Recent Submissions

  • Effectiveness of modified vaccinia Ankara-Bavarian Nordic vaccine against mpox infection: emulation of a target trial

    Navarro, Christine; Lau, Cindy; Buchan, Sarah A; Burchell, Ann N; Nasreen, Sharifa; Friedman, Lindsay; Okpokoro, Evaezi; Austin, Peter C; Tan, Darrell H S; Gubbay, Jonathan B; et al. (BMJ, 2024-09-11)
    Objective: To estimate the real world effectiveness of modified vaccinia Ankara-Bavarian Nordic (MVA-BN) vaccine against mpox infection. Design: Emulation of a target trial. Setting: Linked databases in Ontario, Canada. Participants: 9803 men aged ≥18 years with a history of being tested for syphilis and a laboratory confirmed bacterial sexually transmitted infection (STI) in the previous year, or who filled a prescription for HIV pre-exposure prophylaxis in the previous year. On each day between 12 June 2022 and 27 October 2022, those who had been vaccinated 15 days previously were matched 1:1 with unvaccinated men by age, geographical region, past HIV diagnosis, number of bacterial STI diagnoses in the previous three years, and receipt of any non-MVA-BN vaccine in the previous year. Main outcome measure: The main outcome measure was vaccine effectiveness ((1-hazard ratio)×100) of one dose of subcutaneously administered MVA-BN against laboratory confirmed mpox infection. A Cox proportional hazards model was used to estimate hazard ratios to compare the rate of laboratory confirmed mpox between the two groups. Results: 3204 men who received the vaccine were matched to 3204 unvaccinated controls. A total of 71 mpox infections were diagnosed, with 0.09 per 1000 person days (95% confidence interval (CI) 0.05 to 0.13) in the vaccinated group and 0.20 per 1000 person days (0.15 to 0.27) in the unvaccinated group over the study period of 153 days. Estimated vaccine effectiveness of one dose of MVA-BN against mpox infection was 58% (95% CI 31% to 75%). Conclusion: The findings of this study, conducted in the context of a targeted vaccination programme and evolving outbreak of mpox, suggest that one dose of MVA-BN is moderately effective in preventing mpox infection.
  • Association between maternal mRNA covid-19 vaccination in early pregnancy and major congenital anomalies in offspring: population based cohort study with sibling matched analysis

    Jorgensen, Sarah C J; Drover, Samantha S M; Fell, Deshayne B; Austin, Peter C; D'Souza, Rohan; Guttmann, Astrid; Buchan, Sarah A; Wilson, Sarah E; Nasreen, Sharifa; Brown, Kevin A; et al. (BMJ, 2024-09-16)
    Objective: To examine the association between maternal mRNA covid-19 vaccination during the first trimester of pregnancy and the prevalence of major congenital anomalies in offspring. Design: Population based cohort study with sibling matched analysis. Setting: Multiple health administrative databases, linked and analysed at ICES, an independent, non-profit research institute that collects and analyses healthcare and demographic data, Ontario, Canada, from 16 October 2021 to 1 May 2023. Population: 174 296 singleton live births >20 weeks' gestation with an expected birth date between 16 October 2021 and 1 May 2023: 34 181 (20%) born to mothers who received one or two doses of an mRNA covid-19 vaccine in the first trimester and 34 951 (20%) born to mothers who did not receive a vaccine before or during pregnancy. The sibling matched analysis included 13 312 infants exposed to a covid-19 vaccine in the first trimester and 15 089 matched older siblings with the same mother, with an expected birth date after 16 October 2016 and no reported in utero exposure to a covid-19 vaccine. Main outcome measures: Major congenital anomalies, overall and grouped by specific organ systems, diagnosed within 28 days of birth. Results: Major congenital anomalies were present in 832 (24.3 per 1000 live births) infants exposed to an mRNA covid-19 vaccine in the first trimester compared with 927 (26.5 per 1000 live births) infants not exposed to a vaccine, resulting in an adjusted prevalence ratio of 0.89 (95% confidence interval (CI) 0.79 to 1.01). Major congenital anomalies were present in 283 (21.3 per 1000 live births) and 343 (22.7 per 1000 live births) infants exposed to an mRNA covid-19 vaccine in the first trimester and their older siblings not exposed to a vaccine, respectively (adjusted prevalence ratio 0.91, 95% CI 0.77 to 1.07). First trimester vaccination was not associated with an increase in major congenital anomalies grouped by specific organ system in the primary or sibling matched analyses. Results were similar across a range of subgroup and sensitivity analyses. Conclusions: In this large population based cohort study and sibling matched analysis, mRNA covid-19 vaccination during the first trimester of pregnancy was not associated with an increase in major congenital anomalies in offspring, overall or grouped by organ system.
  • COVID-19 vaccine effectiveness against severe omicron-related outcomes in children aged 5 to 11 years in Ontario: A Canadian immunization research network (CIRN) study

    Piché-Renaud, Pierre-Philippe; Drover, Samantha S.M.; Austin, Peter C.; Morris, Shaun K.; Buchan, Sarah A.; Nasreen, Sharifa; Schwartz, Kevin L.; Tadrous, Mina; Thampi, Nisha; Wilson, Sarah E.; et al. (Elsevier BV, 2025-01)
    Background: Understanding how the efficacy of COVID-19 vaccines translates from clinical trials to real-world settings is critical to inform evolving vaccination policies. The objective of this study was to assess COVID-19 vaccine effectiveness (VE) against severe COVID-19-related outcomes in children aged 5-11 years, including COVID-19-related hospital admissions and multisystem inflammatory syndrome in children (MIS-C). Methods: We conducted a retrospective, population-based cohort study using linked health administrative data in the first year following the emergence of the Omicron variant (January 2 to December 31, 2022) in Ontario, Canada. Baseline differences between subgroups of interest were compared using standardized differences. We used multivariable Cox proportional hazard regression models to estimate VE by time since last vaccine dose by treating vaccination as a time-varying exposure, compared to unvaccinated children. Results: We included a total of 1,058,740 children, of which 583,867 (55.1 %) had received at least one vaccine dose by the end of the study period. In total, there were 185 COVID-19-related hospital admissions and 39 cases of MIS-C. The rate of COVID-19-related admission was substantially higher in children with an underlying comorbid condition compared to children who were previously healthy (adjusted hazard ratio [aHR] = 4.77, 95 %CI, 3.56-6.38). VE against COVID-19-related admission ranged from 93 % (95 %CI, 52-99 %) 7-29 days after a second dose to 63 % (95 %CI; 41-77 %) ≥120 days after a second dose. There was no statistically significant difference in the rate of MIS-C in children who received at least one dose of the vaccine compared to unvaccinated children (aHR = 0.71; 95 %CI, 0.38-1.34). Conclusions: We found that, for children aged 5-11 years, VE against COVID-19-related hospitalization was high in the first four months after a second dose. Children with comorbid conditions were found to be at much higher risk of COVID-19-related severe outcomes and thus may benefit most from COVID-19 vaccination.
  • Optimising early detection of degenerative cervical myelopathy: a systematic review of quantitative screening tools for primary care

    Inzerillo, Sean; Jagtiani, Pemla; Jones, Salazar (BMJ, 2025-01-11)
    Background: Early diagnosis of degenerative cervical myelopathy (DCM) is often challenging due to subtle, non-specific symptoms, limited disease awareness and a lack of definitive diagnostic criteria. As primary care physicians are typically the first to encounter patients with early DCM, equipping them with effective screening tools is crucial for reducing diagnostic delays and improving patient outcomes. This systematic review evaluates the efficacy of quantitative screening methods for DCM that can be implemented in primary care settings. Methods: A systematic search following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines was conducted across PubMed, Embase and Cochrane Library up to July 2024 using keywords relevant to DCM screening. Studies were included if they evaluated the sensitivity and specificity of DCM screening tools applicable to primary care settings. Study quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. Results: The search identified 14 studies evaluating 18 screening methods for DCM. Questionnaires consistently showed high diagnostic accuracy, with Youden indices exceeding 0.60, while only three out of nine conventional physical performance tests met the same threshold. Sensor-assisted tests, particularly those using advanced technology like finger-wearable gyro sensors, exhibited the highest diagnostic accuracy but present challenges related to accessibility and learning curves. Conclusion: This review highlights the potential of quantitative screening methods for early DCM detection in primary care. While questionnaires and conventional tests are effective and accessible, sensor-assisted tests offer greater accuracy but face implementation challenges. A tailored, multifaceted approach is crucial for improving outcomes. Future research should focus on validating these tools in diverse populations and standardising diagnostic criteria.
  • Antibiotic-mediated gut microbiota depletion affects behavioral and neuronal correlates of spatial cognition

    Chanovas Colome, jordi (2024-09-27)
    Trillions of microbes —comprising communities of bacteria, viruses, fungi, and other microorganisms— inhabit the gastrointestinal tract. Known as gut microbiota, these microorganisms are central to human health. Growing evidence connects imbalances of microbial communities with several neurological and psychiatric conditions, and signals to the modulating role of gut bacteria on brain function, including learning and memory. However, how the microbiota influences cognitive behavior remains unclear. Here, we investigated whether gut microbiota depletion disrupts the behavioral and neuronal correlates of spatial cognition. We depleted gut bacteria in mice by giving a cocktail of non-absorbable antibiotics and characterized 1) learning and memory performance in modalities of a place avoidance task with different cognitive demands and 2) Arc and c-Fos-expressing neuronal ensembles in the supra and infrapyramidal blades of the dentate gyrus. We found impaired learning and memory performance in place avoidance tasks that require high, but not low, cognitive demand and blade-specific alterations in Arc and c-Fos-expressing granule cell ensembles with microbiota depletion. Our results suggest that gut microbial signaling may regulate the expression of neural mechanisms underlying cognitive control.
  • Age-dependent and multifaceted effects of methylphenidate in pre- and peri-adolescent rats.

    Zhu, Ning (2011-10-12)
    Attention deficit hyperactivity disorder (ADHD) is a developmental disorder affecting approximately 9% of school-aged children. A number of therapeutic options exist to treat ADHD. One, methylphenidate (MPD), improves behavior of patients with ADHD in real life, as well as their performance in laboratory settings. Recent evidence indicates that MPD affects healthy humans in a manner similar to that in ADHD patients. However, data regarding the mechanisms and effects of MPD are inconsistent, particularly with respect to age groups (i.e., children, adolescents, adults). Using the rat as a model, we conducted several studies to clarify the effects of oral MPD on cognitive function, activity and anxiety during pre- and peri-adolescence in animals. A preliminary study revealed apparent age-dependent and multifaceted effects of oral MPD in pre- and peri-adolescent rats. There, we found that daily oral administration of MPD at 3 mg/kg resulted in: (1) improved performance from postnatal day (PND) 22 to 24 but impaired performance from PND 32-34; (2) increased locomotor activity and reduced anxiety-like behaviors at PND 22-23; and (3) improved performance in an attentional task. Moreover, we found that responses to oral MPD at 3 mg/kg varied among rats, specifically in locomotor activity and performance in the attention task. Here, we examined the following three hypotheses stemming from these preliminary data: (1) the effects of oral MPD at clinically-relevant doses on performance in cognitive tasks are age-dependent; (2) oral MPD alters anxiety-related behaviors and locomotor activity in an age-dependent manner, both of which contribute to the effects of MPD on performance in cognitive tasks; (3) individual responses to novelty (i.e., new environments) in rats predict individual behavioral responses to oral MPD, including cognitive performance, anxiety-like behaviors, and locomotor activity. To test our hypotheses, we tested drug-naive pre- and peri-adolescent rats over three age ranges (PND 23 to 28, PND 29 to 34 and PND 36 to 41) for (1) the effects of oral MPD in a radial arm maze (RAM) task; (2) the effects of oral MPD on anxiety-related behaviors and locomotor activity on the elevated plus-maze; and (3) interaction effects between treatment conditions and individual responses to novelty on measures from the behavioral tests. Plasma and brain levels of MPD were also measured to examine the absorption and metabolism of the drug across these age ranges. We found that (1) oral MPD at 3 mg/kg improved RAM performance during PND 36-41 but not during PND 23-28 and PND 29-34; (2) oral MPD at 3 mg/kg reduced anxiety-related behaviors across all ages and increased locomotor activity only on PND 24 and 37; and (3) the MPD-induced increase in locomotor activity correlated with individual response to novelty. Brain and plasma levels of MPD were highest on PND 24, lower on PND 30, and lowest on PND 37. In addition, the higher dose of oral MPD improved RAM performance significantly during PND 36-37. These results suggest that, during pre- and peri-adolescence: (1) oral MPD at a clinically-relevant dose produces multifaceted effects that change across ages, which together contribute to the improved performance on cognitive tasks; (2) these effects, according to known brain circuit information, are likely through independent circuits; and (3) the effects of oral MPD on cognitive performance vary according to the testing procedures and the state of maturation.
  • Early thrombopoietin intervention in stroke: Requirement for thrombopoietin receptor for brain and behavioral protection.

    Zhuang, Jian (2013-12-18)
    Cerebral ischemia results in brain injury. The brain inflammatory response to ischemia contributes significantly to stroke-induced ischemic injury. Thrombopoietin (TPO), a hematopoietic growth factor that regulates platelet production, has recently been reported to protect the rat from ischemic stroke brain injury and associated behavioral deficits. My research has focused on early post-stroke TPO intervention using a mouse stroke model. Here I present data that addresses TPOs brain- and behavioral-protective effects in mouse ischemic stroke. I found that TPO treatment in WT mice produces significant protection from ischemic hemisphere swelling (57.4 ± 6.5%), hemisphere infarction (43.2 ± 5.2%), neurological deficits (36.9 ± 12.6%) and provides significant preservation of post-stroke cognitive functioning. No TPO protection was observed in TPOR KO mice. IHC data indicates that TPO significantly reduced peri-infarct up-regulation of TNF(40.4 ± 10.2%) and MMP-9 (27.1 ± 11.1%) in microvascular endothelium. Furthermore, peri-infarct vascular neutrophil infiltration into brain (25.1 ± 3.4%) and microgliosis (35.4 ± 6.5%) were inhibited by TPO. In addition to reducing brain infarction and swelling, TPO also acts to reduce microvascular inflammation and thereby to preserve the blood brain barrier. In TPOR KO mice, TPO treatment neither attenuated increased microvascular TNFor MMP-9, nor inhibited neutrophil infiltration or microgliosis. Thus, I have demonstrated that the TPOR is required for TPO brain protection from stroke-induced injury and behavioral deficits. Mechanistically, TPO treatment reduces the microvascular and parenchymal brain inflammatory response to stroke resulting in protection of the brain and blood brain barrier resulting in preservation of functioning on all levels examined.
  • Control of GABAA Receptor α4 Subunit Expression In a Human Neuroblastoma Cell Line

    Zhou, Xiangping (2006-05-09)
    The GABAA receptor (GABAR) is a pentameric ligand-gated Cl- channel, normally composed of two α, two β and one γ subunit. α4 expression has been shown to increase in response to extended periods of in vivo administration of positive modulators of the GABAR, such as neurosteroids, benzodiazepines (BZs) and ethanol. However, the mechanism of α4 upregulation is not clear. Therefore, the purpose of this study was to establish an in vitro model to study neurosteroid regulation of α4 expression. I proposed two potential initial triggers for α4 upregulation: i.) increased current produced by a positive GABA modulator or ii.) allosteric events produced by ligand-receptor interactions. To this end, I used the human neuroblastoma IMR-32 cell line to demonstrate increases in α4 expression by 48 h administration of the GABA-modulatory steroid 3α,5β-THP (3α -OH-5β -pregnan-20-one) following differentiation by nerve growth factor (NGF) in serum-free medium. Functional expression of α4βγ2 GABAR was validated by the unique pharmacology characteristic of this GABAR isoform, which include a near complete insensitivity to BZ agonists, but potent positive modulation by the BZ antagonist flumazenil and the BZ partial inverse agonist RO15-4513. THP induction of α4 expression was a result of its enhancement of GABA-gated current because co-application of the GABAR blocker picrotoxin prevented this effect. Moreover, 48 h administration of compounds which decrease GABA-gated current, such as submaximal concentrations of the GABAR open channel blocker penicillin G or the competitive GABAR antagonist gabazine, decreased α4 expression, as did the BZ inverse agonist DMCM. However, 48 h exposure to the BZ antagonist flumazenil, which allosterically binds to the GABAR but has no effect on GABA-gated current, did not change α4 expression. These findings suggest that α4 expression is correlated with the change in GABA-gated current. The results from my study may have implications for alterations in α4 expression across naturally occurring fluctuations in endogenous steroids.
  • Identification of MicroRNAs Regulating Apolipoprotein Secretion.

    Zhou, Liye (2017-04-07)
    A major risk factor for cardiovascular disease (CVD) is plasma cholesterol that is carried in plasma by two lipoproteins LDL and HDL. Currently available therapies lower LDL and attempts are undergoing to enhance HDL levels for therapeutic purposes. With advances in microRNAs (miRs), small non-coding RNAs that affect multiple pathways, we hypothesize that there might be miRs that regulate plasma lipoproteins. The major proteins in LDL and HDL are apoB and apoA1. To identify miRs that regulate both LDL and HDL, I took two different approaches. In the first approach, I searched for miRs that could potentially modulate apoB and apoA1 expression. From TargetScan, I found that human apoB mRNA contained two predicted targeting sites for miR-548p. In both Huh-7 and HepG2 cells, overexpression of miR-548p significantly decreased intracellular and secreted medium apoB without affecting apoAI. Mechanistic studies revealed that miR-548p decreased apoB expression levels by destabilizing its mRNA. Luciferase reporter assay indicated that miR-548p interacts with apoB 3'-UTR. Site-directed mutagenesis revealed that both seed and supplementary sequences on Site II, not Site I, were required for the targeting of miR-548p on apoB 3'-UTR. Additionally, the cholesterol and fatty acid syntheses rates were decreased in miR-548p overexpressing Huh-7 cells. We also found that Hmgcr and ACSL4, two important enzymes in cholesterol and fatty acid synthesis process, were predicted targets of miR-548p. Indeed, the mRNA and protein levels of Hmgcr and ACSL4 were significantly lowered by miR-548p. The direct targeting of miR-548p on the 3'-UTR of Hmgcr and ACSL4 was studied by luciferase reporter assay. In summary, a novel miR-548p decreased apoB secretion from Huh-7 cells by targeting apoB 3'-UTR and promoting its posttranscriptional degradation. MiR-548p also reduces cholesterol and fatty acid synthesis by down-regulating Hmgcr and ACSL4 expression levels. In the second approach, I took an unbiased global approach to identify miRs that individually and simultaneously affect apoB and apoA1. I transfected human hepatoma Huh7 cells in duplicate with a library of 1237 miRs and measured apoB and apoA1 in the media. This resulted in the identification of 13 and 28 miRs that increased and decreased apoB secretion. In addition, I identified 11 and 23 miRs that increased and decreased apoA1 secretion. Most interestingly, I identified 3 miRs that simultaneously increased apoB and reduced apoA1 levels. I further characterized miR-1200 and explained mechanisms how it regulates apoB and apoA1 secretion. Mechanistic studies showed that miR-1200 reduced apoB secretion by lowering the mRNA levels of apoB. In addition, post-transcriptional degradation of apoB was promoted in miR-1200 transfected cells. Bioinformatic analysis revealed apoB as a predicted target of miR-1200. Luciferase reporter assay and site-directed mutagenesis were used to confirm the direct miR-mRNA interaction. In contrast, miR-1200 increased apoAI protein and mRNA levels by increasing its de novo transcription. The mechanistic studies resulted in the identification of a novel transcription repressor of apoAI, BCL11B. Therefore, miR-1200 increases the transcription of apoAI by reducing the expression levels of BCL11B, and promotes the secretion of apoAI. I also observed that the overexpression of miR-1200 in Huh-7 cells decreased lipid synthesis, which might avoid the lipid accumulation in the liver. In summary, these studies identified a novel human microRNA-1200 that could potentially reduce apoB-containing lipoproteins and increase HDL production from the liver, as well as decreasing lipid synthesis in human hepatoma cell line. These three different roles of miR-1200 in lipid metabolism regulation suggest that it may have protective function against atherosclerosis. In short, I have identified two novel miRs-548p and 1200 as potent regulators of apolipoprotein and lipid metabolism in hepatoma cells. Both have the potential to be developed as therapeutic agents for the treatment of dyslipidemia and atherosclerosis while avoiding hepatic steatosis. As these are primate specific miRs, the physiological effect of overexpressing or inhibiting endogenous miR-548p or miR-1200 could be tested in humanized mice or non-human primate models.
  • ComplementC3 regulated cell death in myocardial ischemia/reperfusion injury

    Fang Chang, Zhou (2023-04-24)
    Ischemic heart disease is a life-threatening condition and requires immediate treatment by unblocking the occluded blood vessel. However, the restoration of blood flow to the ischemic area causes additional damage, named ischemia/reperfusion injury (I/R injury).Currently, there is no clinically approved therapy available to reduce I/R injury. Basic research suggests that myocardial cell death, i.e., necrosis and apoptosis, are key events in I/R injury. Our group previously found that complement and ROS were involved in different stages of necrosis development during the early phase of I/R injury in a murine myocardial model. In particular, SOD1 overexpression significantly reduced necrosis at 1 hour of reperfusion, while catalase overexpression reduced necrosis at 3 hours’ reperfusion. The inhibition of I/R injury by SOD1 and catalase was transient, and I/R injury appeared to be restored at 24 hours’ reperfusion. Meanwhile, complement C3 deposition became significant at 3 hours’ reperfusion and reached the peak at 24 hours of reperfusion. Therefore, we hypothesized that SOD1 and catalase are important in regulation of ROS mediated I/R injury during the first 3 hours of reperfusion, while complement may contribute to I/R injury thereafter. This thesis investigated how complement C3 regulates cell death during I/R injury after 3 hours of reperfusion. In Aim 1 of this study using the I/R mouse model, we found that after 1 hour ischemia/ 24 hours reperfusion, the level of necrosis in C3-/- mice was significantly lower than that in WT mice, while the level of apoptosis in C3-/- mice was significantly higher than that in WT mice. Furthermore, we found that 4 weeks after the initial 1 hour of ischemia, C3-/- mice had significantly less cardiac fibrosis and better cardiac function than WT mice. Our comparative proteomics analyses showed that Cyt c, a key factor in the intrinsic apoptotic pathway, was preferentially present in the C3-binding complexes in WT mice after 3 hours of reperfusion. These results indicate that C3 may promote necrosis and inhibit apoptosis in myocardial I/R injury. In Aim 2 of our study, we further explored the mechanism of C3-mediated apoptosis using an in vitro system of AC16 human ventricular cardiomyocyte cell line. We found that although AC16 cells do not express C3, they can uptake exogenous C3 when exposed to it at 37°C. The uptake of C3 was not significant at low temperature(4°C), suggesting a receptor-mediated uptake of C3. However, AC16 cells do not express the known complement receptors 1, 2, 3 and 4. Thus, C3 uptake in AC16cells is likely through unknown receptor(s). Incubation of exogenous C3 could significantly reduce H2O2-induced-apoptosis in AC16cells. In a cell free apoptosis system, we found C3 could significantly reduce the intrinsic pathway apoptosis, possibly through interaction with factor(s)downstream of Cyt c. In a cell free pull-down assay, pro-C3 was able to bind with the apoptotic factor pro-caspase 3. In summary, our results showed that 1)At early stage of I/R injury, complement C3 can promote necrosis and apoptosis. At the late stage of I/R injury, C3 promotes cardiac fibrosis and causes worse cardiac function. 2) Human cardiomyocytes (AC16 cells), which did not expressC3, readily uptake the exogenous C3 from extracellular milieu. The uptake of C3into AC16 cells is likely through a receptor-mediated endocytosis, although the identity of the receptor is still unknown. 3) The inhibition of oxidative-related apoptosis by exogenous C3 in AC16 cells is very likely through the binding with apoptotic factor(s) in the intrinsic apoptosis pathway.
  • Determinants for Alzheimer’s Disease: An Exploration of Metabolic and Clinical Risk Factors.

    Buitrago-Soto, Luna (2023-04-14)
    Alzheimer’s Disease (AD) is characterized by the pathological deposition of amyloid plaques or tau tangles in the brain. Currently, most therapeutic treatments for AD focus on arresting and reversing amyloid and tau pathologies. To date, unfortunately, clinical trials for drugs targeting amyloid or tau depositions have proven ineffective. Genome-wide and transcriptome-wide association studies have identified various genes and genetic variants in lipid metabolism that are associated with AD. Particularly, dysregulation of sphingomyelin (SM) metabolism has been implicated in AD, leading to the speculation that modulators of the SM pathway could emerge as candidate targets for therapeutic AD treatment. However, the effectiveness of modulators of the SM pathway remains understudied. This study tested whether the SM pathway activator Fingolimod (FTY720), a Sphingosine-1-Phosphate (S1P) signaling modulator, can rescue the behavioral and functional deficits associated with the pathology observed in mouse models of AD. We examined two mouse models, one expressing the amyloid pathology (APP/PS1) and the other the tau pathology (Tg4510), which are known to exhibit severe cognitive and synaptic plasticity impairments. We found that after two months of exposure to Fingolimod in drinking water, the mice showed recovered cognitive function measured in novel object recognition and Barnes maze memory tasks and rescued expression of synaptic plasticity measured in long-term synaptic potentiation in the entorhinal cortex and hippocampal synapses. Our findings suggest that upregulation of the SM pathway via modulation of S1P signaling may have protective effects in AD.
  • The role of hepatic phospholipid transfer protein in lipoprotein metabolism.

    Yazdanyar, Amirfarbod (2013-04-19)
    Atherosclerosis is the leading cause of death in western countries. Lipoprotein metabolism is closely related with the disease. ApoB-containing lipoproteins (BLp) are atherogenic particles, while, high density lipoproteins (HDL) are anti-atherogenic. Phospholipids Transfer Protein (PLTP) activity has important impacts on BLp and HDL homeostasis. Animal studies, including general knock out (KO) and transgenic approaches; have shown pro-atherogenic properties for PLTP. However, the tissue specific function of PLTP remains mostly unknown. The liver is one of the critical organs for lipoprotein metabolism and is also a major source for PLTP expression. To address the impact of liver-specific PLTP on lipoprotein metabolism, we created two mouse models which either express PLTP specifically in the liver on a PLTP-null background or lack PLTP in the liver (liver-specific PLTP KO) in a wild type background. In both models, our findings show that the liver has a 25% contribution into total plasma PLTP activity and liver-specific PLTP is highly involved in BLp and HDL production by hepatocytes. We found that hepatic ablation of PLTP leads to a significant decrease in plasma PLTP activity, HDL-cholesterol, HDL-phospholipids, non-HDL-cholesterol, non-HDL-phospholipids, apoA-I, and apoB levels. In addition, nuclear magnetic resonance (NMR) examination of lipoproteins showed that hepatic PLTP deficiency decreases HDL and apoB-containing particle numbers, as well as the VLDL particle size which was confirmed by electron microscopy. However, liver-specific PLTP expression results in a remarkable increase in plasma levels of BLp, as well as a slight increase in HDL levels. In order to unravel the mechanism, we evaluated the apoB and triglyceride production after blocking the clearance of the particles. We found that liver-specific PLTP increases apoB (both apo48 and apoB100) and triglyceride (TG) secretion. To investigate the role of liver PLTP in HDL production, we cultured primary hepatocytes from our knockout and wild type mice and tested their ability to form nascent HDL particles in the presence and absence of exogenous PLTP. We found that PLTP activity, as opposed to heat inactivated PLTP, promotes nascent HDL production through ABCA1-mediated pathway since ablation of ABCA1 tremendously diminished HDL production and the presence of active PLTP had no effect. We also showed that PLTP closely interact with ABCA1 in order to enhance HDL formation. Taken together, our results indicate that liver-generated PLTP promotes both BLp and HDL production by hepatocytes. In addition, the liver makes a 25% contribution to the total plasma PLTP activity, and thus ablation of liver PLTP may influence lipoprotein metabolism in the plasma.
  • Regional Variations in Microstructure and Biomechanical Properties of the Human Vertebral Endplate and Trabecular Bone.

    Xavier, Fred (2015-08-04)
    Vertebral compression fractures represent one of the most common injuries resulting from osteoporosis with an incidence rate of 700,000 per year in the United States. Spinal osteoporosis affects the anterior part of the vertebrae, leading to its collapse and kyphosis. However, there is limited data on the regional differences in the microstructure of the vertebral endplate and cancellous bone and their relationships with degenerative disc diseases and vertebral fractures. Does bone resorption occur at a higher rate in the anterior than in the lateral region of the vertebra? We conducted shear testing experiments on isolated cylindrical bone cores of thoracic vertebrae. Our data showed a 5-fold increase in mechanical strength from the anterior to the lateral regions of both endplate and cancellous bone (p=0.04). Material and ash density measurements correlated positively with shear strength. We further studied the regional variation in biomechanical properties inside human and bovine thoracic vertebrae using surgical anterior self-tapping cancellous screws. Our results showed a trend for increased insertion torque, pullout strength, and stiffness from divergent (laterally oriented) to convergent (centrally oriented) screws. Radiological image analyses from Faxitron X-ray, CT scan, and microCT confirmed that density was higher in the central than in the lateral regions of the cancellous bone. However, we observed the opposite relationship at the cervical and lumbar levels where the divergent screw configuration was mechanically stronger than the convergent one. According to our histological analyses, the density of microvessels at the vertebral endplate decreased from central to lateral regions by 15%. The ratio bone volume over total volume (BV/TV) increased by 31% from superior to inferior bony endplates. There was a decrease in the ratio osteoid matrix surface/bone surface (OS/BS) by 20% from superior to inferior bony endplates. We also observed that older individuals with increased degenerative disc diseases (DDD) had lower cancellous bone density, but higher bone remodeling activity than younger ones. The biomechanical properties of both endplate and cancellous bone vary considerably within the human spinal column. The lateral region of the vertebral body (EP and cancellous) is denser and stronger at the cervical and lumbar levels. The opposite relationship is observed in the thoracic segment. Newer implants could prioritize a lateral or central loading accordingly; thus, reducing the likelihood of screw loosening and the subsidence of disc replacement devices. Moreover, the superior endplate is weaker than the inferior, but more porous, and probably more important for the diffusion of nutrients to the nucleus pulposus. Therefore, the likelihood of disc degeneration might increase with superior endplate injuries.
  • Inhibition of Connective Tissue Growth Factor prevents increase in extracellular matrix molecules in a rodent model of Diabetic Retinopathy.

    Winkler, Jennifer (2012-02-08)
    Connective tissue growth factor (CTGF) is a profibrotic factor that induces extracellular matrix (ECM) production and angiogenesis, two processes involved in diabetic retinopathy (DR). In this study, we examined whether insulin therapy or a CTGF specific siRNA administered to diabetic rats decreased the levels of CTGF and of selected putative downstream genes in retina. Rats with streptozotocin-induced diabetes were used for these studies. Animals received either no treatment or were administered constant insulin therapy for 12 weeks. mRNA and protein levels of CTGF and select ECM genes were determined using Real-Time PCR and western blotting of retina. Localization of CTGF, GFAP (Glial Fibrillary Acidic Protein), a marker of astrocytes and activated Müller cells, and Vascular Endothelial Growth Factor, (VEGF), a well studied growth factor in the pathologies of DR, were visualized in the retina using immunohistochemistry. CTGF mRNA and protein significantly increased in retina of diabetic rats at 8 and 12 weeks of hyperglycemia. There was also an increase CTGF, GFAP and VEGF immunostaining during hyperglycemia. Double label immunohistochemistry indicated that retinal Müller cells of diabetic rats expressed CTGF. Hyperglycemia also upregulated mRNA levels of fibronectin, laminin ß1, collagen IVa3 and VEGF, and this increase was prevented by insulin therapy. Treatment of diabetic rats with CTGF siRNA decreased laminin ß1, collagen IVa3 mRNAs and CTGF mRNA and protein but did not affect fibronectin or VEGF mRNA levels. Since insulin therapy is associated with oscillations in blood glucose levels, we sought to develop a supplementary gene therapy to inhibit CTGF in DR. Prior to its use in vivo, we sought to determine whether a gene therapy tool could inhibit CTGF in a rat cell culture model that expresses CTGF. First, we tested the efficiency of an adenovirus encoding for a CTGF antisense oligonucleotide and three CTGF siRNA's in vitro to decrease the levels of Transforming Growth Factor-β (TGF-β)-induced CTGF expression in Rat 2 fibroblasts. The adenovirus and siRNA both decreased CTGF in Rat 2 fibroblasts. To circumvent any possible immune responses caused by the adenovirus we chose to use the siRNA for our in vivo studies to inhibit CTGF in the diabetic retina. A subset of diabetic rats received a single intravitreal injection of CTGF siRNA in one eye and a scrambled, control, siRNA in the contralateral eye. Retinas were examined three and ten days after the injection. CTGF siRNA significantly inhibited CTGF mRNA and protein 3 days after injection. CTGF siRNA also induced a significant decrease in laminin β1, collagen IV α3 and GFAP mRNA but had no effect on fibronectin mRNA levels. The level of CTGF mRNA was similar in retina of control and CTGF siRNA injected animals 10 days after injection In conclusion, these studies indicate that CTGF and ECM genes are regulated with insulin therapy. Importantly, CTGF siRNA regulates changes in ECM molecules and reduced GFAP expression suggesting that the treatment decreased the level of glial cell activation and reactivity in DR. Taken together, our results indicate that CTGF is an important target for the treatment of DR.
  • Interaction of eukaryotic initiation factors eIF1A and eIF4G with the ribosome and assembly of ribosomal initiation complexes on Picornavirus IRESs.

    Yu, Yingpu (2010-04-27)
    Ribosomes serve as the platform for decoding the genetic information carried in mRNA and translating it into polypeptides; they are a major target for natural and synthetic antibiotics. Although fundamental to gene expression, many aspects of translation in eukaryotes are still not well understood, particularly translation initiation. The goal of my thesis was to yield insights of the interplay of mRNA, tRNA, cellular factors with the ribosome that are critical for the initiation of polypeptide synthesis. Specifically, my work focused on the interactions of translation initiation factors eIF1A and eIF4G with the 40S ribosomal subunit and the interactions of picornavirus RNAs with cellular factors that help them to bypass the regulation of canonical translation initiation. Initiation is the key rate-limiting stage in the eukaryotic translation process and more than 12 eukaryotic initiation factors (eIFs) are involved in this step. Most eukaryotic cellular mRNAs utilize the cap-dependent initiation pathway, in which the ribosomal preinitiation complex first binds to the ‘cap’ structure of the mRNA and then scans to select the AUG start codon to initiate the translation process. eIF4G and eIF1A are two factors that are required for steps in the canonical translation initiation process and they have distinct functions: eIF4G promotes attachment of mRNA to the ribosome and facilitates scanning by ribosomal complexes on the mRNA. eIF1A is involved in tRNA recruitment, scanning, start codon selection and subunit joining. Instead of eIF4F binding to the 5’-end of capped mRNA, some viral RNAs contain an internal ribosomal entry site (IRES) in their 5’UTR; this structure allows the viral RNA to recruit the 40S ribosomal subunit to an internal site on the mRNA independently of the cap and of the 5’-end of the mRNA. Four types of IRESs that have been identified in viral mRNAs can bypass one or all steps of the cap-dependent initiation pathway. Type 1 IRESs are found in picornaviruses such as poliovirus; type 2 IRESs are located in the 5’ UTRs of picornaviruses of the Cardiovirus and Aphthovirus genera, such as Encephalomyocarditis virus (EMCV) and Foot-and-mouth disease virus (FMDV). These two IRESs bind directly to eIF4G and eIF4A subunits of eIF4F, don’t need eIF4E, and can bypass the scanning step in initiation. Although they do share other steps with and use many of the same canonical factors as the cap-dependent initiation pathway, these IRESs also need IRES trans-acting factors (ITAFs) to fulfill their function. Different IRESs in the same family have different requirements for the ITAFs. The reason for this, and the mechanism by which ITAFs promote IRES function have both been obscure. To answer these questions, the specific aims of my thesis are: (1) To locate the position of eIF4G on the 40S ribosomal subunit; (2) To characterize the mechanism of action of eIF1A and to determine its location on the 40S ribosomal subunit; (3) To characterize aspects of the mechanism of translation initiation on Type 1 and Type 2 picornavirus IRESs. By using the technique of directed hydroxyl radical cleavage, we determined the positions of eIF4G/eIF4A on the ribosome, near the exit (E) site of the mRNA-binding channel, suggesting that these subunits of eIF4F are situated at the trailing edge of the ribosome and that eIF4A may unwind secondary structure in the cap-proximal region of the 5’UTR to promote “landing” on them by ribosomal preinitiation complexes. We positioned all domains of eIF1A on the 40S subunit and correlated these positions with the functions of eIF1A in promoting recruitment of initiator tRNA to the ribosomal peptidyl (P) site, preventing premature binding of elongator tRNA to the aminoacyl (A) site, coordinating sequential adoption by the 40S subunit of the ‘open’ conformation (to permit binding of mRNA) and the ‘closed’ conformation (on initiation codon recognition), and in contributing to the stability of ribosomal complexes during scanning. We determined that Type 2 IRESs bound to eIF4G adopt a compact side-by-side orientation, and that ITAFs promote this orientation. We also observed that eIF4G binds with similar orientations to domain V of Type 1 IRESs and to the J-K domain of Type 2 IRESs, suggesting that this factor plays a similar mechanistic role in initiation on Type 1 and Type 2 IRESs. Together, these results have yielded insights into how canonical initiation factors interact with the ribosome to promote the recruitment of mRNA, how they promote ribosomal scanning and how IRESs interact with ITAFs, a subset of initiation factors and ribosomal complexes to bypass early steps in the canonical eukaryotic initiation pathway.
  • Identification of Functional Domains in Sphingomyelin Synthase (SMS) and Further Investigation into the Role of Phospholipid Transfer Protein (PLTP) in Lipid Metabolism and Atherosclerosis

    Yeang, Calvin (2010-09-16)
    Lipid metabolism is closely related to the development of atherosclerosis. The work presented in this thesis involves two genes which are important for lipid metabolism and atherosclerosis: sphingomyelin synthase (SMS) and phospholipid transfer protein (PLTP). Sphingomyelin synthase (SMS) utilizes ceramide and phosphatidylcholine as substrates to produce sphingomyelin (SM) and diacylglycerol, thereby regulating important lipid messengers. There are two isoforms of the enzyme, SMS1 and SMS2. It has been speculated that the active site of both SMS1 and SMS2, containing two evolutionary conserved histidines and one aspartic acid, is identical to that of lipid phosphate phosphatase (LPP). We systematically mutated these amino acids by site-directed mutagenesis and found that each point mutation abolished SMS activity without altering cellular distribution. Despite sharing 77% sequence homology to each other, SMS1 is located in the trans-Golgi while SMS2 is located in the plasma membrane as well as the Golgi. This prompted us to explore what targeting signals exist in SMS1 and SMS2. Conventional motifs responsible for protein targeting to the plasma membrane or Golgi are either not present in, or unique to, SMS1 and SMS2. Therefore, we determined the targeting signals in SMS1 and SMS2 through a combination of creating truncations of non-homologous amino acids from SMS1 and SMS2 and creating SMS chimeras by swapping non-homologous amino acid sequences between SMS1 and SMS2. We found that SMS1 and SMS2 transit through the classical secretory pathway. Furthermore, SMS1 contains a C-terminal Golgi targeting signal and SMS2 contains a C-terminal plasma membrane targeting domain. (PLTP) is an ubiquitously expressed, secreted protein which functions in the blood and in tissues. A high saturated fat diet induces free cholesterol and phospholipid accumulation in the plasma of phospholipid transfer protein (Pltp) deficient mice. We examined the atherogenic consequence of this phenomenon and investigated the possible mechanism(s). To study atherosclerosis, we bred Pltp KO mice with Apoe KO mice, a mouse model of atherosclerosis. Pltp KO/Apoe KO mice, fed a coconut oil-enriched high-fat diet (COD) for 7 weeks, had higher plasma free cholesterol (149%), phospholipids (15%), and sphingomyelin (54%) than Apoe KO controls. In contrast to chow-fed animals, COD-fed Pltp KO/Apoe KO mice had the same atherosclerotic lesion size as that in Apoe KO mice. Similar to Pltp KO mice, plasma from COD fed Pltp KO/Apoe KO mice contained VLDL/LDL sized lamellar particles. Bile measurement indicated that COD-fed PltpKO mice have 33% less hepatic cholesterol output than controls. In conclusion, COD-fed Pltp deficient mice are no longer protected from atherosclerosis and have impaired biliary lipid secretion associated with free cholesterol and phospholipid accumulation.
  • Mechanism of Action of Novel Anti-Cancer Peptide, PNC-27, that Selectively Kills Cancer Cells.

    Yazdi, Ehsun (2011-02-03)
    In vivo and in vitro studies have shown the efficiency of anti-cancer peptides PNC-27 and PNC-28 in killing cancer cells by necrosis without affecting normal cells (1, 2 and 5). These peptides were derived from the hdm2-binding domain of p53 (PNC-27: aa12-26 and PNC-28: aa17-26) and linked to a membrane residency peptide (MRP). Here I provide morphological and biochemical evidence of the effect of PNC-27 on a wide range of cancer cells including primary cancer cells, and the absence of any effect on untransformed cells. I also demonstrate that PNC-27-induced cytotoxicity is time, dose and temperature dependent. Moreover, I demonstrate the role of HDM2 as a possible tumor marker and a target for PNC-27 in the tumor cell’s plasma membrane which is confirmed by: immunofluorescence, exogenous expression of HDM2 in normal cell plasma membrane, competition experiments and immunoblot of isolated plasma membrane. I also evaluate sequence of cellular events following the exposure of tumor cells to PNC-27 using live cell imaging spinning disc confocal microscopy. Furthermore, using Immuno-gold TEM and SEM I show that gold-conjugated antibodies to HDM2 and PNC-27 decorated ring-like pore structures in the plasma membrane of tumor cells exposed to PNC-27, further confirming the co-localization of HDM2 and PNC-27 in tumor cell membrane. These results strongly suggest that the highly amphipatic PNC-27 inserts itself into the plasma membrane of only cancer cells upon binding to HDM2, where, by random movement, the HDM2-PNC-27 complexes assemble into oligomers and eventually into membrane pores. Influx of free PNC-27 molecules through the newly formed membrane pores into the cell’s cytoplasm leads to the assembly of pores in mitochondrial membranes and rapid cell death.
  • Association between ambulatory blood pressure and alternative formulations of job strain.

    Landsbergis, Paul A; Schnall, Peter L; Warren, Katherine; Pickering, Thomas G; Schwartz, Joseph E (Scandinavian Journal of Work, Environment and Health, 1994-10)
    Objectives: The goal of the study was to determine whether alternative formulations of Karasek & Theorell's job-strain construct are associated with ambulatory blood pressure and the risk of hypertension. Methods: Full-time male employees (N = 262) in eight worksites completed a casual blood pressure screening, medical examinations, and questionnaires and wore an ambulatory blood pressure monitor for 24 h on a workday. Cases of hypertension were ascertained from casual blood pressure readings for a case-referent analysis. A cross-sectional analysis was also conducted, ambulatory (continuous) blood pressure measurements being used as the outcome. Results: All formulations of job strain exhibited significant associations with systolic blood pressure at work and home, but not with diastolic blood pressure. Employees experiencing job strain had a systolic blood pressure that was 6.7 mm Hg (approximately 0.89 kPa) higher and a diastolic blood pressure that was 2.7 mm Hg (approximately 0.36 kPa) higher at work than other employees, and the odds of hypertension were increased [odds ratio (OR) 2.9, 95% confidence interval (95% CI) 1.3-6.6]. Using national means for decision latitude and demands to define job strain increased the systolic and diastolic blood pressure associations to 11.5 mm Hg (approximately 1.53 kPa) and 4.1 mm Hg (approximately 0.54 kPa), respectively. Adding organizational influence to the task-level decision latitude variable produced a stronger association for hypertension with job strain (OR 3.7, 95% CI 1.6-8.5). Adding social support to the job-strain model also slightly increased the hypertension risk. Conclusions: The impact of job strain, at least on systolic blood pressure, is consistent and robust across alternative formulations, more restrictive cut points tending to produce stronger effects.
  • Clinical experience and results of a Sentinel Health Investigation related to indoor fungal exposure.

    Johanning, E; Landsbergis, P; Gareis, M; Yang, C S; Olmsted, E (Environmental Health Perspectives, 1999-06)
    This is a review of exposure conditions, clinical presentation, and morbidity of children and adults with indoor fungal exposure such as toxic Stachybotrys chartarum. Indoor exposure was characterized using different methods including microscopic, culture, cytotoxicity screening tests, and chemical analyses. Clinical case histories and physical and laboratory findings are presented of children (age < 18 years, n = 22; mean age 9 years; 60% females) and adults (age >18 years, n = 125; mean age 39 years, 67% females) who consulted an environmental health specialty clinic. In the pediatric patients' exposure history, widespread fungal contamination of water-damaged building materials with known toxic or allergic fungi was identified. Primarily disorders of the respiratory system, skin, mucous membranes, and central nervous system were reported. Some enumeration and functional laboratory abnormalities, mainly of the lymphatic blood cells, were observed, although no statistically significant differences were found. IgE or IgG fungi-specific antibodies, used as exposure markers, were positive in less than 25% of all tested cases. In an evaluation of a symptomatic girl 11 years of age (sentinel case investigation) living in an apartment with verified toxigenic fungi (i.e., S. chartarum), several health indicators showed improvement after exposure cessation.
  • Evaluation and management of chronic work-related musculoskeletal disorders of the distal upper extremity

    Piligian, George; Herbert, Robin; Hearns, Michael; Dropkin, Jonathan; Landsbergis, Paul; Cherniack, Martin (Wiley, 2000-01)
    This clinical review will describe the epidemiology, clinical presentation, and management of the following work-related musculoskeletal disorders (WMSDs) of the distal upper extremity: deQuervain's disease, extensor and flexor forearm tendinitis/tendinosis, lateral and medial epicondylitis, cubital tunnel syndrome, and hand-arm vibration syndrome (HAVS). These conditions were selected for review either because they were among the most common WMSDs among patients attending the New York State Occupational Health Clinics (NYSOHC) network, or because there is strong evidence for work-relatedness in the clinical literature. Work-related carpal tunnel syndrome is discussed in an accompanying paper. In an attempt to provide evidence-based treatment recommendations, literature searches on the treatment of each condition were conducted via Medline for the years 1985-1999. There was a dearth of studies evaluating the efficacy of specific clinical treatments and ergonomic interventions for WMSDs. Therefore, many of the treatment recommendations presented here are based on a consensus of experienced public health-oriented occupational medicine physicians from the NYSOHC network after review of the pertinent literature. A summary table of the clinical features of the disorders is presented as a reference resource.

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