Temporal associations between the different domains of rheumatoid arthritis disease activity and the onset of patient-reported depressive symptoms
BACKGROUND: Depression is a frequently occurring comorbid condition in patients with rheumatoid arthritis (RA), and research into the temporal relationships regarding its onset has mainly focused on functional status. The study aim was to examine temporal associations of the diverse measures of RA disease activity with incident self-reports of depressive symptoms.
METHODS: RA patients from the Consortium of Rheumatology Researchers of North America (CORRONA) registry were utilized. Cox regression was used to assess the lagged time-varying association of RA disease activity with the incident onset of depressive symptoms as measured using a single-item depression question. Predictor variables included joint counts, global assessments, pain, function, serum biomarkers, and composite disease activity. Hazard ratios (HRs) comparing categorical quintiles were estimated with 95 % confidence intervals.
RESULTS: Every metric of disease activity, except inflammatory markers, were significantly associated with the self-reported onset of depressive symptoms. Adjusted HRs comparing fifth quintiles to first quintiles were the following: CDAI = 2.3 [2.1-2.7]; pain = 2.3 [2.0-2.6]; SJC = 1.4 [1.4-1.6]. When examining successive self-reports (two consecutive), the magnitude of the associations greatly increased: CDAI = 3.6 [2.5-5.0].
CONCLUSIONS: The data suggest depressive symptom onset in RA patients is related to measures reported by the patient: pain, functional status, and global disease activity; and measures reported by providers, rather than biological markers. The magnitude of the associations, however, were greater for the patient-reported measures when compared to physician assessments, implying that patients' experience of their disease activity may be a precipitating factor of depression onset.
Disclosure of adverse events in the United States and Canada: an update, and a proposed framework for improvement
There is consensus that physicians, health professionals and health care organizations should discuss harm that results from health care delivery (adverse events), including the reasons for harm, with patients and their families. Thought leaders and policy makers in the USA and Canada support this goal. However, there are gaps in both countries between patients and physicians in their attitudes about how errors should be handled, and between disclosure policies and their implementation in practice. This paper reviews the state of disclosure policy and practice in the two countries, and the barriers to full disclosure. Important barriers include fear of consequences, attitudes about disclosure, lack of skill and role models, and lack of peer and institutional support. The paper also describes the problem of the second victim, a corollary of disclosure whereby health care workers are also traumatized by the same events that harm patients. The presence of multiple practical and personal barriers to disclosure suggests the need for a comprehensive solution directed at multiple levels of the health care system, including health departments, institutions, local managers, professional staff, patients and families, and including legal, health system and local institutional support. At the local level, implementation could be based on a translating-evidence-into-practice framework. Applying this framework would involve the formation of teams, training, measurement and identification of local barriers to achieving universal disclosure of adverse events. Significance for public healthIt is inevitable that some patients will be harmed rather than helped by health care. There is consensus that patients and their families must be told about these harmful events. However, there are gaps between patient and physician attitudes about how errors should be handled, and between disclosure policies and their implementation. There are important barriers that impede disclosure, including fear of consequences, attitudes about disclosure, lack of skill, and lack of institutional support. A related problem is that of the second victim, whereby health care workers are traumatized by the same harmful events. This can impair their performance and further compromise safety. The problem is unlikely to be solved by focusing solely on increasing disclosure. A comprehensive solution is needed, directed at multiple levels of the health care system, including health departments, institutions, local managers, professional staff, patients and families, and including legal, health system and local institutional support.
Patient report of guideline-congruent gestational weight gain advice from prenatal care providers: differences by prepregnancy BMI
BACKGROUND: Prenatal care provider weight gain advice consistent with the Institute of Medicine recommendations is related to guideline-adherent gestational weight gain (GWG), yet many women may not receive guideline-congruent advice. We examined pregnant women's recall of prenatal care provider GWG advice in relation to prepregnancy body mass index (BMI).
METHODS: We conducted a prospective cohort study of women (n = 149) receiving prenatal care for a singleton pregnancy at a large academic medical center in 2010. Data were collected via a survey during late pregnancy and medical record abstraction.
RESULTS: Thirty-three percent of women did not recall receiving the provider GWG advice; 33 percent recalled advice consistent with 2009 Institute of Medicine recommendations. Recalled advice differed by prepregnancy BMI; 29 percent of normal weight, 26 percent of overweight, and 45 percent of obese women reported not receiving advice, and 6, 37, and 39 percent, respectively, recalled advice exceeding Institute of Medicine recommendations. Among the 62 percent who recalled that their provider had labeled their prepregnancy BMI, 100 percent of normal weight, 32 percent of overweight, and 23 percent of obese women recalled the labels "normal weight," "overweight," and "obese," respectively.
CONCLUSIONS: Helping providers give their patients memorable and guideline-consistent GWG advice is an actionable step toward preventing excessive GWG and associated maternal and child health consequences.
Mechanically ventilated critically ill patients treated in the intensive care unit (ICU) require enteral feedings to maintain adequate nutrition during critical illness. Delivery of adequate enteral nutrition is also critical to the recovery of critically ill patients. Enteral nutrition has been shown to decrease length of time on the ventilator, decrease length of stay and ICU and decrease mortality. Despite all the evidence regarding the benefits of enteral nutrition, critically ill patients continue to receive less than their prescribed calories and protein. Nurses are in a unique position to influence the delivery of enteral nutrition. Nursing practices that contribute to underfeeding must be identified and corrected to ensure adequate delivery of nutrients is achieved. The purpose of the study was to describe the professional practice of critical care nurses regarding enteral feeding in mechanically ventilated critically ill patients. Several barriers were identified by the participants in the study that contributed to underfeeding including inconsistent practice regarding gastric residual volume, holding feeds when changing patient position and lack of a standardized protocol for enteral feeding. Also identified in the study was the idea that nurses do not see enteral feeding as a life-saving intervention. It is not the “sexy part” of what ICU nurses do. Enteral feeding guidelines need to be developed to include those interventions that are important to nursing practice in order to increase enteral feeding times and improve patient outcomes.
Amphiphilic Degradable Polymer/Hydroxyapatite Composites as Smart Bone Tissue Engineering Scaffolds: A Dissertation
Over 600,000 bone-grafting operations are performed each year in the United States. The majority of the bone used for these surgeries comes from autografts that are limited in quantity or allografts with high failure rates. Current synthetic bone grafting materials have poor mechanical properties, handling characteristics, and bioactivity. The goal of this dissertation was to develop a clinically translatable bone tissue engineering scaffold with improved handling characteristics, bioactivity, and smart delivery modalities. We hypothesized that this could be achieved through the rational selection of Food and Drug Administration (FDA) approved materials that blend favorably with hydroxyapatite (HA), the principle mineral component in bone. This dissertation describes the development of smart bone tissue engineering scaffolds composed of the biodegradable amphiphilic polymer poly(D,L-lactic acid-co-ethylene glycol-co- D,L-lactic acid) (PELA) and HA. Electrospun nanofibrous HA-PELA scaffolds exhibited improved handling characteristics and bioactivity over conventional HApoly( D,L-lactic acid) composites. Electrospun HA-PELA was hydrophilic, elastic, stiffened upon hydration, and supported the attachment and osteogenic differentiation of rat bone marrow stromal cells (MSCs). These in vitro properties translated into robust bone formation in vivo using a critical-size femoral defect model in rats. Spiral-wrapped HA-PELA scaffolds, loaded with MSCs or a lowdose of recombinant human bone morphogenetic protein-2, templated bone formation along the defect. As an alternate approach, PELA and HA-PELA were viii rapid prototyped into three-dimensional (3-D) macroporous scaffolds using a consumer-grade 3-D printer. These 3-D scaffolds have differential cell adhesion characteristics, swell and stiffen upon hydration, and exhibit hydration-induced self-fixation in a simulated confined defect. HA-PELA also exhibits thermal shape memory behavior, enabling the minimally invasive delivery and rapid (>3 sec) shape recovery of 3-D scaffolds at physiologically safe temperatures (~ 50ºC). Overall, this dissertation demonstrates how the rational selection of FDA approved materials with synergistic interactions results in smart biomaterials with high potential for clinical translation.
Mobile genetic elements represent a large portion of the genome in many species. Posing a danger to the integrity of genetic information, silencing and structural machinery has evolved to suppress the mobility of foreign and transposable elements within the genome. Condensin proteins – which regulate chromosome structure to promote chromosome segregation – have been demonstrated to function in repetitive gene regulation and transposon silencing in several species. In model system Caenorhabditis elegans, microarray analysis studies have implicated Condensin II subunit HCP-6 in the silencing of multiple loci, including DNA transposon MIRAGE. To address the hypothesis that HCP-6 has a direct function in transcriptional gene silencing of the MIRAGE transposon, we queried MIRAGE expression and chromatin profiles in wild-type and hcp-6 mutant animals. Our evidence confirms that HCP-6 does indeed function during silencing of MIRAGE. However, we found no significant indication that HCP-6 binds to MIRAGE, nor that HCP-6 mediates MIRAGE enrichment of H3K9me3, the repressive heterochromatin mark observed at regions undergoing transcriptional silencing. We suggest that the silencing of MIRAGE, a newly evolved transposon and the only tested mobile element considerably derepressed upon loss of HCP-6, is managed by HCP-6 indirectly.
Measurement properties of the Western Ontario and McMaster Universities Osteoarthritis Index: a systematic review
OBJECTIVE: To conduct a systematic review of the measurement properties of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and to evaluate the quality of WOMAC measurement studies using COSMIN (Consensus-Based Standards for the Selection of Health Measurement Instruments) criteria.
METHODS: A search was conducted in the MEDLINE, CINAHL, Embase, PsycINFO, Scopus, and SPORTDiscus databases through September 2013. Data that assessed the WOMAC measurement model, reliability, validity, respondent burden, and equivalence across methods of administration were extracted. Overall study quality was rated following COSMIN criteria.
RESULTS: A total of 76 articles from 22 countries were included. Internal consistency reliability was consistently high (≥0.90) for the function scale and acceptable (≥0.70) for the pain and stiffness scales. Test-retest reliability was acceptable. Score equivalence was demonstrated across paper and electronic methods of data collection. Floor and ceiling effects were low except for notable (24-38%) proportions of patients achieving the best possible scores on the pain and stiffness scales 1-23 years after arthroplasty. Five exploratory factor analyses did not support a measurement model in which the pain and function items were distinct. Correlations between the WOMAC pain and function scales were high (median 0.79). The WOMAC pain and function scales had similar correlations with other pain measures, and therefore the WOMAC pain scale did not show divergent validity. COSMIN criteria were not fully met in most studies.
CONCLUSION: The WOMAC scales were reliable, but its pain scale was highly related to physical function. Further research into joint-specific pain measures that have broader content validity is needed.
Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive human malignancies, is thought to be initiated by KRAS activation. Here, we find that transcriptional activation mediated by the GLI family of transcription factors, although dispensable for pancreatic development, is required for KRAS induced pancreatic transformation. Inhibition of GLI using a dominant-negative repressor (Gli3T) inhibits formation of precursor Pancreatic Intraepithelial Neoplasia (PanIN) lesions in mice, and significantly extends survival in a mouse model of PDAC. Further, ectopic activation of the GLI1/2 transcription factors in mouse pancreas accelerates KRAS driven tumor formation and reduces survival, underscoring the importance of GLI transcription factors in pancreatic tumorigenesis. Interestingly, we find that although canonical GLI activity is regulated by the Hedgehog ligands, in the context of PDAC, GLI transcription factors initiate a unique ligand-independent transcriptional program downstream of KRAS, that involves regulation of the RAS, PI3K/AKT, and NF-кB pathways.
We identify I-kappa-B kinase epsilon (IKBKE) as a PDAC specific target of GLI, that can also regulate GLI transcriptional activity via positive feedback mechanism involving regulation of GLI subcellular localization. Using human PDAC cells, and an in vivo model of pancreatic neoplasia, we establish IKBKE as a novel regulator pf pancreatic tumorigenesis that acts as an effector of KRAS/GLI, and mediates pancreatic transformation. We show that genetic knockout of Ikbke leads to a dramatic inhibition of initiation and progression of pancreatic intraepithelial viii neoplasia (PanIN) lesions in mice carrying pancreas specific activation of oncogenic Kras. Furthermore, we find that although IKBKE is a known NF-кB activator, it only modestly regulates NF-кB activity in PDAC. Instead, we find that IKBKE strongly promotes AKT phosphorylation in PDAC in vitro and in vivo, and that IKBKE mediates reactivation of AKT post-inhibition of mTOR. We also show that while mTOR inhibition alone does not significantly affect pancreatic tumorigenesis, combined inhibition of IKBKE and mTOR has a synergistic effect leading to significant decrease tumorigenicity of PDAC cells.
Together, our findings identify GLI/IKBKE signaling as an important oncogenic effector pathway of KRAS in PDAC that regulates tumorigenicity, cell proliferation, and apoptosis via regulation of AKT and NF-кB signaling. We provide proof of concept for therapeutic targeting of GLI/IKBKE in PDAC, and support the evaluation of IKBKE as a therapeutic target in treatment of pancreatic cancer, and IKBKE inhibition as a strategy to improve efficacy of mTOR inhibitors in the clinic.
Psychometric Evaluation of Joint-Specific Patient-Reported Outcome Measures Before and After Total Knee Replacement: A Dissertation
Background: Patient reports of pain and function are used to inform the need for and timing of total knee replacement (TKR) and evaluate TKR outcomes. This dissertation compared measurement properties of commonly-used patient surveys in TKR and explored ways to develop more efficient knee-specific function measures.
Methods: 1,179 FORCE-TJR patients (mean age=66.1, 61% female) completed questionnaires before and 6 months after TKR. Patient surveys included the knee-specific Knee injury and Osteoarthritis Outcome Score (KOOS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and generic SF-36 Health Survey. Tests of KOOS and WOMAC measurement properties included evaluations of scaling assumptions and reliability. Item response theory methods were used to calibrate 22 KOOS function items in one item bank; simulated computerized adaptive tests (CAT) then were used to evaluate shorter function scores customized for each patient. Validity and responsiveness of measures varying in attributes (knee-specific versus generic, longer versus shorter, CAT versus fixed-length) were compared.
Results: KOOS and WOMAC scales generally met tests of scaling assumptions, although many pain items were equally strong measures of pain and physical function. Internal consistency reliability of KOOS and WOMAC scales exceeded minimum levels of 0.70 recommended for group-level comparisons across sociodemographic and clinical subgroups. Function items could be calibrated in one item bank. CAT simulations indicated that reliable knee-specific function scores could be estimated for most patients with a 55-86% reduction in respondent burden, but one-third could not achieve a reliable (≥ 0.95) CAT score post-TKR because the item bank did not include enough items vi measuring high function levels. KOOS and WOMAC scales were valid and responsive. Short function scales and CATs were as valid and responsive as longer KOOS and WOMAC function scales. The KOOS Quality of Life (QOL) scale and SF-36 Physical Component Summary discriminated best among groups evaluating themselves as improved, same or worse at 6 months.
Conclusions: Results support use of the KOOS and WOMAC in TKR. Improved knee-specific function measures require new items that measure higher function levels. TKR outcomes should be evaluated with a knee-specific quality of life scale such as KOOS QOL, as well as knee-specific measures of pain and function and generic health measures.
This is the July 2015 issue of the UMass Center for Clinical and Translational Science Newsletter containing news and events of interest.
Fluorescence-based imaging techniques provide a simple, highly sensitive method of studying live cells and whole organisms in real time. Without question, fluorophores such as GFP, fluorescein, and rhodamines have contributed vastly to our understanding of both cell biology and biochemistry. However, most of the fluorescent molecules currently utilized suffer from one major drawback, the use of visible light. Due to cellular autofluorescence and the absorbance of incident light by cellular components, fluorescence imaging with visible wavelength fluorophores often results in high background noise and thus a low signal-to-noise ratio. Fortunately, this situation can be ameliorated by altering the wavelength of light used during imaging. Near-infrared (NIR) light (650-900 nm) is poorly absorbed by cells; therefore, fluorophores excited by this light provide a high signal-to-noise ratio and low background in cellular systems. While these properties make NIR fluorophores ideal for cellular imaging, most currently available NIR molecules cannot be used in live cells. The first half of this thesis addresses the synthetic difficulties associated with preparing NIR fluorophores that can be used within living systems. Small molecule NIR fluorophores are inherently hydrophobic which makes them unsuitable for use in the aqueous environment of the cell. Water-solubility is imparted to these dyes through highly polar sulfonates, which subsequently prevents the dyes from entering the cell. The novel work presented here details vii synthetic routes to aid in the development of sulfonated NIR fluorophores, which can be delivered into live cells through the inclusion of an esterase-labile sulfonate protecting group. Application of these synthetic techniques should allow for the development of novel NIR fluorophores with intracellular applications. The second half of this thesis addresses the need for novel NIR imaging reagents. Although several classes of NIR scaffolds do exist, most NIR probes are derivatives of a single class, heptamethine indocyanines. The work described here increases this palette by displaying the ability of NIR oxazines to function as an imaging reagent in live cells and in vivo and as a molecular sensor of biologically-relevant environmental conditions. Combined, the work contained herein has the capacity to not only advance the current NIR toolkit, but to expand it so that fluorescence imaging can move out of the dark and into the NIR light.
How does a nervous system orchestrate compound behaviors? Finding the neural basis of behavior requires knowing which neurons control the behavior and how they are connected. To accomplish this we measured and manipulated neural activity in a live, behaving animal with a completely defined connectome. The C. elegans escape response is a compound behavior consisting of a sequence of behavioral motifs. Gentle touch induces a reversal and suppression of head movements, followed by a deep turn allowing the animal to navigate away from the stimulus. The connectome provides a framework for the neural circuit that controls this behavior. We used optical physiology to determine the activity patterns of individual neurons during the behavior. Calcium imaging of locomotion interneurons and motor neurons reveal unique activity profiles during different motifs of the escape response. Furthermore, we used optogenetics and laser ablations to determine the contribution of individual neurons to each motif. We show these that the suppression of head movements and turning motifs are distinct motor programs and can be uncoupled from the reversal. The molecular mechanisms that regulate these motifs involve from signaling with the neurotransmitter tyramine. Tyramine signaling and gap junctions between locomotion interneurons and motor neurons regulate the temporal orchestration of the turning motif with the reversal. Additionally, tyramine signaling through a GPCR in GABAergic neurons facilitates the asymmetric turning during forward viii locomotion. The combination of optical tools and genetics allows us to dissect a how a neural circuit converts sensory information into a compound behavior.
Several rAAV vectors efficiently cross the blood-brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system
Noninvasive systemic gene delivery to the central nervous system (CNS) has largely been impeded by the blood-brain barrier (BBB). Recent studies documented widespread CNS gene transfer after intravascular delivery of recombinant adeno-associated virus 9 (rAAV9). To investigate alternative and possibly more potent rAAV vectors for systemic gene delivery across the BBB, we systematically evaluated the CNS gene transfer properties of nine different rAAVEGFP vectors after intravascular infusion in neonatal mice. Several rAAVs efficiently transduce neurons, motor neurons, astrocytes, and Purkinje cells; among them, rAAVrh.10 is at least as efficient as rAAV9 in many of the regions examined. Importantly, intravenously delivered rAAVs did not cause abnormal microgliosis in the CNS. The rAAVs that achieve stable widespread gene transfer in the CNS are exceptionally useful platforms for the development of therapeutic approaches for neurological disorders affecting large regions of the CNS as well as convenient biological tools for neuroscience research.
Global CNS transduction of adult mice by intravenously delivered rAAVrh.8 and rAAVrh.10 and nonhuman primates by rAAVrh.10
Some recombinant adeno-associated viruses (rAAVs) can cross the neonatal blood-brain barrier (BBB) and efficiently transduce cells of the central nervous system (CNS). However, in the adult CNS, transduction levels by systemically delivered rAAVs are significantly reduced, limiting their potential for CNS gene therapy. Here, we characterized 12 different rAAVEGFPs in the adult mouse CNS following intravenous delivery. We show that the capability of crossing the adult BBB and achieving widespread CNS transduction is a common character of AAV serotypes tested. Of note, rAAVrh.8 is the leading vector for robust global transduction of glial and neuronal cell types in regions of clinical importance such as cortex, caudate-putamen, hippocampus, corpus callosum, and substantia nigra. It also displays reduced peripheral tissue tropism compared to other leading vectors. Additionally, we evaluated rAAVrh.10 with and without microRNA (miRNA)-regulated expressional detargeting from peripheral tissues for systemic gene delivery to the CNS in marmosets. Our results indicate that rAAVrh.8, along with rh.10 and 9, hold the best promise for developing novel therapeutic strategies to treat neurological diseases in the adult patient population. Additionally, systemically delivered rAAVrh.10 can transduce the CNS efficiently, and its transgene expression can be limited in the periphery by endogenous miRNAs in adult marmosets.
Making the White Matter Matters: Progress in Understanding Canavan's Disease and Therapeutic Interventions Through Eight Decades
Canavan's disease (CD) is a fatal autosomal recessive pediatric leukodystrophy in which patients show severe neurodegeneration and typically die by the age of 10, though life expectancy in patients can be highly variable. Currently, there is no effective treatment for CD; however, gene therapy seems to be a feasible approach to combat the disease. Being a monogenic defect, the disease provides an excellent model system to develop gene therapy approaches that can be extended to other monogenic leukodystrophies and neurodegenerative diseases. CD results from mutations in a single gene aspartoacylase which hydrolyses N-acetyl aspartic acid (NAA) which accumulates in its absences. Since CD is one of the few diseases that show high NAA levels, it can also be used to study the enigmatic biological role of NAA. The disease was first described in 1931, and this review traces the progress made in the past 8 decades to understand the disease by enumerating current hypotheses and ongoing palliative measures to alleviate patient symptoms in the context of the latest advances in the field.
Neuronal regeneration in C. elegans requires subcellular calcium release by ryanodine receptor channels and can be enhanced by optogenetic stimulation
Regulated calcium signals play conserved instructive roles in neuronal repair, but how localized calcium stores are differentially mobilized, or might be directly manipulated, to stimulate regeneration within native contexts is poorly understood. We find here that localized calcium release from the endoplasmic reticulum via ryanodine receptor (RyR) channels is critical in stimulating initial regeneration following traumatic cellular damage in vivo. Using laser axotomy of single neurons in Caenorhabditis elegans, we find that mutation of unc-68/RyR greatly impedes both outgrowth and guidance of the regenerating neuron. Performing extended in vivo calcium imaging, we measure subcellular calcium signals within the immediate vicinity of the regenerating axon end that are sustained for hours following axotomy and completely eliminated within unc-68/RyR mutants. Finally, using a novel optogenetic approach to periodically photo-stimulate the axotomized neuron, we can enhance its regeneration. The enhanced outgrowth depends on both amplitude and temporal pattern of excitation and can be blocked by disruption of UNC-68/RyR. This demonstrates the exciting potential of emerging optogenetic technology to beneficially manipulate cell physiology in the context of neuronal regeneration and indicates a link to the underlying cellular calcium signal. Taken as a whole, our findings define a specific localized calcium signal mediated by RyR channel activity that stimulates regenerative outgrowth, which may be dynamically manipulated for beneficial neurotherapeutic effects.
Understanding how an organism's nervous system transforms sensory input into behavioral outputs requires recording and manipulating its neural activity during unrestrained behavior. Here we present an instrument to simultaneously monitor and manipulate neural activity while observing behavior in a freely moving animal, the nematode Caenorhabditis elegans. Neural activity is recorded optically from cells expressing a calcium indicator, GCaMP3. Neural activity is manipulated optically by illuminating targeted neurons expressing the optogenetic protein Channelrhodopsin. Real-time computer vision software tracks the animal's behavior and identifies the location of targeted neurons in the nematode as it crawls. Patterned illumination from a DMD is used to selectively illuminate subsets of neurons for either calcium imaging or optogenetic stimulation. Real-time computer vision software constantly updates the illumination pattern in response to the worm's movement and thereby allows for independent optical recording or activation of different neurons in the worm as it moves freely. We use the instrument to directly observe the relationship between sensory neuron activation, interneuron dynamics and locomotion in the worm's mechanosensory circuit. We record and compare calcium transients in the backward locomotion command interneurons AVA, in response to optical activation of the anterior mechanosensory neurons ALM, AVM or both.
Graded levels of IRF4 regulate CD8+ T cell differentiation and expansion, but not attrition, in response to acute virus infection
In response to acute virus infections, CD8(+) T cells differentiate to form a large population of short-lived effectors and a stable pool of long-lived memory cells. The characteristics of the CD8(+) T cell response are influenced by TCR affinity, Ag dose, and the inflammatory cytokine milieu dictated by the infection. To address the mechanism by which differences in TCR signal strength could regulate CD8(+) T cell differentiation, we investigated the transcription factor, IFN regulatory factor 4 (IRF4). We show that IRF4 is transiently upregulated to differing levels in murine CD8(+) T cells, based on the strength of TCR signaling. In turn, IRF4 controls the magnitude of the CD8(+) T cell response to acute virus infection in a dose-dependent manner. Modest differences in IRF4 expression dramatically influence the numbers of short-lived effector cells at the peak of the infection, but have no impact on the kinetics of the infection or on the rate of T cell contraction. Furthermore, the expression of key transcription factors such as T cell factor 1 and Eomesodermin are highly sensitive to graded levels of IRF4. In contrast, T-bet expression is less dependent on IRF4 levels and is influenced by the nature of the infection. These data indicate that IRF4 is a key component that translates the strength of TCR signaling into a graded response of virus-specific CD8(+) T cells.
Astrocytic uptake of GABA through GABA transporters (GATs) is an important mechanism regulating excitatory/inhibitory balance in the nervous system; however, mechanisms by which astrocytes regulate GAT levels are undefined. We found that at mid-pupal stages the Drosophila melanogaster CNS neuropil was devoid of astrocyte membranes and synapses. Astrocyte membranes subsequently infiltrated the neuropil coordinately with synaptogenesis, and astrocyte ablation reduced synapse numbers by half, indicating that Drosophila astrocytes are pro-synaptogenic. Shortly after synapses formed in earnest, GAT was upregulated in astrocytes. Ablation or silencing of GABAergic neurons or disruption of metabotropic GABA receptor 1 and 2 (GABA(B)R1/2) signaling in astrocytes led to a decrease in astrocytic GAT. Notably, developmental depletion of astrocytic GABA(B)R1/2 signaling suppressed mechanosensory-induced seizure activity in mutants with hyperexcitable neurons. These data reveal that astrocytes actively modulate GAT expression via metabotropic GABA receptor signaling and highlight the importance of precise regulation of astrocytic GAT in modulation of seizure activity.
rAAV-Mediated Gene Transfer For Study of Pathological Mechanisms and Therapeutic Intervention in Canavan's Disease: A Dissertation
Canavan’s Disease is a fatal Central Nervous System disorder caused by genetic defects in the enzyme – aspartoacylase and currently has no effective treatment options. We report additional phenotypes in a stringent preclinical aspartoacylase knockout mouse model. Using this model, we developed a gene therapy strategy with intravenous injections of the aspartoacylase gene packaged in recombinant adeno associated viruses (rAAVs). We first investigated the CNS gene transfer abilities of rAAV vectors that can cross the blood-brain-barrier in neonatal and adult mice and subsequently used different rAAV serotypes such as rAAV9, rAAVrh.8 and rAAVrh.10 for gene replacement therapy. A single intravenous injection rescued lethality, extended survival and corrected several disease phenotypes including motor dysfunctions. For the first time we demonstrated the existence of a therapeutic time window in the mouse model. In order to limit off-target effects of viral delivery we employed a synthetic strategy using microRNA mediated posttranscriptional detargeting to restrict rAAV expression in the CNS. We followed up with another approach to limit peripheral tissue distribution. Strikingly, we demonstrate that intracerebroventricular administration of a 50-fold lower vectors dose can rescue lethality and extend survival but not motor functions. We also study the contributions of several peripheral tissues in a primarily CNS disorder and examine several molecular attributes behind pathogenesis of Canavan’s disease using primary neural cell cultures. In summary, this thesis describes the potential of novel rAAV-mediated gene replacement therapy in Canavan’s disease and the use of rAAVs as a tool to tease out its pathological mechanism.