Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1)
In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8(+) T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses.
Oncogenic RAS directs silencing of tumor suppressor genes through ordered recruitment of transcriptional repressors
We previously identified 28 cofactors through which a RAS oncoprotein directs transcriptional silencing of Fas and other tumor suppressor genes (TSGs). Here we performed RNAi-based epistasis experiments and found that RAS-directed silencing occurs through a highly ordered pathway that is initiated by binding of ZFP354B, a sequence-specific DNA-binding protein, and culminates in recruitment of the DNA methyltransferase DNMT1. RNAi and pharmacological inhibition experiments reveal that silencing requires continuous function of RAS and its cofactors and can be rapidly reversed, which may have therapeutic implications for reactivation of silenced TSGs in RAS-positive cancers.
Relation of N-terminal pro-B-type natriuretic peptide with diastolic function in hypertensive heart disease
BACKGROUND: Elevated natriuretic peptide levels in asymptomatic individuals without heart failure are associated with increased risk of adverse cardiovascular outcomes and may reflect subclinical cardiac dysfunction.
METHODS: In a sample of 313 asymptomatic individuals (51% women, mean age 61 years) with hypertension and diastolic dysfunction, we examined the association of plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) with both conventional and advanced echocardiographic measures of systolic and diastolic function, including myocardial strain, using speckle-tracking-based analyses.
RESULTS: In univariate analyses, higher NT-proBNP was associated with greater left ventricular mass index (P = 0.003), left atrial volume index (P = 0.007), lateral E' velocity (P < 0.0001), E/E' ratio (P < 0.0001), peak global longitudinal systolic strain (P = 0.015), systolic strain rate (P = 0.021), and early diastolic strain rate (P < 0.0001). In multivariable analyses, NT-proBNP remained associated with measures of diastolic dysfunction, including lateral E' velocity (P = 0.013) and the E/E' ratio (P = 0.008). However, early diastolic strain rate was the echocardiographic parameter most strongly associated with NT-proBNP (P = 0.003).
CONCLUSIONS: In the setting of asymptomatic hypertensive heart disease and preserved ejection fraction, elevation in natriuretic peptide levels is predominantly associated with subclinical diastolic dysfunction.
Generation and destruction of antigenic peptides by ER resident aminopeptidases ERAP1 and ERAP2 have been shown in the last few years to be important for the correct functioning and regulation of the adaptive immune response. These two highly homologous aminopeptidases appear to have evolved complex mechanisms well suited for their biological role in antigen presentation. Furthermore, polymorphic variability in these enzymes appears to affect their function and predispose individuals to disease. This review discusses our current understanding of the molecular mechanisms behind ERAP1/2 function as suggested by several recently determined crystallographic structures of these enzymes.
Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE-DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo.
Removal of introns from nascent transcripts (pre-mRNAs) by the spliceosome is an essential step in eukaryotic gene expression. Previous studies have suggested that the earliest steps in spliceosome assembly in yeast are highly ordered and the stable recruitment of U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site necessarily precedes recruitment of U2 snRNP to the branch site to form the "prespliceosome." Here, using colocalization single-molecule spectroscopy to follow initial spliceosome assembly on eight different S. cerevisiae pre-mRNAs, we demonstrate that active yeast spliceosomes can form by both U1-first and U2-first pathways. Both assembly pathways yield prespliceosomes functionally equivalent for subsequent U5.U4/U6 tri-snRNP recruitment and for intron excision. Although fractional flux through the two pathways varies on different introns, both are operational on all introns studied. Thus, multiple pathways exist for assembling functional spliceosomes. These observations provide insight into the mechanisms of cross-intron coordination of initial spliceosome assembly.
The training of health care providers has been identified as key to resolving the health disparities experienced by persons with disabilities. We contend that: 1) cultural competency provides a useful conceptual framework for teaching disability-related content to health professions students; 2) educational experiences can be structured to reflect the socio-cultural complexity of the 'disability culture;' 3) desired competencies associated with culture can be defined with regard to professionals' approach to patients with disabilities; 4) exposure to persons who have disabilities in their homes allows the student to make connections between the nuances of daily life with a disability and one's health care needs; 5) the framework allows the disability culture to be integrated with other cultural contexts, including race and ethnicity; and 6) the framework acknowledges the potential impact of providers' conscious or unconscious recognition of their potential membership in the disability culture on their approach to patients with disabilities.
NK cells have direct activity against fungal pathogens. Using an unbiased systematic approach, Li et al. (2013) find that NKp30 is a major NK cell receptor responsible for fungal recognition. Moreover, diminished NKp30 expression is associated with reduced antifungal activity in NK cells isolated from HIV-infected persons.
Differentiating reversible cerebral vasoconstriction syndrome with subarachnoid hemorrhage from other causes of subarachnoid hemorrhage
IMPORTANCE: Reversible cerebral vasoconstriction syndrome (RCVS) is a clinical-angiographic syndrome characterized by recurrent thunderclap headaches and reversible segmental multifocal cerebral artery narrowing. More than 30% of patients with RCVS develop subarachnoid hemorrhage (SAH). Patients with RCVS with SAH (RCVS-SAH) are often misdiagnosed as having potentially ominous conditions such as aneurysmal SAH (aSAH) or cryptogenic "angiogram-negative" SAH (cSAH) owing to overlapping clinical and imaging features.
OBJECTIVE: To identify predictors that can distinguish RCVS-SAH from aSAH and cSAH at the time of clinical presentation.
DESIGN: Retrospective analysis of 3 patient cohorts: patients with RCVS (1998-2009), patients with aSAH (1995-2003), and patients with cSAH (1995-2003).
SETTING: Academic hospital and tertiary referral center.
PARTICIPANTS: Consecutive patients with RCVS-SAH (n = 38), aSAH (n = 515), or cSAH (n = 93) whose conditions were diagnosed using standard criteria.
MAIN OUTCOMES AND MEASURES: Multivariate logistic regression analysis was used to identify predictors that differentiate RCVS-SAH from aSAH and cSAH.
RESULTS: Predictors differentiating RCVS-SAH from aSAH were younger age, chronic headache disorder, prior depression, prior chronic obstructive pulmonary disease, lower Hunt-Hess grade, lower Fisher SAH group, higher number of affected arteries, and the presence of bilateral arterial narrowing. Predictors differentiating RCVS-SAH from cSAH were younger age, female sex, prior hypertension, chronic headache disorder, lower Hunt-Hess grade, lower Fisher SAH group, and the presence of bilateral arterial narrowing.
CONCLUSIONS AND RELEVANCE: We identified important clinical and imaging differences between RCVS-SAH, aSAH, and cSAH that may be useful for improving diagnostic accuracy, clinical management, and resource utilization.
Syntaxin 13, a genetic modifier of mutant CHMP2B in frontotemporal dementia, is required for autophagosome maturation
Phagophore maturation is a key step in the macroautophagy pathway, which is critical in many important physiological and pathological processes. Here we identified Drosophila N-ethylmaleimide-sensitive fusion protein 2 (dNSF2) and soluble NSF attachment protein (Snap) as strong genetic modifiers of mutant CHMP2B, an ESCRT-III component that causes frontotemporal dementia and autophagosome accumulation. Among several SNAP receptor (SNARE) genes, Drosophila syntaxin 13 (syx13) exhibited a strong genetic interaction with mutant CHMP2B. Knockdown of syntaxin 13 (STX13) or its binding partner Vti1a in mammalian cells caused LC3-positive puncta to accumulate and blocks autophagic flux. STX13 was present on LC3-positive phagophores induced by rapamycin and was highly enriched on multilamellar structures induced by dysfunctional ESCRT-III. Loss of STX13 also caused the accumulation of Atg5-positive puncta and the formation of multilamellar structures. These results suggest that STX13 is a genetic modifier of ESCRT-III dysfunction and participates in the maturation of phagophores into closed autophagosomes.
Analysis of in vitro insulin-resistance models and their physiological relevance to in vivo diet-induced adipose insulin resistance
Diet-induced obesity (DIO) predisposes individuals to insulin resistance, and adipose tissue has a major role in the disease. Insulin resistance can be induced in cultured adipocytes by a variety of treatments, but what aspects of the in vivo responses are captured by these models remains unknown. We use global RNA sequencing to investigate changes induced by TNF-alpha, hypoxia, dexamethasone, high insulin, and a combination of TNF-alpha and hypoxia, comparing the results to the changes in white adipose tissue from DIO mice. We found that different in vitro models capture distinct features of DIO adipose insulin resistance, and a combined treatment of TNF-alpha and hypoxia is most able to mimic the in vivo changes. Using genome-wide DNase I hypersensitivity followed by sequencing, we further examined the transcriptional regulation of TNF-alpha-induced insulin resistance, and we found that C/EPBbeta is a potential key regulator of adipose insulin resistance.
An often-overlooked aspect of neural plasticity is the plasticity of neuronal composition, in which the numbers of neurons of particular classes are altered in response to environment and experience. The Drosophila brain features several well-characterized lineages in which a single neuroblast gives rise to multiple neuronal classes in a stereotyped sequence during development. We find that in the intrinsic mushroom body neuron lineage, the numbers for each class are highly plastic, depending on the timing of temporal fate transitions and the rate of neuroblast proliferation. For example, mushroom body neuroblast cycling can continue under starvation conditions, uncoupled from temporal fate transitions that depend on extrinsic cues reflecting organismal growth and development. In contrast, the proliferation rates of antennal lobe lineages are closely associated with organismal development, and their temporal fate changes appear to be cell cycle-dependent, such that the same numbers and types of uniglomerular projection neurons innervate the antennal lobe following various perturbations. We propose that this surprising difference in plasticity for these brain lineages is adaptive, given their respective roles as parallel processors versus discrete carriers of olfactory information.
Impact of mirabegron extended-release on the treatment of overactive bladder with urge urinary incontinence, urgency, and frequency
Overactive bladder is a highly prevalent disorder with a significant impact on quality of life. Antimuscarinic agents are commonly used, but persistence is limited due to unsatisfactory efficacy and/or tolerability. Mirabegron is the first beta-3 adrenoceptor agonist approved for the treatment of overactive bladder syndrome. This paper reviews the pharmacology, mechanism of action, efficacy, and safety of mirabegron. A PubMed search of all English articles pertaining to mirabegron was performed. An alternative to antimuscarinics, mirabegron has a unique mechanism, improves overactive bladder symptoms and quality of life, and has limited adverse effects and few contraindications.
The processes of development, repair, and remodeling of virtually all tissues and organs, are dependent upon mechanical signals including external loading, cell-generated tension, and tissue stiffness. Over the past few decades, much has been learned about mechanotransduction pathways in specialized two-dimensional culture systems; however, it has also become clear that cells behave very differently in two- and three-dimensional (3D) environments. Three-dimensional in vitro models bring the ability to simulate the in vivo matrix environment and the complexity of cell-matrix interactions together. In this review, we describe the role of tension in regulating cell behavior in three-dimensional collagen and fibrin matrices with a focus on the effective use of global boundary conditions to modulate the tension generated by populations of cells acting in concert. The ability to control and measure the tension in these 3D culture systems has the potential to increase our understanding of mechanobiology and facilitate development of new ways to treat diseased tissues and to direct cell fate in regenerative medicine and tissue engineering applications.
Maternally supplied mRNAs encode proteins that pattern early embryos in many species. In the nematode Caenorhabditis elegans, a suite of RNA-binding proteins regulates expression of maternal mRNAs during oogenesis, the oocyte to embryo transition, and early embryogenesis. To understand how these RNA-binding proteins contribute to development, it is necessary to determine how they select specific mRNA targets for regulation. OMA-1 and OMA-2 are redundant proteins required for oocyte maturation--an essential part of meiosis that prepares oocytes for fertilization. Both proteins have CCCH type tandem zinc finger RNA-binding domains. Here, we define the RNA binding specificity of OMA-1 and demonstrate that OMA-1/2 are required to repress the expression of a glp-1 3'-UTR reporter in developing oocytes. OMA-1 binds with high affinity to a conserved region of the glp-1 3'-UTR previously shown to interact with POS-1 and GLD-1, RNA-binding proteins required for glp-1 reporter repression in the posterior of fertilized embryos. Our results reveal that OMA-1 is a sequence-specific RNA-binding protein required to repress expression of maternal transcripts during oogenesis and suggest that interplay between OMA-1 and other factors for overlapping binding sites helps to coordinate the transition from oocyte to embryo.
Hematocrit alters VerifyNow P2Y12 assay results independently of intrinsic platelet reactivity and clopidogrel responsiveness
BACKGROUND: The VerifyNow P2Y12 assay assesses the adequacy of clopidogrel therapy by measuring ADP-induced platelet activation in whole blood. Low hematocrit is associated with high clopidogrel on-treatment platelet reactivity (HTPR) defined by this assay.
OBJECTIVES: To characterize the effect of hematocrit on VerifyNow values and determine if it is due to hematocrit-dependent changes in intrinsic platelet reactivity or an in vitro assay phenomenon.
PATIENTS/METHODS: Adenosine diphosphate-induced platelet activation was measured using the VerifyNow P2Y12 assay, whole blood impedance and light transmission platelet aggregometry (LTA) before and after clopidogrel loading in 113 patients undergoing elective cardiac catheterization. Iso-TRAP-induced platelet activation was additionally measured using the VerifyNow device. Multivariate modeling employing clinical and laboratory variables was used to investigate the association between hematocrit and VerifyNow values.
RESULTS: VerifyNow P2Y12 reaction units (PRU) and iso-TRAP Base units before and after clopidogrel loading, but not their relative change, exhibited strong negative correlation with hematocrit (P < /= 0.0005 for both). While hematocrit remained a strong predictor of post-clopidogrel PRU (P = 0.001) in multivariate modeling, it was independent of post-clopidogrel ADP-induced platelet reactivity as measured by LTA (P = 0.001). Correcting for the effects of hematocrit resulted in a 15-39% reduction in the prevalence of HTPR defined by thresholds of 208-236 PRU.
CONCLUSIONS: The effect of hematocrit on VerifyNow PRU values is an in vitro phenomenon that is independent of intrinsic change in ADP-induced platelet reactivity and clopidogrel responsiveness. Correcting for hematocrit when using this assay may more accurately identify patients with HTPR that may benefit from alternative antiplatelet therapy.
Recent observations have uncovered multiple pathways whereby CD4 T cells can contribute to protective immune responses against microbial threats. Incorporating the generation of memory CD4 T cells into vaccine strategies thus presents an attractive approach toward improving immunity against several important human pathogens, especially those against which antibody responses alone are inadequate to confer long-term immunity. Here, we review how memory CD4 T cells provide protection against influenza viruses. We discuss the complexities of protective memory CD4 T cell responses observed in animal models and the potential challenges of translating these observations into the clinic. Specifically, we concentrate on how better understanding of organ-specific heterogeneity of responding cells and defining multiple correlates of protection might improve vaccine-generated memory CD4 T cells to better protect against seasonal, and more importantly, pandemic influenza.
Efficacy and safety of natalizumab in Crohn's disease patients treated at 6 Boston academic hospitals
BACKGROUND: Despite trials demonstrating its efficacy, many physicians harbor concerns regarding the use of natalizumab in the treatment of patients with refractory Crohn's disease (CD). The purpose of this study was to perform a descriptive analysis of a series of CD patients not currently enrolled in a clinical trial.
METHODS: A retrospective case review of patients treated with natalizumab at 6 sites in Massachusetts: Boston Medical Center, Beth Israel Deaconess Medical Center, Brigham and Women's Hospital, Lahey Clinic, Massachusetts General Hospital, and UMass Medical Center.
RESULTS: Data on 69 CD patients on natalizumab were collected. At the start of treatment, patients' disease duration was 12 years. A high proportion of patients were women (68%), presented with perianal disease (65%) and upper gastrointestinal tract involvement (14%). Prior nonbiologic therapies were steroids (96%), thiopurines (94%), antibiotics (74%), methotrexate (58%), and at least two anti-tumor necrosis factor agent failures (81%). Sixty-nine percent (44 of 64 patients) with available medical evaluation had a partial or complete clinical response. Loss of response was 13% after an average of 1 year of treatment. Adverse events were infusion reactions, headaches, fever, and infections. No case of progressive multifocal leukoencephalopathy was observed.
CONCLUSIONS: In our clinical experience outside the context of a clinical trial, natalizumab is largely reserved for CD patients with extensive ileocolonic disease who have failed conventional immunosuppressants and of at least 2 anti-tumor necrosis factor agents. This drug is, however, well tolerated and offers significant clinical improvement for more than a year in one-third of these difficult-to-treat CD patients.
Immunodepletion plasma proteomics by tripleTOF 5600 and Orbitrap elite/LTQ-Orbitrap Velos/Q exactive mass spectrometers
Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered > 250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.
BACKGROUND: Both chronic and acute pain have been cited as the most common symptoms amongst patients with multiple sclerosis (MS), with recent prevalence estimates as high as 83 %. The evidence for spasticity and trigeminal neuralgia pharmacological treatments in MS has been systematically reviewed, but no equivalent reviews have been published concerning MS pain unrelated to these two conditions.
OBJECTIVE: Our objective was to systematically review pain management strategies for the reduction of non-spastic and non-trigeminal neuralgic pain in MS patients.
DATA SOURCES: Experimental studies published after 1965 were chosen for review by searching electronic databases (e.g. PubMed, Cumulative Index to Nursing and Allied Health Literature, Science Citation Index Expanded, Conference Proceedings Citation Index-Science, and clinicaltrials.gov) and bibliographies/citations of previously published reviews.
STUDY SELECTION: Studies were included if all participants were adults clinically diagnosed with MS, study sample was not restricted to participants with spasticity or trigeminal neuralgia, and participant-reported pain was a primary or secondary outcome measured with a validated tool.
STUDY APPRAISAL AND SYNTHESIS METHODS: Records were screened and methodological qualities of included studies were assessed independently by two reviewers under the supervision of another reviewer using the principles recommended in the Cochrane Handbook for Systematic Review of Interventions and the levels of evidence espoused by the American Academy of Neurology.
RESULTS: Fifteen studies met the inclusion and exclusion criteria for review; interventions included antidepressants, anticonvulsants, dextromethorphan/quinidine, cannabinoids, and opioids/opioid antagonists. The pooled effect size for anticonvulsants (4 studies, 78 participants) was -1.88 (95 % CI: -3.13 to -0.64). The pooled effect size for cannabinoids (3 studies, 565 participants) was 0.08 (95 % CI: -0.74 to 0.89). Overall, only four trials reported Class 1 evidence. For these trials, dizziness was the most commonly reported adverse event, followed by nausea and somnolence.
LIMITATIONS: The relatively small number of trials in MS patients with chronic pain precludes specific recommendations for treatment strategies. The review did not reveal any studies of drug combinations.
CONCLUSIONS: More trials with rigorous design and reporting are needed to determine effective treatments for specific pain types presenting in people living with MS.