cell aplasia maintenance. In mice exhibiting a dramatic B cell reduction we identified a small population of progenitor B cells in the bone marrow that may serve as a reservoir for long-term CAR-modified T cell stimulation. Lastly, we determine that infusion of CD8+ CAR-modified T cells alone is sufficient to maintain long-term B cell eradication. The mouse model we report here should prove valuable for investigating CAR-based and other therapies for adult B-ALL.
The goals of asthma treatment include preventing recurrent exacerbations. Yet there is no consensus about the terminology for describing or defining “exacerbation,” or about how to characterize an episode’s severity.
National Institutes of Health (NIH) institutes and other federal agencies convened an expert group to propose how asthma exacerbation should be assessed as a standardized asthma outcome in future asthma clinical research studies.
We utilized comprehensive literature reviews and expert opinion to compile a list of asthma exacerbation outcomes, and classified them as either core (required in future studies), supplemental (used according to study aims and standardized), or emerging (requiring validation and standardization). This work was discussed at an NIH-organized workshop in March 2010 and finalized in September 2011.
No dominant definition of “exacerbation” was found. The most widely used definitions included 3 components, all related to treatment, rather than symptoms: (1) systemic use of corticosteroids, (2) asthma-specific emergency department visits or hospitalization, and (3) use of short-acting β-agonists (SABAs) as quick-relief (sometimes referred to as “rescue” or “reliever”) medications.
The working group participants propose that the definition of “asthma exacerbation” be “a worsening of asthma requiring the use of systemic corticosteroids to prevent a serious outcome.” As core outcomes, they propose inclusion and separate reporting of several essential variables of an exacerbation. Further, they propose the development of a standardized, component-based definition of “exacerbation” with clear thresholds of severity for each component.
Asthma exacerbations; severity of acute asthma; asthma outcomes; urgent asthma care
During chronic viral infections, responses by virus-specific CD8+ T cells become marginalized by the acquisition of functional defects and reduced cell numbers in a process defined as T cell exhaustion. T cell tolerance to self-antigen is also characterized by impaired effector function and eventual deletion of self-reactive T cells. Induction of both tolerance and exhaustion involve many shared inhibitory mechanisms, thus similar therapeutic approaches have proven effective in these distinct environments. We previously demonstrated that tolerant self-reactive CD8+ T cells expressing dual-T cell receptors (i.e. dual-TCR) could be rescued by immunization through a second TCR specific for a foreign antigen. These data revealed that T cell tolerance was regulated at the level of the self-reactive TCR. Here, dual-TCR CD8+ T cells were used to examine if exhaustion during persistent viral infection could be rescued by an analogous strategy of immunization through a second TCR not involved in recognition of virus. In direct contrast to the rescue achievable in tolerant CD8+ T cells, exhausted T cells were equally impaired through both TCR. These findings suggest that exhaustion is maintained by defects downstream of the virus-specific TCR, and establish that exhaustion and tolerance are distinctly regulated states of T cell dysfunction.
CD8+ T cells; Dual-T cell Receptor; Chronic Viral Infection; Exhaustion; Tolerance
The Ikaros family of transcription factors is critical for normal T cell development while limiting malignant transformation. Mature CD8 T cells express multiple Ikaros family members, yet little is known about their function in this context. To test the functions of this gene family, we used retroviral transduction to express a naturally occurring, dominant negative (DN) isoform of Ikaros in activated CD8 T cells. Notably, expression of DN Ikaros profoundly enhanced the competitive advantage of activated CD8 T cells cultured in IL-12, such that by 6 days of culture, DN Ikaros-transduced cells were 100-fold more abundant than control cells. Expression of a DN isoform of Helios, a related Ikaros-family transcription factor, conferred a similar advantage to transduced cells in IL-12. While DN Ikaros-transduced cells had higher expression of the IL-2 receptor alpha chain, DN Ikaros-transduced cells achieved their competitive advantage through an IL-2 independent mechanism. Finally, the competitive advantage of DN Ikaros-transduced cells was manifested in vivo, following adoptive transfer of transduced cells. These data identify the Ikaros family of transcription factors as regulators of cytokine responsiveness in activated CD8 T cells, and suggest a role for this family in influencing effector and memory CD8 T cell differentiation.
The National Heart, Lung, and Blood Institute Severe Asthma Research Program (SARP) has characterized over the past 10 years 1,644 patients with asthma, including 583 individuals with severe asthma. SARP collaboration has led to a rapid recruitment of subjects and efficient sharing of samples among participating sites to conduct independent mechanistic investigations of severe asthma. Enrolled SARP subjects underwent detailed clinical, physiologic, genomic, and radiological evaluations. In addition, SARP investigators developed safe procedures for bronchoscopy in participants with asthma, including those with severe disease. SARP studies revealed that severe asthma is a heterogeneous disease with varying molecular, biochemical, and cellular inflammatory features and unique structure–function abnormalities. Priorities for future studies include recruitment of a larger number of subjects with severe asthma, including children, to allow further characterization of anatomic, physiologic, biochemical, and genetic factors related to severe disease in a longitudinal assessment to identify factors that modulate the natural history of severe asthma and provide mechanistic rationale for management strategies.
asthma; remodeling; inflammation; bronchoscopy; imaging
The adoptive transfer of antigen-specific effector T cells is being used to treat human infections and malignancy. T cell persistence is a prerequisite for therapeutic efficacy, but reliably establishing a high-level and durable T cell response by transferring cultured CD8+ T cells remains challenging. Thus, strategies that promote a transferred high-level T cell response may improve the efficacy of T cell therapy. Lymphodepletion enhances persistence of transferred T cells in mice in part by reducing competition for IL-15, a common γ-chain cytokine that promotes T cell memory, but lymphodepleting regimens have toxicity. IL-15 can be safely administered and has minimal effects on CD4+ regulatory T cells at low doses, making it an attractive adjunct in adoptive T cell therapy. Here, we show in lymphoreplete macaca nemestrina, that proliferation of adoptively transferred central memory-derived CD8+ effector T (TCM/E) cells is enhanced in vivo by administering IL-15. TCM/E cells migrated to memory niches, persisted, and acquired both central memory and effector memory phenotypes regardless of the cytokine treatment. Unexpectedly, despite maintaining T cell proliferation, IL-15 did not augment the magnitude of the transferred T cell response in blood, bone marrow, or lymph nodes. T cells induced to proliferate by IL-15 displayed increased apoptosis demonstrating that enhanced cycling was balanced by cell death. These results suggest that homeostatic mechanisms that regulate T cell numbers may interfere with strategies to augment a high-level T cell response by adoptive transfer of CD8+ TCM/E cells in lymphoreplete hosts.
Mature mammalian sperm contain a complex population of RNAs some of which might regulate spermatogenesis while others probably play a role in fertilization and early development. Due to this limited knowledge, the biological functions of sperm RNAs remain enigmatic. Here we report the first characterization of the global transcriptome of the sperm of fertile stallions. The findings improved understanding of the biological significance of sperm RNAs which in turn will allow the discovery of sperm-based biomarkers for stallion fertility. The stallion sperm transcriptome was interrogated by analyzing sperm and testes RNA on a 21,000-element equine whole-genome oligoarray and by RNA-seq. Microarray analysis revealed 6,761 transcripts in the sperm, of which 165 were sperm-enriched, and 155 were differentially expressed between the sperm and testes. Next, 70 million raw reads were generated by RNA-seq of which 50% could be aligned with the horse reference genome. A total of 19,257 sequence tags were mapped to all horse chromosomes and the mitochondrial genome. The highest density of mapped transcripts was in gene-rich ECA11, 12 and 13, and the lowest in gene-poor ECA9 and X; 7 gene transcripts originated from ECAY. Structural annotation aligned sperm transcripts with 4,504 known horse and/or human genes, rRNAs and 82 miRNAs, whereas 13,354 sequence tags remained anonymous. The data were aligned with selected equine gene models to identify additional exons and splice variants. Gene Ontology annotations showed that sperm transcripts were associated with molecular processes (chemoattractant-activated signal transduction, ion transport) and cellular components (membranes and vesicles) related to known sperm functions at fertilization, while some messenger and micro RNAs might be critical for early development. The findings suggest that the rich repertoire of coding and non-coding RNAs in stallion sperm is not a random remnant from spermatogenesis in testes but a selectively retained and functionally coherent collection of RNAs.
NOD2 is an intracellular pattern recognition receptor that provides innate sensing of bacterial muramyl dipeptide by host cells, such as dendritic cells, macrophages and epithelial cells. While NOD2's role as an innate pathogen sensor is well established, NOD2 is also expressed at low levels in T cells and there are conflicting data as to whether NOD2 plays an intrinsic role in T cell function. Here we show that following adoptive transfer into WT hosts, NOD2−/− OT-I T cells show a small decrease in the number of OVA-specific CD8 T cells recovered at the peak of the response to respiratory influenza virus infection. On the other hand, no such defect was observed upon intranasal immunization with a replication defective adenovirus carrying the OVA epitope recognized by OT-I, or when OVA was delivered with LPS subcutaneously, or when influenza-OVA was delivered intraperitoneally. Thus we observed a selective defect in NOD2-deficient T cell responses only during a live viral infection. Moreover, there was no apparent defect when NOD2−/− OT-I T cells were stimulated in vitro. Finally, this selective defect in recovery of NOD2-deficient CD8 T cells was not observed in a non-transgenic respiratory infection model in which mixed bone marrow chimeras were used such that the NOD2−/− T cells were allowed to develop and respond in a NOD2-sufficient host. Taken together our data indicate that T cell intrinsic NOD2 is not required for CD8 T cell responses to antigen delivered under a variety of conditions in vitro and in vivo. However, CD8 T cells that have developed in the absence of NOD2 show a selective and modest impairment in their response to live respiratory influenza infection.
asthma genetics; atopy; C11orf30; LRRC32; total serum IgE levels
TCR-induced NF-AT activation leads to the expression of both activating and inhibitory proteins. Previously, we had identified Egr-2 and Egr-3 as NF-AT-induced transcription factors which promote the inhibition of T cell activation. In this report we identify Sprouty1 as a downstream target of Egr-3. CD4+ T cells lacking Spry1 demonstrate enhanced proliferation and cytokine production. Likewise, Spry1Flox/Flox Lck Cre CD8+ T cells display increased cytolytic activity. Mechanistically, Spry1 acts at the level of PLC-γ promoting the inhibition of both Ca++ induced NF-AT activation and MAP-kinase induced AP-1 activation while sparing NF-κB signaling. In vivo, mice in which Spry1 is selectively deleted in T cells demonstrate enhanced responses to a tumor vaccine and subsequently reject tumors more robustly than Wt mice. These findings suggest that targeting Spry1 might prove to be a novel means of enhancing tumor immunotherapy.
Although savings would be substantial, and numerous drug shortages could be relieved if all medication in the vial were used for patient care, USP <797> is the standard by which all CSPs should be prepared.
The update of US Pharmacopeia Chapter <797> in 2008 included guidelines stating that single-dose vials (SDVs) opened and maintained in an International Organization for Standardization Class 5 environment can be used for up to 6 hours after initial puncture. A study was conducted to evaluate the cost of discarding vials after 6 hours and to further test sterility of vials beyond this time point, subsequently defined as the beyond-use date (BUD).
Financial determination of SDV waste included 2 months of retrospective review of all doses prescribed. Additionally, actual waste log data were collected. Active and control vials (prepared using sterilized trypticase soy broth) were recovered, instead of discarded, at the defined 6-hour BUD.
The institution-specific waste of 19 selected SDV medications discarded at 6 hours was calculated at $766,000 annually, and tracking waste logs for these same medications was recorded at $770,000 annually. Microbiologic testing of vial extension beyond 6 hours showed that 11 (1.86%) of 592 samples had one colony-forming unit on one of two plates. Positive plates were negative at subsequent time points, and all positives were single isolates most likely introduced during the plating process.
The cost of discarding vials at 6 hours was significant for hazardous medications in a large academic medical center. On the basis of microbiologic data, vial BUD extension demonstrated a contamination frequency of 1.86%, which likely represented exogenous contamination; vial BUD extension for the tested drugs showed no growth at subsequent time points and could provide an annual cost savings of more than $600,000.
Infiltration of Ly6Chi monocytes from the blood is a hallmark of viral encephalitis. In mice with lethal encephalitis caused by West Nile virus (WNV), an emerging neurotropic flavivirus, inhibition of Ly6Chi monocyte trafficking into the brain by anti-very late antigen (VLA)-4 integrin antibody blockade at the time of first weight loss and leukocyte influx resulted in long-term survival of up to 60% of infected mice, with subsequent sterilizing immunity. This treatment had no effect on viral titers but appeared to be due to inhibition of Ly6Chi macrophage immigration. Although macrophages isolated from the infected brain induced WNV-specific CD4+ T-cell proliferation, T cells did not directly contribute to pathology, but are likely to be important in viral control, as antibody-mediated T-cell depletion could not reproduce the therapeutic benefit of anti-VLA-4. Instead, 70% of infiltrating inflammatory monocyte-derived macrophages were found to be making nitric oxide (NO). Furthermore, aminoguanidine-mediated inhibition of induced NO synthase activity in infiltrating macrophages significantly prolonged survival, indicating involvement of NO in the immunopathology. These data show for the first time the therapeutic effects of temporally targeting pathogenic NO-producing macrophages during neurotropic viral encephalitis.
Neurotropic virus; Flavivirus; Inflammatory monocytes; West Nile virus encephalitis; Macrophage infiltration; VLA-4; Integrins; Nitric oxide
Like all DNA viruses, human cytomegalovirus (HCMV) infection is known to result in profound effects on host cell cycle. Infection of fibroblasts with HCMV is known to induce an advance in cell cycle through the G0-G1 phase and then a subsequent arrest of cell cycle in early S-phase, presumably resulting in a cellular environment optimum for high levels of viral DNA replication whilst precluding replication of cellular DNA. Although the exact mechanisms used to arrest cell cycle by HCMV are unclear, they likely involve a number of viral gene products and evidence points to the ability of the virus to prevent licensing of cellular DNA synthesis. One viral protein known to profoundly alter cell cycle is the viral immediate early 86 (IE86) protein - an established function of which is to initially drive cells into early S phase but then inhibit cellular DNA synthesis. Here we show that, although IE86 interacts with the cellular licensing factor Cdt1, it does not inhibit licensing of cellular origins. Instead, IE86-mediated inhibition of cellular DNA synthesis requires mini-chromosome-maintenance 3 (MCM3) associated protein (MCM3AP), which can cause subsequent inhibition of initiation of cellular DNA synthesis in a licensing-independent manner.