ABSTRACT
Tests that detect the presence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigen in clinical specimens from the upper respiratory tract can provide a rapid means of coronavirus disease 2019 (COVID-19) diagnosis and help identify individuals who may be infectious and should isolate to prevent SARS-CoV-2 transmission. This systematic review assesses the diagnostic accuracy of SARS-CoV-2 antigen detection in COVID-19 symptomatic and asymptomatic individuals compared to quantitative reverse transcription polymerase chain reaction (RT-qPCR) and summarizes antigen test sensitivity using meta-regression. In total, 83 studies were included that compared SARS-CoV-2 rapid antigen-based lateral flow testing (RALFT) to RT-qPCR for SARS-CoV-2. Generally, the quality of the evaluated studies was inconsistent; nevertheless, the overall sensitivity for RALFT was determined to be 75.0% (95% confidence interval: 71.0-78.0). Additionally, RALFT sensitivity was found to be higher for symptomatic vs. asymptomatic individuals and was higher for a symptomatic population within 7 days from symptom onset compared to a population with extended days of symptoms. Viral load was found to be the most important factor for determining SARS-CoV-2 antigen test sensitivity. Other design factors, such as specimen storage and anatomical collection type, also affect the performance of RALFT. RALFT and RT-qPCR testing both achieve high sensitivity when compared to SARS-CoV-2 viral culture.
ABSTRACT
BACKGROUND: Individuals can test positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by molecular assays following the resolution of their clinical disease. Recent studies indicate that SARS-CoV-2 antigen-based tests are likely to be positive early in the disease course, when there is an increased likelihood of high levels of infectious virus. METHODS: Upper respiratory specimens from 251 participants with coronavirus disease 2019 symptoms (≤7 days from symptom onset) were prospectively collected and tested with a lateral flow antigen test and a real-time polymerase chain reaction (rt-PCR) assay for detection of SARS-CoV-2. Specimens from a subset of the study specimens were utilized to determine the presence of infectious virus in the VeroE6TMPRSS2 cell culture model. RESULTS: The antigen test demonstrated a higher positive predictive value (90%) than rt-PCR (70%) when compared to culture-positive results. The positive percentage agreement for detection of infectious virus for the antigen test was similar to rt-PCR when compared to culture results. CONCLUSIONS: The correlation between SARS-CoV-2 antigen and SARS-CoV-2 culture positivity represents a significant advancement in determining the risk for potential transmissibility beyond that which can be achieved by detection of SARS-CoV-2 genomic RNA. SARS-CoV-2 antigen testing can facilitate low-cost, scalable, and rapid time-to-result, while providing good risk determination of those who are likely harboring infectious virus, compared to rt-PCR.
Subject(s)
COVID-19 , SARS-CoV-2 , Antigens, Viral , Humans , Real-Time Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
OBJECTIVE: The aim of the study was to examine whether high-grade cervical intraepithelial neoplasia (CIN) was more closely associated with human papillomavirus (HPV) same-genotype persistence (SGTP) versus clearance of prior infection with a subsequent infection by a new genotype (genotype switch [GS]), clearance of HPV infection, or acquisition of a new HPV infection after a negative infection status, during a follow-up testing subsequent to abnormal screening results. MATERIALS AND METHODS: MEDLINE, Cochrane Library, Health Technology Assessment, and clinicaltrials.gov were searched from January 2000 to July 2019 for prospective controlled trials and observational studies of women and retrospective studies using HPV assays with extended- or full-genotype reporting. The primary outcome was high-grade CIN after at least 2 rounds of testing. Overall quality of evidence for the risk estimate outcomes was assessed. Of the 830 identified abstracts, 66 full-text articles were reviewed, and 7 studies were included in the synthesis. The study protocol was registered with the PROSPERO International Prospective Register of Systematic Reviews (CRD42018091093). RESULTS: Continued HPV-positive women falls in 2 equally large groups: SGTP and GS. Sensitivity, positive predictive value, and positive likelihood ratio of SGTP were significantly higher than for GS. Human papillomavirus genotypes may be ranked into 3 tiers (immediate colposcopy, follow-up testing, return to routine screening), according to associated risk of persistence for high-grade CIN and to prevailing clinical action thresholds. CONCLUSIONS: There is moderately high-quality evidence to support the clinical utility of SGTP to improve risk discrimination for high-grade CIN compared with qualitative HPV testing without genotype-specific information.
Subject(s)
Papillomaviridae/genetics , Papillomavirus Infections/epidemiology , Papillomavirus Infections/genetics , Uterine Cervical Dysplasia/virology , Uterine Cervical Neoplasms/virology , Adult , Colposcopy , Early Detection of Cancer/methods , Female , Genotype , Humans , Meta-Analysis as Topic , Middle Aged , Papillomaviridae/isolation & purification , Risk Factors , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Young Adult , Uterine Cervical Dysplasia/genetics , Uterine Cervical Dysplasia/pathologyABSTRACT
OBJECTIVE: Thirteen human papillomavirus (HPV) genotypes are associated with the highest risk of cervical disease/cancer; however, the risk of disease progression and cancer is genotype dependent. The objective of this systematic review was to examine evidence for high-grade cervical intraepithelial neoplasia (≥CIN 3) risk discrimination using HPV genotyping. MATERIALS AND METHODS: A systematic review of English and non-English articles through MEDLINE, Cochrane, clinicaltrials.gov, and abstracts presented at relevant professional society conferences were searched from 2000 to 2019. Search terms included: cervical cancer screening, HPV genotyping, CIN, HPV persistence, humans, and colposcopy; prospective, controlled trials, observational studies, and retrospective studies of residual specimens; evidence included HPV genotyping (beyond genotypes 16/18/45) results. Data were obtained independently by authors using predefined fields. Risk of bias was evaluated with a modified Newcastle-Ottawa Scale. The Grading of Recommendations, Assessment, Development and Evaluation methodology facilitated overall quality of evidence evaluation for risk estimation. The study protocol was registered with the PROSPERO International Prospective Register of Systematic Reviews (CRD42018091093). The primary outcome was CIN 3 or worse risk both at baseline and at different follow-up periods. RESULTS: Of 236 identified sources, 60 full texts were retrieved and 16 articles/sources were included. Risk of bias was deemed low; the overall quality of evidence for CIN 3 or worse risk with negative for intraepithelial lesions or malignancies or low-grade squamous intraepithelial cytology was assessed as moderate; that with atypical squamous cells-undetermined significance and "all cytology" was assessed as high. Clinical and methodological heterogeneity precluded meta-analysis. Human papillomavirus genotyping discriminated risk of CIN 3 or worse to a clinically significant degree, regardless of cytology result. CONCLUSIONS: The evidence supports a clinical utility for HPV genotyping in risk discrimination during cervical cancer screening.
Subject(s)
Early Detection of Cancer/methods , Genotyping Techniques/methods , Neoplasm Grading/methods , Papillomaviridae/classification , Papillomaviridae/isolation & purification , Squamous Intraepithelial Lesions/diagnosis , Uterine Cervical Neoplasms/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Middle Aged , Papillomaviridae/genetics , Young AdultABSTRACT
OBJECTIVE: To systematically examine human papillomavirus (HPV) genotyping compared with qualitative high-risk HPV result during follow-up after treatment of high-grade cervical intraepithelial neoplasia (CIN), for risk estimation of posttreatment high-grade CIN. DATA SOURCES: MEDLINE, Cochrane, and ClinicalTrials.gov were searched from January 2000 to April 2019 for prospective studies of women and retrospective studies of residual specimens from women, tested using HPV assays with genotype reporting. METHODS OF STUDY SELECTION: The primary outcome was posttreatment high-grade CIN after treatment of high-grade CIN. Risk of bias (individual study quality) was evaluated with a modified Newcastle-Ottawa Scale. Overall quality of evidence for the risk estimate outcomes was evaluated using modified GRADE methodology for observational diagnostic studies. TABULATION, INTEGRATION, AND RESULTS: Of the 233 identified abstracts, 33 full-text articles were retrieved, and seven studies were included in the synthesis. The risk of bias was deemed to be low. Either a positive qualitative HPV test result or a positive test result for the same genotype that was present pretreatment have a sensitivity for predicting posttreatment high-grade CIN that approaches 100%. However, the positive predictive value (PPV) for the same genotype result pretreatment and posttreatment (median 44.4%) is about double the PPV (median 22.2%) for qualitative HPV results. The PPV of a new HPV infection posttreatment approximates zero. Human papillomavirus genotyping discriminated risk of posttreatment high-grade CIN to a clinically significant degree for women after treatment procedures for high-grade CIN lesions, when same-genotype persistence was compared with new genotype infection. CONCLUSION: There is moderately high-quality evidence to support the improved clinical utility of HPV genotyping compared with qualitative HPV positivity to follow-up after treatment of high-grade CIN. SYSTEMATIC REVIEW REGISTRATION: PROSPERO: CRD42018091095. FUNDING SOURCE: Becton, Dickinson and Company, BD Life Sciences-Diagnostic Systems.
Subject(s)
Genotyping Techniques/statistics & numerical data , Papillomaviridae/genetics , Papillomavirus Infections/virology , Uterine Cervical Dysplasia/virology , Uterine Cervical Neoplasms/virology , Adult , Female , Genotype , Genotyping Techniques/methods , Humans , Middle Aged , Predictive Value of Tests , Prospective Studies , Retrospective Studies , Risk Assessment , Uterine Cervical Neoplasms/therapy , Uterine Cervical Dysplasia/therapyABSTRACT
OBJECTIVES: Previous "Treatment of Severe Childhood Aggression" (TOSCA) reports demonstrated that many children with severe physical aggression and attention-deficit/hyperactivity disorder (ADHD) responded well to two randomized treatments (parent training [PT]+stimulant+placebo = Basic vs. PT+stimulant+risperidone = Augmented) for 9 weeks. An important clinical question is whether these favorable outcomes are maintained over longer times. METHODS: Clinical responders to the 9-week trial (n = 103/168), defined as Clinical Global Impressions (CGI)-Improvement of much/very much improved plus substantial reduction in parent ratings of disruptiveness, were followed another 12 weeks (21 weeks total) while remaining on blinded treatment. Outcome measures included Clinical Global Impressions scale, Nisonger Child Behavior Rating Form (NCBRF), other parent/teacher-rated scales, laboratory tests, clinician ratings of abnormal movement, and other adverse events (AEs). RESULTS: Parent ratings of problem behavior showed minimal worsening of behavior from end of the 9-week acute trial (expected from regression to the mean after selecting best responders), but outcomes at Extension endpoint were meaningfully improved compared with acute study baseline. As expected, outcomes for Basic and Augmented treatment did not differ among these children selected for good clinical response. During Extension, more Augmented subjects had elevated prolactin; there were no clinically confirmed cases of tardive dyskinesia. Delayed sleep onset was the most frequent Basic AE. We also conducted a last-observation-carried-forward analysis, which included both nonresponders and responders. We found that, at the end of Extension, Augmented subjects had more improvement than Basic subjects on the NCBRF Positive Social subscale (p = 0.005; d = 0.44), the Antisocial Behavior Scale Reactive Aggression subscale (p = 0.03; d = 0.36), and marginally so on the Disruptive Behavior Total subscale (p = 0.058; d = 0.29, the primary outcome). CONCLUSIONS: The medium-term outcomes were good for the participants in both treatment groups, perhaps because they were selected for good response. When nonresponders were included in ITT analyses, there was some indication that Augmented surpassed Basic treatment.
Subject(s)
Aggression/drug effects , Attention Deficit Disorder with Hyperactivity/drug therapy , Central Nervous System Stimulants/administration & dosage , Risperidone/administration & dosage , Aggression/psychology , Antipsychotic Agents/administration & dosage , Antipsychotic Agents/therapeutic use , Attention Deficit Disorder with Hyperactivity/psychology , Central Nervous System Stimulants/therapeutic use , Child , Combined Modality Therapy , Double-Blind Method , Female , Humans , Male , Parents/education , Psychiatric Status Rating Scales , Risperidone/therapeutic use , Severity of Illness Index , Time Factors , Treatment OutcomeABSTRACT
CNS damage often results in demyelination of spared axons due to oligodendroglial cell death and dysfunction near the injury site. Although new oligodendroglia are generated following CNS injury and disease, the process of remyelination is typically incomplete resulting in long-term functional deficits. Chondroitin sulfate proteoglycans (CSPGs) are upregulated in CNS grey and white matter following injury and disease and are a major component of the inhibitory scar that suppresses axon regeneration. CSPG inhibition of axonal regeneration is mediated, at least in part, by the protein tyrosine phosphatase sigma (PTPσ) receptor. Recent evidence demonstrates that CSPGs inhibit OL process outgrowth, however, the means by which their effects are mediated remains unclear. Here we investigate the role of PTPσ in CSPG inhibition of OL function. We found that the CSPGs, aggrecan, neurocan and NG2 all imposed an inhibitory effect on OL process outgrowth and myelination. These inhibitory effects were reversed by degradation of CSPGs with Chondroitinase ABC prior to OL exposure. RNAi-mediated down-regulation of PTPσ reversed the inhibitory effect of CSPGs on OL process outgrowth and myelination. Likewise, CSPG inhibition of process outgrowth and myelination was significantly reduced in cultures containing PTPσ(-/-) OLs. Finally, inhibition of Rho-associated kinase (ROCK) increased OL process outgrowth and myelination during exposure to CSPGs. These results suggest that in addition to their inhibitory effects on axon regeneration, CSPGs have multiple inhibitory actions on OLs that result in incomplete remyelination following CNS injury. The identification of PTPσ as a receptor for CSPGs, and the participation of ROCK downstream of CSPG exposure, reveal potential therapeutic targets to enhance white matter repair in the damaged CNS.
Subject(s)
Chondroitin Sulfate Proteoglycans/pharmacology , Gene Expression Regulation/drug effects , Myelin Sheath/drug effects , Oligodendroglia/drug effects , Receptor-Like Protein Tyrosine Phosphatases, Class 2/metabolism , Aggrecans/pharmacology , Animals , Animals, Newborn , Antigens/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Chondroitin ABC Lyase/pharmacology , Ganglia, Spinal/cytology , Gangliosides/metabolism , Myelin Basic Protein/metabolism , Neurofilament Proteins/metabolism , Proteoglycans/pharmacology , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Stem Cells , rho-Associated Kinases/metabolismABSTRACT
Oligodendrocyte (OLG) death plays a major role in white matter dysfunction and demyelination following injury to the CNS. Axonal contact, communication, and neuronal activity appear to promote OLG survival and function in cell culture and during development. The application of electrical stimulation to mixed neural cultures has been shown to promote OLG differentiation and the formation of myelin in vitro. Here we show that OLG viability can be significantly enhanced in mixed cortical cultures by applying biphasic pulses of electrical stimulation (ESTIM). Enhanced survival via ESTIM requires the presence of neurons and is suppressed by inhibition of voltage-gated sodium channels. Additionally, contact between the axon and OLG is necessary for ESTIM to promote OLG survival. This report suggests that patterned neuronal activity could repress delayed progression of white matter injury and promote CNS repair in neurological conditions that involve white matter damage.
Subject(s)
Electric Stimulation/methods , Oligodendroglia/physiology , Amino Acid Chloromethyl Ketones/pharmacology , Analysis of Variance , Anesthetics, Local/pharmacology , Animals , Antigens/metabolism , Biophysics , Bromodeoxyuridine/metabolism , Caspase 3/metabolism , Cell Count , Cell Differentiation , Cell Proliferation , Cell Survival/physiology , Cells, Cultured , Cerebral Cortex/cytology , Cysteine Proteinase Inhibitors/pharmacology , Female , Galactosylceramidase/metabolism , Glial Fibrillary Acidic Protein/metabolism , Myelin Basic Protein/metabolism , Neurons/physiology , Oligodendroglia/drug effects , Phosphopyruvate Hydratase/metabolism , Pregnancy , Proteoglycans/metabolism , Rats , Rats, Sprague-Dawley , Stem Cells , Tetrodotoxin/pharmacologyABSTRACT
Functional electrical stimulation (FES) can restore control and offset atrophy to muscles after neurological injury. However, FES has not been considered as a method for enhancing CNS regeneration. This paper demonstrates that FES dramatically enhanced progenitor cell birth in the spinal cord of rats with a chronic spinal cord injury (SCI). A complete SCI at thoracic level 8/9 was performed on 12 rats. Three weeks later, a FES device to stimulate hindlimb movement was implanted into these rats. Twelve identically-injured rats received inactive FES implants. An additional control group of uninjured rats were also examined. Ten days after FES implantation, dividing cells were marked with bromodeoxyuridine (BrdU). The "cell birth" subgroup (half the animals in each group) was sacrificed immediately after completion of BrdU administration, and the "cell survival" subgroup was sacrificed 7 days later. In the injured "cell birth" subgroup, FES induced an 82-86% increase in cell birth in the lumbar spinal cord. In the injured "cell survival" subgroup, the increased lumbar newborn cell counts persisted. FES doubled the proportion of the newly-born cells which expressed nestin and other markers suggestive of tripotential progenitors. In uninjured rats, FES had no effect on cell birth/survival. This report suggests that controlled electrical activation of the CNS may enhance spontaneous regeneration after neurological injuries.
Subject(s)
Adult Stem Cells/physiology , Electric Stimulation/methods , Neurogenesis/physiology , Spinal Cord Injuries/therapy , Analysis of Variance , Animals , Antigens/metabolism , Biophysics/methods , Bromodeoxyuridine/metabolism , CD11b Antigen/metabolism , Cell Survival , Disease Models, Animal , Female , Glial Fibrillary Acidic Protein/metabolism , Phosphopyruvate Hydratase/metabolism , Proteoglycans/metabolism , Rats , Rats, Long-Evans , Spinal Cord Injuries/pathology , Spinal Cord Injuries/physiopathologyABSTRACT
Neonatal stroke presents with seizures and results in neurologic morbidity, including epilepsy, hemiparesis, and cognitive deficits. Stem cell-based therapy offers a possible therapeutic strategy for neonatal stroke. We developed an immature mouse model of stroke with acute seizures and ischemic brain injury. Postnatal day 12 CD1 mice received right-sided carotid ligation. Two or 7 days after ligation, mice received an intrastriatal injection of B5 embryonic stem cell-derived neural stem cells. Four weeks after ligation, hemispheric brain atrophy was measured. Pups receiving stem cells 2 days after ligation had less severe hemispheric brain atrophy compared with either noninjected or vehicle-injected ligated controls. Transplanted cells survived, but 3 out of 10 pups injected with stem cells developed local tumors. No difference in hemispheric brain atrophy was seen in mice injected with stem cells 7 days after ligation. Neural stem cells have the potential to ameliorate ischemic injury in the immature brain, although tumor development is a serious concern.
Subject(s)
Brain Ischemia/therapy , Carotid Arteries/physiology , Neurons/transplantation , Stem Cell Transplantation , Stroke/therapy , Animals , Atrophy , Brain Ischemia/etiology , Brain Ischemia/mortality , Brain Neoplasms/pathology , Cell Survival , Ligation , Mice , Neurons/drug effects , Seizures/etiology , Stem Cell Transplantation/adverse effects , Stem Cells/drug effects , Stereotaxic Techniques , Stroke/etiology , Stroke/mortality , Teratoma/pathology , Tretinoin/pharmacologyABSTRACT
The use of RNA interference (RNAi) to suppress the expression of genes has drastically improved the ability to examine gene function and is now being considered as a therapeutic approach for many diseases including genetic forms of neurodegenerative disease. Recently, research has focused on RNAi for the treatment of Huntington's and other polyglutamine diseases. In this work we explored the efficacy and specificity of short hairpin RNAs to target human huntingtin mRNA. We found two sequences that are specific for, and efficiently suppress human huntingtin mRNA. Mouse cell lines that stably harbored human short hairpin RNA constructs specifically inhibited the expression of human huntingtin supplied by transfected expression plasmids. However, these same constructs were unable to stably suppress endogenous human huntingtin when stably transfected into human 293 cells, despite effectively knocking down expression of huntingtin in transient transfection. These results demonstrate the efficacy and specificity of RNAi as a tool to target human huntingtin in RNAi-based therapies but point toward potential problems, possibly cell-type specific, regarding stable suppression of human huntingtin.
Subject(s)
Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Peptide Fragments/metabolism , RNA Interference , Animals , Base Sequence , Cell Line , Humans , Huntingtin Protein , Mice , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Peptide Fragments/genetics , RNA/chemistry , RNA/genetics , RNA/metabolismABSTRACT
Although neural regeneration is an active research field today, no current treatments can aid regeneration after spinal cord injury. This article reviews the feasibility of spinal cord repair and provides an overview of the range of strategies scientists are taking toward regeneration. The major focus of this article is the future role of stem cell transplantation and similar rehabilitative restorative approaches designed to optimize spontaneous regeneration by mobilizing endogenous stem cells and facilitating other cellular mechanisms of regeneration, such as axonal growth and myelination.
Subject(s)
Nerve Regeneration/physiology , Recovery of Function/physiology , Spinal Cord Injuries/physiopathology , Spinal Cord Injuries/therapy , Stem Cell Transplantation , Animals , Electric Stimulation Therapy , Humans , Spinal Cord Injuries/pathology , Stem Cells/physiologyABSTRACT
Mutations in the presenilin-1 (PS1) gene are causally linked to early-onset Alzheimer's disease (AD). Studies of neurons suggest that PS1 mutations result in a gain-of-function, which perturbs calcium regulation and increases cell vulnerability to apoptosis. Alterations in immune cell function have also been demonstrated in AD, and a role for PS1 in immune regulation has been suggested recently. We now report that splenocytes from PS1-mutant (M146V) knockin mice exhibit increased caspase activity, abnormal calcium regulation and aberrant mitochondrial function. Isolated splenic T cells from PS1-mutant mice respond poorly to proliferative signals and have downregulated cluster designation 3 and interleukin (IL)- 2-receptor expression necessary for a normal T-cell immune response. Thus, adverse effects of a mutation that causes AD on immune function that involves perturbed calcium regulation and cytokine signaling in lymphocytes, and associated sensitivity of lymphocytes to apoptosis are demonstrated. These findings suggest that abnormalities in immune function might play major roles in the pathogenesis of AD.
Subject(s)
Alzheimer Disease/immunology , Presenilin-1/genetics , Alzheimer Disease/genetics , Animals , Apoptosis , Calcium Signaling , Cells, Cultured , Cytokines/biosynthesis , Lymphocytes/immunology , Lymphocytes/metabolism , Membrane Potential, Mitochondrial , Mice , Mice, Mutant Strains , Mitochondria/physiology , Mutation , Reactive Oxygen Species/metabolismABSTRACT
The activation of the transcription factor nuclear factor-kappaB (NF-kappaB) by growth factors, cytokines, and cellular stress can prevent apoptosis, but the underlying mechanism is unknown. Here we provide evidence for an action of NF-kappaB on calcium signaling that accounts for its anti-apoptotic function. Embryonic fibroblasts lacking the transactivating subunit of NF-kappaB RelA (p65) exhibit enhanced inositol 1,4,5-trisphosphate (IP(3)) receptor-mediated calcium release and increased sensitivity to apoptosis, which are restored upon re-expression of RelA. The size of the endoplasmic reticulum (ER) calcium pool and the number of IP(3) receptors per cell are decreased in response to stimuli that activate NF-kappaB and are increased when NF-kappaB activity is suppressed. The selective antagonism of IP(3) receptors blocks apoptosis in RelA-deficient cells, whereas activation of NF-kappaB in normal cells leads to decreased levels of the type 1 IP(3) receptor and decreased calcium release. Overexpression of Bcl-2 normalizes ER calcium homeostasis and prevents calcium-mediated apoptosis in RelA-deficient cells. These findings establish an ER calcium channel as a pivotal target for NF-kappaB-mediated cell survival signaling.
Subject(s)
Apoptosis , Calcium/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , NF-kappa B/metabolism , Adenosine Triphosphate/metabolism , Animals , Blotting, Western , Calcium Channels/metabolism , Calcium-Transporting ATPases/metabolism , Cell Survival , Ceramides/pharmacology , Cytosol/metabolism , DNA/metabolism , Endoplasmic Reticulum/metabolism , Immunoblotting , Immunohistochemistry , Inositol 1,4,5-Trisphosphate Receptors , Lipid Metabolism , Mice , Mice, Transgenic , Microscopy, Fluorescence , Microsomes/metabolism , NF-kappa B/chemistry , Oligonucleotides, Antisense/chemistry , Oxidative Stress , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Signal Transduction , Time Factors , Transcription Factor RelA , Transcriptional ActivationABSTRACT
First discovered in plants the nematode Caenorhabditis elegans, the production of small interfering RNAs (siRNAs) that bind to and induce the degradation of specific endogenous mRNAs is now recognized as a mechanism that is widely employed by eukaryotic cells to inhibit protein production at a post-transcriptional level. The endogenous siRNAs are typically 19- to 23-base double-stranded RNA oligonucleotides, produced from much larger RNAs that upon binding to target mRNAs recruit RNases to a protein complex that degrades the targeted mRNA. Methods for expressing siRNAs in cells in culture and in vivo using viral vectors, and for transfecting cells with synthetic siRNAs, have been developed and are being used to establish the functions of specific proteins in various cell types and organisms. RNA interference methods provide several major advantages over prior methods (antisense DNA or antibody-based techniques) for suppressing gene expression. Recent preclinical studies suggest that RNA interference technology holds promise for the treatment of various diseases. Pharmacologists have long dreamed of the ability to selectively antagonize or eliminate the function of individual proteins--RNAi technology may eventually make that dream a reality.
Subject(s)
Drug Therapy , Pharmacology/trends , RNA Interference/physiology , Technology, Pharmaceutical/methods , Animals , Cell Death , Humans , Neoplasms/therapy , RNA Interference/drug effects , Signal Transduction , Technology, Pharmaceutical/trendsABSTRACT
Activation of integrin receptors in neurons can promote cell survival and synaptic plasticity, but the underlying signal transduction pathway(s) is unknown. We report that integrin signaling prevents apoptosis of embryonic hippocampal neurons by a mechanism involving integrin-linked kinase (ILK) that activates Akt kinase. Activation of integrins using a peptide containing the amino acid sequence EIKLLIS derived from the alpha chain of laminin protected hippocampal neurons from apoptosis induced by glutamate or staurosporine, and increased Akt activity in a beta1 integrin-dependent manner. Transfection of neurons with a plasmid encoding dominant negative Akt blocked the protective effect of the integrin-activating peptide, as did a chemical inhibitor of Akt. Although inhibitors of phosphoinositide-3 (PI3) kinase blocked the protective effect of the peptide, we found no increase in PI3 kinase activity following integrin stimulation suggesting that PI3 kinase was necessary for Akt activity but was not sufficient for the increase in Akt activity following integrin activation. Instead, we show a requirement for ILK in integrin receptor-induced Akt activation. ILK was activated following integrin stimulation and dominant negative ILK blocked integrin-mediated Akt activation and cell survival. Activation of ILK and Akt were also required for neuroprotection by substrate-associated laminin. These results establish a novel pathway that signals cell survival in neurons in response to integrin receptor activation.
Subject(s)
Hippocampus/metabolism , Integrins/metabolism , Neurons/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Apoptosis/drug effects , Cell Survival/physiology , Cells, Cultured , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Genes, Dominant , Hippocampus/cytology , Neurons/cytology , Neuroprotective Agents/pharmacology , Oligopeptides/pharmacology , Peptides/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt , Rats , Signal Transduction/physiologyABSTRACT
Cells in the brain deploy multiple mechanisms to maintain the integrity of nerve cell circuits, and to facilitate responses to environmental demands and promote recovery of function after injury. The mechanisms include production of neurotrophic factors and cytokines, expression of various cell survival-promoting proteins (e.g. protein chaperones, antioxidant enzymes, Bcl-2 and inhibitor of apoptosis proteins), protection of the genome by telomerase and DNA repair proteins, and mobilization of neural stem cells to replace damaged neurons and glia. The aging process challenges such neuroprotective and neurorestorative mechanisms, often with devastating consequences as in Alzheimer's disease (AD), Parkinson's and Huntington's diseases and stroke. Genetic and environmental factors superimposed upon the aging process can determine whether brain aging is successful or unsuccessful. Mutations in genes that cause inherited forms of AD (amyloid precursor protein (APP) and presenilins), Parkinson's disease (alpha-synuclein and parkin) and trinucleotide repeat disorders (e.g. huntingtin and the androgen receptor) overwhelm endogenous neuroprotective mechanisms. On the other hand, neuroprotective mechanisms can be bolstered by dietary (caloric restriction, and folate and antioxidant supplementation) and behavioral (cognitive and physical activities) modifications. At the cellular and molecular levels, successful brain aging can be facilitated by activating a hormesis response to which neurons respond by upregulating the expression of neurotrophic factors and stress proteins. Neural stem cells that reside in the adult brain are also responsive to environmental demands, and appear capable of replacing lost or dysfunctional neurons and glial cells, perhaps even in the aging brain. The recent application of modem methods of molecular and cellular biology to the problem of brain aging is revealing a remarkable capacity within brain cells for adaptation to aging and resistance to disease.
Subject(s)
Aging/physiology , Brain Diseases/genetics , Brain Diseases/physiopathology , Brain/physiology , Signal Transduction/physiology , Aged , Brain Diseases/diet therapy , Caloric Restriction , Diet , Health Behavior , HumansABSTRACT
The matrix metalloproteinases (MMPs) are a family of structurally related metalloendopeptidases so named due to their propensity to target extracellular matrix (ECM) proteins. Accumulating evidence, however, suggests that these proteases cleave numerous non-ECM substrates including enzymes and cell surface receptors. MMPs may also bind to cell surface receptors, though such binding has typically been thought to mediate internalization and degradation of the bound protease. More recently, it has been shown that MMP-1 coimmunoprecipitates with the alpha2beta1 integrin, a receptor for collagen. This association may serve to localize the enzymatic activity of MMP-1 so that collagen is cleaved and cell migration is facilitated. In other studies, however, it has been shown that integrin engagement may be linked to the activation of signaling cascades including those mediated by Gialpha containing heterotrimers. As an example, alpha2beta1 can form a complex with CD47 that may associate with Gialpha. In the present study we have therefore investigated the possibility that MMP-1 may affect intracellular changes that are linked to the activation of a Gi protein-coupled receptor. We show that treatment of neural cells with MMP-1 is followed by a rapid reduction in cytosolic levels of cAMP. Moreover, MMP-1 potentiates proteinase activated receptor-1 (PAR-1) agonist-linked increases in intracellular calcium, an effect which is often observed when an agonist of a Gi protein-coupled receptor is administered in association with an agonist of a Gq coupled receptor. In addition, MMP-1 stimulates pertussis toxin sensitive release ofMMP-9 both from cultured neural cells and monocyte/macrophages. Together, these results suggest that MMP-1 signals through a pertussis toxin-sensitive G protein-coupled receptor.
Subject(s)
Matrix Metalloproteinase 1/pharmacology , Matrix Metalloproteinase 9/metabolism , Pertussis Toxin , Signal Transduction/drug effects , Signal Transduction/physiology , Virulence Factors, Bordetella/pharmacology , Animals , Antibodies/pharmacology , Calcium/metabolism , Cells, Cultured , Cyclic AMP/metabolism , Cytosol/metabolism , GTP-Binding Protein alpha Subunit, Gi2 , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Humans , In Vitro Techniques , Integrin beta1/metabolism , Matrix Metalloproteinase Inhibitors , Monocytes/cytology , Monocytes/drug effects , Monocytes/metabolism , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Precipitin Tests , Proto-Oncogene Proteins/metabolism , Rats , Rats, Sprague-Dawley , Receptor, PAR-1 , Receptors, Cell Surface/metabolism , Receptors, Thrombin/metabolismABSTRACT
Mutations in the presenilin-1 (PS1) gene cause early onset familial Alzheimer's disease (FAD) by a mechanism believed to involve perturbed endoplasmic reticulum (ER) function and altered proteolytic processing of the amyloid precursor protein. We investigated the molecular mechanisms underlying cell death and ER dysfunction in cultured cells and knock-in mice expressing FAD PS1 mutations. We report that PS1 mutations cause a marked increase in basal protein levels of the pro-apoptotic transcription factor Gadd153. PS1 mutations increase Gadd153 protein translation without affecting mRNA levels, while decreasing levels of the anti-apoptotic protein Bcl-2. Moreover, an exaggerated Gadd153 response to stress induced by ER stress agents was observed in PS1 mutant cells. Cell death in response to ER stress is enhanced by PS1 mutations, and this endangering effect is attenuated by anti-sense-mediated suppression of Gadd153 production. An abnormality in the translational regulation of Gadd153 may sensitize cells to the detrimental effects of ER stress and contribute to the pathogenic actions of PS1 mutations in FAD.
Subject(s)
CCAAT-Enhancer-Binding Proteins/metabolism , Membrane Proteins/genetics , Transcription Factors/metabolism , Alzheimer Disease/metabolism , Animals , Apoptosis/physiology , CCAAT-Enhancer-Binding Proteins/antagonists & inhibitors , CCAAT-Enhancer-Binding Proteins/genetics , Calcium/metabolism , Calcium-Transporting ATPases/antagonists & inhibitors , Cell Line , Cerebral Cortex/metabolism , Clone Cells , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Hippocampus/metabolism , Humans , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Mutation , Oligonucleotides, Antisense/pharmacology , PC12 Cells , Presenilin-1 , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/metabolism , Rats , Stress, Physiological/metabolism , Thapsigargin/pharmacology , Transcription Factor CHOP , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Up-Regulation/drug effects , Up-Regulation/physiologyABSTRACT
The olfactory system is functionally linked to the hippocampus, and odors can modify the activity of hippocampal neurons. Because hippocampal neurons are selectively vulnerable to death in several prominent neurodegenerative conditions, we tested the hypothesis that activity in olfactory pathways can modify the sensitivity of hippocampal neurons to excitotoxic damage. We report that rats subjected to olfactory bulbectomy exhibit a decrease in the vulnerability of hippocampal pyramidal neurons to excitotoxic injury. Four-month-old male Sprague-Dawley rats were subjected to bilateral olfactory bulbectomy or a sham operation. Three months later the rats were given a unilateral infusion of kainic acid in the dorsal hippocampus and were euthanized 24 h later. There was a threefold increase in the number of CA3 neurons that survived kainic acid administration in the bulbectomized rats compared to sham-operated rats. These findings provide the first evidence that olfactory input affects the vulnerability of neurons to excitotoxic death.