Uncontrolled Replication Research Articles

P0012 The nanoparticle Quillaja saponin KGI exerts anti-proliferative effects by down-regulation of cell cycle molecules in U937 and HL-60 human leukaemia cells

Background Cancer cells are characterised by uncontrolled replication involving loss of control of cyclin-dependent kinases (CDKs) and cyclins, and by abolished differentiation. In this study we introduce KGI, a nanoparticle with a Quillaja saponin as an active molecule. Methods By the use of RNA array analysis and confirmation at the protein level, we studied the effects of KGI on myeloid leukaemia cells. Findings KGI affects myeloid leukaemia cells (particularly the U937 monoblast cancer cell) by the following mechanisms: ceasing cell replication via proteasome degradation; down-regulation of key molecules at check points between G 1 /S and G 2 /M phases; reduction of thymidine kinase activity; followed by exit to differentiation and production of IL-8, eventually leading to apoptosis. Leukaemia cell lines (U937 and HL-60 cells) were exposed to KGI for 8 h, after which the drug was removed. The cancer cells did not revert to replication over the following 10 days. Interpretation Our findings suggest that the nanoparticle KGI inhibits proliferation and promotes differentiation in leukaemic cells by interfering with the cell cycle process.

Deficient IFN signaling by myeloid cells leads to MAVS-dependent virus-induced sepsis.

The type I interferon (IFN) signaling response limits infection of many RNA and DNA viruses. To define key cell types that require type I IFN signaling to orchestrate immunity against West Nile virus (WNV), we infected mice with conditional deletions of the type I IFN receptor (IFNAR) gene. Deletion of the Ifnar gene in subsets of myeloid cells resulted in uncontrolled WNV replication, vasoactive cytokine production, sepsis, organ damage, and death that were remarkably similar to infection of Ifnar −/− mice completely lacking type I IFN signaling. In Mavs−/−×Ifnar−/− myeloid cells and mice lacking both Ifnar and the RIG-I-like receptor adaptor gene Mavs, cytokine production was muted despite high levels of WNV infection. Thus, in myeloid cells, viral infection triggers signaling through MAVS to induce proinflammatory cytokines that can result in sepsis and organ damage. Viral pathogenesis was caused in part by massive complement activation, as liver damage was minimized in animals lacking complement components C3 or factor B or treated with neutralizing anti-C5 antibodies. Disease in Ifnar −/− and CD11c Cre+ Ifnar f/f mice also was facilitated by the proinflammatory cytokine TNF-α, as blocking antibodies diminished complement activation and prolonged survival without altering viral burden. Collectively, our findings establish the dominant role of type I IFN signaling in myeloid cells in restricting virus infection and controlling pathological inflammation and tissue injury.

Construction of a live-attenuated HIV-1 vaccine through genetic code expansion.

A safe and effective vaccine against human immunodeficiency virus type 1 (HIV-1) is urgently needed to combat the worldwide AIDS pandemic, but still remains elusive. The fact that uncontrolled replication of an attenuated vaccine can lead to regaining of its virulence creates safety concerns precluding many vaccines from clinical application. We introduce a novel approach to control HIV-1 replication, which entails the manipulation of essential HIV-1 protein biosynthesis through unnatural amino acid (UAA*)-mediated suppression of genome-encoded blank codon. We successfully demonstrate that HIV-1 replication can be precisely turned on and off in vitro.

Construction of a Live‐Attenuated HIV‐1 Vaccine through Genetic Code Expansion

AbstractA safe and effective vaccine against human immunodeficiency virus type 1 (HIV‐1) is urgently needed to combat the worldwide AIDS pandemic, but still remains elusive. The fact that uncontrolled replication of an attenuated vaccine can lead to regaining of its virulence creates safety concerns precluding many vaccines from clinical application. We introduce a novel approach to control HIV‐1 replication, which entails the manipulation of essential HIV‐1 protein biosynthesis through unnatural amino acid (UAA*)‐mediated suppression of genome‐encoded blank codon. We successfully demonstrate that HIV‐1 replication can be precisely turned on and off in vitro.

Liver fibrosis progression despite HCV cure with antiviral therapy in HIV-HCV-coinfected patients.

Accelerated liver fibrosis and more frequent hepatic decompensation events and liver-related deaths are characteristically seen in chronic hepatitis C patients coinfected with HIV compared with HCV-monoinfected individuals. Quantitative estimates of long-term clinical benefits derived from curing HCV with antiviral therapy in coinfected patients are scarce, despite being needed for accurate cost-effectiveness decisions using expensive direct-acting antivirals in this population. We retrospectively examined all HIV-HCV-coinfected patients followed at one reference clinic in Madrid since 2004. Liver fibrosis was measured longitudinally using elastometry; changes above 30% in kilopascal units were considered as significant. A total of 568 HIV-HCV-coinfected patients were examined. Pegylated interferon/ribavirin therapy had been given to 396 (69.7%) of whom 138 (34.8%) had achieved sustained virological response (SVR). Mean follow-up was of 6.8 (±1.5) years for hepatic events and 4.4 (±0.8) years for liver fibrosis. Hepatic decompensation events, liver-related deaths and significant liver fibrosis progression occurred less frequently in SVR than in non-treated/treatment failures. Although regression of liver fibrosis occurred in most SVR patients, fibrosis significantly progressed in 7.2% of them, in association with higher plasma HIV RNA (P=0.005) and longer exposure to HIV protease inhibitors (P=0.009). Achievement of SVR dramatically reduces the risk of hepatic decompensation events and liver-related deaths in HIV-HCV-coinfected patients. Although liver fibrosis generally improves following HCV cure, worsening may occur in association with uncontrolled HIV replication and prolonged exposure to protease inhibitors. Thus, periodic assessment of liver fibrosis is warranted after SVR and screening for liver cancer should continue in coinfected patients with advanced liver fibrosis.

Risk factors for invasive pneumococcal disease in HIV-infected adults in France in the highly active antiretroviral therapy era

Invasive pneumococcal diseases remain frequent and severe in HIV-infected subjects. To identify opportunities for prevention, we assessed risk factors of invasive pneumococcal diseases (IPD) in HIV-infected patients over a 10-year period in France. We performed a retrospective case-control study in a reference centre of HIV management in Paris. All HIV-infected patients having suffered from IPD between 2000 and 2011 were included. Control subjects were HIV-infected with no history of IPD or pneumonia, matched by date of diagnosis of HIV with controls. Two controls were randomly selected for each subject. In all, 42 HIV-infected patients presented 44 IPD episodes during the study period and were compared to 84 controls. In the multivariate analysis, patients with IPD were more likely than controls to have a Charlson Comorbidity Index ≥2 (adjusted OR = 7.07, 95% CI 1.99-25.1, p = 0.003), CD4-cell count <200/cells/µL (aOR = 6.93, 95% CI 1.80-26.7, p = 0.005), HIV-RNA viral load >400 copies/mL (aOR = 5.56, 95% CI 1.58-19.5, p = 0.007) and a non-European origin (aOR = 4.26, 95% CI 1.02-17.9, p = 0.047). HIV-infected patients with a higher burden of comorbidities, uncontrolled HIV replication, low CD4-cell counts and/or of non-European origin are at higher risk of developing IPD. Better screening for and management of HIV infection is necessary to reduce the risk of IPD.

Stem cells, colorectal cancer and cancer stem cell markers correlations.

: The idea of stem cells as being progenitors of cancer was initially controversial, but later supported by research in the field of leukemia and solid tumors. Afterwards, it was established that genetic abnormalities can affect the stem and progenitor cells, leading to uncontrolled replication and deregulated differentiation. These alterations will cause the changeover to cancerous stem cells (CSC) having two main characteristics: tumor initiation and maintenance. This review will focus on the colorectal cancer stem cell (CR-CSCs) theory which provides a better understanding of different tumor processes: initiation, aggressive growth, recurrence, treatment resistance and metastasis. A search in PubMed/Medline was performed using the following keywords: colorectal cancer stem cells (CR-CSCs), colorectal neoplasms stem cells, colorectal cancer stem cell (CR-CSCs) markers, etc. Electronic searches were supplemented by hand searching reference lists, abstracts and proceedings from meetings. Isolation of CR-CSCs can be achieved by targeting and selecting subpopulation of tumor cells based on expression of one or multiple cell surface markers associated with cancer self-renewal, markers as: CD133, CD166, CD44, CD24, beta1 integrin-CD29, Lgr5, EpCAM (ESA), ALDH-1, Msi-1, DCAMLK1 or EphB receptors. The identification and localization of CR-CSCs through different markers will hopefully lead to a better stratification of prognosis and treatment response, as well as the development of new effective strategies for cancer management.

Spatial modeling of HIV cryptic viremia and 2-LTR formation during raltegravir intensification

Combination Antiretroviral Therapy (cART) can suppress plasma HIV below the limit of detection in normal assays. Recently reported results suggest that viral replication may continue in some patients, despite undetectable levels in the blood. It has been suggested that the appearance of the circularized episomal HIV DNA artifact 2-LTR following treatment intensification with the integrase inhibitor raltegravir is a marker of ongoing viral replication. Other work has suggested that lymphoid organs may be a site of reduced antiviral penetration and increased viral production. In this study we model the hypothesis that this ongoing replication occurs in lymphoid follicle sanctuary sites and investigate the patterns of 2-LTR formation expected after raltegravir application. Experimental data is used to estimate the reaction and diffusion parameters in the model, and Monte-Carlo simulations are used to explore model behavior subject to variation in these rates. The results suggest that conditions for the formation of an observed transient peak in 2-LTR formation following raltegravir intensification include a sanctuary site diameter larger than 0.2mm, a viral basic reproductive ratio within the site larger than 1, and a total volume of active sanctuary sites above 20mL. Significant levels of uncontrolled replication can occur in the sanctuary sites without measurable changes in the plasma viral load. By contrast, subcritical replication (where the basic reproductive ratio of the virus is less than 1 in all sites) always results in monotonic increases of measured 2-LTR following raltegravir intensification, occurring at levels below the limit of detection.

Immune surveillance and response to JC virus infection and PML

The ubiquitous human polyomavirus JC virus (JCV) is the established etiological agent of the debilitating and often fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Most healthy individuals have been infected with JCV and generate an immune response to the virus, yet remain persistently infected at subclinical levels. The onset of PML is rare in the general population, but has become an increasing concern in immunocompromised patients, where reactivation of JCV leads to uncontrolled replication in the CNS. Understanding viral persistence and the normal immune response to JCV provides insight into the circumstances which could lead to viral resurgence. Further, clues on the potential mechanisms of reactivation may be gleaned from the crosstalk among JCV and HIV-1, as well as the impact of monoclonal antibody therapies used for the treatment of autoimmune disorders, including multiple sclerosis, on the development of PML. In this review, we will discuss what is known about viral persistence and the immune response to JCV replication in immunocompromised individuals to elucidate the deficiencies in viral containment that permit viral reactivation and spread.

The nanoparticulate Quillaja saponin KGI exerts anti-proliferative effects by down-regulation of cell cycle molecules in U937 and HL-60 human leukemia cells

Cancer cells are characterized by uncontrolled replication involving loss of control of cyclin dependent kinases (CDKs) and cyclins, and by abolished differentiation. In this study we introduce KGI, which is a nanoparticle with a Quillaja saponin as an active molecule. By the use of RNA array analysis and confirmation at the protein level, we show that KGI affects myeloid leukemia cells (in particular, the U937 monoblast cancer cell) by the following mechanisms: (A) ceasing cell replication via proteasome degradation, (B) down-regulation of key molecules at check points between G1/S and G2/M phases, (C) reduction of thymidine kinase activity, followed by (D) exit to differentiation and production of interleukin-8 (IL-8), eventually leading to apoptosis. Leukemia cell lines (U937 and HL-60 cells) were exposed to KGI for 8 h, after which the drug was removed. The cancer cells did not revert to replication over the following 10 days. Thus our findings suggest that the nanoparticle KGI inhibits proliferation and promotes differentiation in leukemic cells by interfering with the cell cycle process.

Ifit2 Deficiency Results in Uncontrolled Neurotropic Coronavirus Replication and Enhanced Encephalitis via Impaired Alpha/Beta Interferon Induction in Macrophages

Type I interferons (IFN-α/β) limit viral dissemination prior to the emergence of adaptive immune responses through the concerted action of interferon-stimulated genes (ISGs). Although IFN-α/β induction by coronaviruses is modest, it effectively limits viral spread within the central nervous system (CNS) and protects against mortality. The protective roles of specific ISGs against the mouse hepatitis virus (MHV) members of the coronaviruses are largely unknown. This study demonstrates a protective role of the ISG Ifit2 in encephalitis induced by the dual hepato- and neurotropic MHV-A59. Contrasting the mild encephalitis and 100% survival of MHV-A59-infected wild-type (wt) mice, nearly 60% of infected Ifit2(-/-) mice exhibited severe encephalitis and succumbed between 6 and 8 days postinfection. Increased clinical disease in Ifit2(-/-) mice coincided with higher viral loads and enhanced viral spread throughout the CNS parenchyma. Ifit2(-/-) mice also expressed significantly reduced IFN-α/β and downstream ISG mRNAs Ifit1, Isg15, and Pkr, while expression of proinflammatory cytokines and chemokines was only modestly affected in the CNS. Impaired IFN-α/β induction in the absence of Ifit2 was confirmed by ex vivo mRNA analysis of microglia and macrophages, the prominent cell types producing IFN-α/β following MHV CNS infection. Furthermore, both IFN-α/β mRNA and protein production were significantly reduced in MHV-infected Ifit2(-/-) relative to wt bone marrow-derived macrophages. Collectively, the data implicate Ifit2 as a positive regulator of IFN-α/β expression, rather than direct antiviral mediator, during MHV-induced encephalitis.

Additive Manufacturing and its Implications for Military Ethics

Recent advances in so-called ‘additive manufacturing’ pose significant, new challenges, in scope if not in kind, for military ethicists. While the problem of dual-use technologies (i.e. technologies that can be used for both good and malevolent purposes) is not new, the possibility of the rapid, uncontrolled replication of highly sophisticated tools of violent action – tools that heretofore have been largely inaccessible to laymen – could vastly expand the number of persons able to commit violent acts, or even wage war, far beyond traditional boundaries. This article explains the nature of additive manufacturing and identifies the challenges it poses for militaries and governments with the de facto responsibility to keep war-making tools out of the wrong hands. In light of the industrial revolution occasioned by the advent of additive manufacturing and the revolution in military affairs that it portends, it proposes a research agenda for military ethicists. In particular, it argues that military ethicists must now expand their scope of inquiry in a way that accords due prominence to the nexus between these technologies and jus ante bellum issues of conflict avoidance.

Factors Associated with Amplified HIV Transmission Behavior Among American Men Who Have Sex with Men Engaged in Care: Implications for Clinical Providers

The HIV epidemic continues unabated in the USA, with men who have sex with men (MSM) being most frequently infected. The purpose of this study is to understand the biological and behavioral risk factors associated with increased HIV transmission efficiency, which is HIV transmission risk behavior in the context of uncontrolled HIV replication or intercurrent sexually transmitted infections. Participants were 201 HIV-infected MSM who received their primary care at an HIV ambulatory care center in Boston. Logistic regression models were conducted to determine factors associated with engaging in behavior associated with potentially amplified transmission. In the final model, heavy alcohol use (AOR, 3.27; 95 % CI 1.37-7.79), as well as stimulant drug use (crystal meth, crack, or other cocaine; AOR, 3.00; CI 1.32-6.84), having at least a college degree (OR, 2.74; CI, 1.15-6.54), and decreased duration of HIV infection (OR, 0.91; CI, 0.85-0.97) were each uniquely associated with enhanced HIV transmission behavior. HIV primary care providers should routinely assess patients for potential HIV transmission behaviors, particularly those who are younger and more recently diagnosed with HIV, who drink alcohol heavily, and who use any nonprescription drugs, particularly stimulants, in order to decrease the spread of HIV.

Association of autoantibodies anti-OxLDL and markers of inflammation with stage of HIV infection

HIV infected individuals show a wide spectrum of changes caused by systemic immunosuppression homing from the virus itself or therapies used to improve this state. Uncontrolled replication of the virus causes changes in lipid profile, such as increased levels of triglycerides, low density lipoprotein and decreased high density lipoprotein, to promote a dyslipidemia condition [1]. Oxidative stress is also elevated in HIV+ patients due to excessive production of free radicals that play a major role in oxidative modification in LDL particle [2]. HIV+ patients with low counts of T CD4 cells show reduced plasma levels of anti-LDLm (LDL modified), and these low levels of antibodies are also associated with the lipodystrophy syndrome[3].SerumlevelsofOxLDLarehigherinHIV+patientsonantiretroviral therapy especially in patients with lipodystrophy syndrome compared with soronegative patients [4]. There are also other types of disorders that are frequent in HIV infection, such as endothelial dysfunction and metabolic disorders including insulin resistance [5]. All these factors increase the risk of HIV+ patients to accelerate the progression of the atherosclerosis. In this cross-section study, we included consecutive 69 adult patients of both gender with sorological positive to HIV infection. In HIV-infectedindividouswereevaluatedimmunemarkers,HIVinfection

Cas3 stimulates runaway replication of a ColE1 plasmid in Escherichia coli and antagonises RNaseHI

Cas3 nuclease-helicase is part of CRISPR immunity systems in many bacteria and archaea. In type I CRISPR, Cas3 nuclease degrades invader DNA that has been base-paired to crRNA as an R-loop within a “Cascade” complex. An R-loop is a DNA-RNA hybrid that includes a displaced single-strand DNA loop. Purified Cas3 from E. coli and the archaeon M. thermautrophicus can process R-loops without DNA/RNA sequence specificity and without Cascade. This has potential to affect other aspects of microbial biology that involve R-loops. Regulatory RNAs and host cell proteins modulate replication of ColE1 plasmids (e.g., pUC) from R-loop primers. We observed that Cas3 could override endogenous control of a ColE1 replicon, stimulating uncontrolled (“runaway”) replication and resulting in much higher plasmid yields. This effect was absent when using helicase-defective Cas3 (Cas3K320L) or a non-ColE1 plasmid, and was dependent on RNaseHI. Cas3 also promoted formation of plasmid multimers or concatemers, a phenotype consistent with deregulated ColE1 replication and typical of cells lacking RNaseHI. These effects of Cas3 on ColE1 plasmids are inconsistent with it unwinding R-loops in vivo, at least in this assay. We discuss a model of how Cas3 might be able to regulate RNA molecules in vivo, unless it is targeted to CRISPR defense by Cascade, or kept in check by RecG and RNaseHI.

An Effective Find and Replicate Strategy for Data Communication in Intermittently Connected Wireless Ad Hoc and Sensor Networks

ICWASNs are a kind of wireless networks where, due to mobility of nodes and lack of connectivity, there may be frequent disconnections among the nodes. Hence, the routing path from the source to destination will not be available always. It is proven that, in these networks, messages are replicated multiple times in order to withstand the maximum delay and to achieve high throughput. But these multiple replication-based protocols result in an increase in network overhead and high resource consumption because of uncontrolled replication. Previous works in ICWASNs assume that networks will always have multiple partitions, and each message will be routed using store and forward mechanism. If source and destination are connected, then it is not needed to replicate multiple copies and waste the resources. We introduce a new simple scheme which applies single-copy routing if destination is available; else it switches to store and forward routing. The proposed system tries to reduce the average number of message replications while increasing the throughput.

Immunopathogenesis of non-healing American cutaneous leishmaniasis and progressive visceral leishmaniasis.

The outcomes of Leishmania infection are determined by host immune and nutrition status, parasite species, and co-infection with other pathogens. While subclinical infection and self-healing cutaneous leishmaniasis (CL) are common, uncontrolled parasite replication can lead to non-healing local lesions or visceral leishmaniasis (VL). It is known that infection control requires Th1-differentiation cytokines (IL-12, IL-18, and IL-27) and Th1 cell and macrophage activation. However, there is no generalized consensus for the mechanisms of host susceptibility. The recent studies on regulatory T cells and IL-17-producing cells help explain the effector T cell responses that occur independently of the known Th1/Th2 cell signaling pathways. This review focuses on the immunopathogenesis of non-healing American CL and progressive VL. We summarize recent evidence from human and animal studies that reveals the mechanisms of dysregulated, hyper-responses to Leishmania braziliensis, as well as the presence of disease-promoting or the absence of protective responses to Leishmania amazonensis and Leishmania donovani. We highlight immune-mediated parasite growth and immunopathogenesis, with an emphasis on the putative roles of IL-17 and its related cytokines as well as arginase. A better understanding of the quality and regulation of innate immunity and T cell responses triggered by Leishmania will aid in the rational control of pathology and the infection.

Thrombocytopenia During Primary HIV-1 Infection Predicts the Risk of Recurrence During Chronic Infection

Thrombocytopenia During Primary HIV-1 Infection Predicts the Risk of Recurrence During Chronic Infection

Prevalence and risk factors associated with pulmonary hypertension in HIV-infected patients on regular follow-up

Pulmonary arterial hypertension (PAH) is uncommon among HIV-positive patients. However, it is a potentially life-threatening condition. Transthoracic echocardiography (TTE) is a noninvasive tool validated for PAH screening. The aim of our study was to establish the prevalence and factors associated with PAH in HIV-infected patients. Consecutive HIV-infected individuals attended at one HIV reference clinic in Madrid, Spain, during year 2011 were examined. Demographics and clinical data were recorded and a Doppler echocardiography was performed in all individuals. PAH was considered when right ventricular pressure was more than 35 mmHg (mild if <40 mmHg, moderate if 40-65 mmHg, and severe if >65 mmHg). Three hundred and ninety-two individuals were examined (83.4% men, median age 47 years, 53% were men who have sex with men and 53% former intravenous drug addicts). Overall, 84% were on HAART, 76% had undetectable HIV viral load and median CD4 cell counts were 577 cells/μl. Cardiovascular risk factors were smoking 50%, arterial hypertension 16% and diabetes mellitus 9%. A total of 28.5 and 4.8% had chronic hepatitis C (CHC) and 4.8% chronic hepatitis B, respectively. PAH was diagnosed in 9.9% of patients (6.4% mild, 2.8% moderate and 0.8% severe). Multivariate logistic regression analysis [odds ratio (OR), 95% confidence interval (CI)] showed that detectable plasma HIV-RNA [OR, 3.3; 95% CI, 1.04-10], CHC [OR, 3.1; 95% CI 1.2-8.2] and female sex [OR, 2.9; 95% CI, 1.04-8.3] were independently associated with PAH. The prevalence of PAH HIV-infected patients on regular follow-up approaches 10%, being moderate-severe in nearly 4% of cases. Patients with CHC and/or uncontrolled HIV replication exhibit a higher risk of PAH.

Senescence‐escape in melanoma

Cellular senescence is an important defense against uncontrolled cellular replication and is a critical tumor suppressor mechanism. Although initially described as an in vitro phenomenon, in recent years, several lines of evidence have indicated that senescence also occurs in vivo. Disparate cellular stresses can provoke senescence including telomere shortening, activation of certain oncogenes, oxidative stress and stalled replication forks. These stresses are best known to engage the p16INK4a-RB and ARF-p53 pathways to effect growth arrest and prevent neoplasia. Among these, ‘oncogene-induced senescence’ (OIS) is particularly known for the prevention of melanoma, resulting from the expression of mutant B-RAF or N-RAS in this disease.