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Habituation and Dishabituation in the Olfactory Bulb: From Neural Responses to Behavior
Get PDFHabituation and dishabituation modulate the neural resources and behavioral significance allocated to incoming stimuli across the sensory systems. The purpose of the research presented in this dissertation was to characterize these processes in the mouse olfactory bulb (OB) and to determine if OB acetylcholine (ACh) has a role in physiological and behavioral olfactory dishabituation. Calcium imaging was used to determine the timecourse and magnitude of habituation in different parts of the OB during and after a prolonged odor presentation. Widefield imaging of the dendritic, or glomerular, response of OB output cells demonstrated that prolonged odor input habituates glomerular responses during the presentation as well as to subsequent presentations of the odor. Manipulation of OB ACh release during prolonged odor presentations using electrical stimulation dishabituated these decreased glomerular odor responses. A novel behavioral investigation paradigm was developed to see how prolonged odor input affects odor salience in awake, behaving mice. Optogenetic stimulation of OB cholinergic neurons rapidly modulated odor salience in this paradigm, causing mice to suddenly investigate a previously ignored odor. Non-olfactory sensory stimulation also dishabituated odor investigation and this increase could be blocked pharmacologically with a cholinergic antagonist in the OB, demonstrating the ecological validity of this ACh effect. Two-photon imaging revealed that, unlike the glomerular responses, soma responses of different OB cell types can be quite different from each other during a prolonged odor presentation. These results highlight the need for future studies that explore the role of different OB cell types in the representation of olfactory information over time and behavioral dishabituation
Design, Validation, and Clinical Testing of a Novel Fastening Device for a Scoliosis Brace
Get PDFEach year, thirty thousand children in the US were put into a scoliosis brace. The primary function of scoliosis braces was to reduce and prevent progression of the spinal deformity as the patient grew by application of corrective forces to the spine. Straps, often made of Velcro, were attached to the back of the brace that applied forces to the spine when the straps were tightened. Braces were prescribed to be worn up to 23 hours a day. Studies have concluded that discomfort increased with increasing strap tension and pad pressure. They have also shown an increase in strap tension occurred during deep breathing and some daily activities which led to discomfort. Consequently, discomfort often led to poor user compliance and reduced brace wear hence decreasing treatment efficacy. Treatment efficacy also decreased due to a loss of strap tension activities of lying down. The overall goals were to develop a novel fastening device, or controlled tension unit (CTU), for a scoliosis brace that allowed the user to set the strap tension to the prescribed value as determined by the orthotist at the time of brace fitting and to maintain the prescribed strap tension during a variety of typical daily living activities. This device should ensure the corrective force capacity of the scoliosis brace was present and the occurrence of strap loosening and tension loss was minimized. In the end, three studies were carried out to design and validate the CTU devices. For Study One, the objective was to determine the force-displacement properties of the controlled tension devices alone. The materials used were: Controlled Tension Units (CTU), Robotic Testing Platform. The methods were as follows: The controlled tension units were mounted in a robotic testing platform that was programmed to displace the CTU device at a set speed (or rate of spring displacement) and measure the force response of the device. Three different speeds and spring tensions were tested. The results were as follows: The CTU were designed with desired load settings of 20N, 30N, and 40N which were confirmed. The units applied a relatively constant tension over a working range of 12.7mm and maintained a load tolerance within ±10%. The force output response and load tolerances were independent of the rate of spring displacement. In conclusion, CTU devices could be fabricated with selectable load settings that held a relatively constant tension throughout a desired range of displacement. For Study Two, the first objective was to validate that the CTU force output was maintained over a finite amount of brace gap opening and closing (as it related to the brace gap separation). The second objective was to evaluate the corrective force capacity and structural stiffness properties of a standard 3-strap brace using either Velcro straps or CTU devices as the fastening system. The materials used were: Controlled Tension Units, Standard (Velcro Strap) Brace, Standard (CTU) Brace, Robotic Testing Platform, Scoliosis Analog Model (SAM), Lab Tensiometers. The methods were as follows: Three brace configurations were tested: Native Standard Brace, Standard (CTU) Brace, and Standard (Velcro Strap) Brace. A low tension CTU (≈20N) was used for all tests. Throughout the movement, the reaction forces and strap tensions in the craniocaudal and mediolateral axes were continuously recorded. The results were as follows: The CTU devices provided a greater range of brace gap displacement compared to the Velcro straps. For the CTU devices, the strap tension was constant over the range of displacement and remained close to the tension value of the CTU device (approximately 20N). However, the tensiometer readings were significantly greater with the Velcro straps being greatest at the top strap and lowest at the bottom. In conclusion, CTU devices could be used to develop a more flexible dynamic brace that allowed for directional movement without compromising the corrective force capacity of the brace. Contrary to the Velcro straps, the CTU strap tension setting would remain present independent of the brace gap allowing for opportunities of deep breathing, increased range of movement, and/or improved brace force correction. For Study Three, the objective was to determine if CTU reduced discomfort while maintaining a constant strap tension. The materials used were: Standard (Velcro Strap) Brace, Standard (CTU) Brace, Portable Tensiometers. The methods were as follows: The discomfort, strap tension, and gap distance were evaluated during typical daily activities as well as the range of motion where applicable using the Standard (Velcro Strap) Brace and the Standard (CTU) Brace. Afterwards, a CTU Increase Test was performed in which the tension in each CTU fastener was incrementally increased until the patient experienced a greater level of discomfort than with the Velcro strap condition at the prescribed tension. In conclusion, CTU improved bracing in scoliosis by maintaining strap tension, improving brace flexibility, decreasing discomfort at similar strap tensions, or by achieving higher strap tensions without increasing discomfort. In conclusion, the CTU devices allowed the user to set the strap tension to the prescribed value as determined by the orthotist at the time of brace fitting and maintained the prescribed strap tension during a variety of typical daily living activities to ensure the corrective force capacity of the scoliosis brace was present and the occurrence of strap loosening and tension loss was minimized
Retrieval Analysis of Necropsy Total Hip Replacements: Considerations Beyond the Implant
Get PDFIntroduction. Total hip arthroplasty (THA) surgery is one of the most commonly performed and successful orthopedic procedures in the United States. More than 300,000 primary THAs and 40,000 revision THAs performed in the United States every year. While the need for revision surgeries can stem from a variety of causes, there have been, to the author’s knowledge, no studies attempting to correlate the concentrations of certain inflammatory cytokines to metal ion concentrations found in the tissue surrounding the implant, amount of polyethylene wear, or strength of the interface of the modular taper. The purpose of this study was to begin to look at those factors to see if any were indicative of implant survivorship, as well as to see if metal ion content contributes to implant longevity. The testing for this group of well-functioning implants will be useful as a baseline when comparing the same types of testing for failed implants.
Methods. A total of nineteen cadaveric total hip implants were obtained from two sources, the Medical Education and Research Institute (Memphis, TN) and RestoreLifeUSA (Elizabethton, TN). The bearings for these implants were either metal on polyethylene or ceramic on polyethylene. Synovial fluid and tissue samples were taken from the joint for testing. Head dissociation was performed, in which an Instron 4505 was used in accordance with ASTM Standard F2009-00 to remove the head from the stem of the implant, recording force. Corrosion scoring was performed on taper surfaces by three scorers. The polyethylene acetabular liner was measured on the superior side with a micrometer to determine how much material loss was evident compared to the inferior side. These three values were then correlated to the testing performed using the synovial fluid and tissue. The synovial fluid was analyzed for inflammatory cytokines IL-6, MCP-1, IL-1β, MIP-3α, M-CSF, IL-8, IL-2, and TNF-α using a premixed Luminex screening assay. These results were given in picograms per milliliter. An anterior and posterior synovial tissue sample was analyzed for the presence of metal ions cobalt, chromium, and titanium using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). All of these results were compiled and analyzed together to search for potential correlations.
Results. There were no significant differences in dissociation forces between the groups of implants with head corrosion score 1 and head corrosion score 2. The comparison of MCP-1 to the dissociation force produced a correlation coefficient of 0.64 (p-value 0.05) and the comparison of MIP-3α to the dissociation force produced a correlation coefficient of 0.67 (p-value 0.03). However, when the graphs of these correlations were observed, it seemed likely that this correlation was due to one sample pulling the graph in a positive direction which is demonstrated by the 95% confidence interval (CI) of the correlation coefficient (0.011 to 0.90 for MCP-1, and 0.069 to 0.91 for MIP-3α). When comparing polyethylene wear to the inflammatory cytokine concentrations, no significant correlations were seen. There was a positive correlation between cobalt and chromium levels and dissociation force (r=0.56 for cobalt, r=0.66 for chromium), and a negative correlation between titanium levels and dissociation force (r=-0.30). The positive relationship was opposite of what was expected, as more metal debris should mean the implant surfaces are losing material, which should therefore decrease the strength of the taper connection. The 95% confidence interval for the correlation coefficients included zero for cobalt and titanium, and was fairly wide for chromium (0.11 to 0.90). When observing cytokines and metal ion presence, most relationships were very scattered with low correlation coefficients. However, for cobalt, strong positive relationships were seen for IL-6 (r=0.67, CI: 0.19 to 0.89), MCP-1 (r=0.76, CI: 0.33 to 0.93), and MIP-3α (r=0.60, CI: 0.066 to 0.86). When looking at confidence intervals, there seemed to be a mild correlation between cobalt and IL-6 and a moderate correlation between cobalt and MCP-1. No meaningful relationships were seen for any cytokines with chromium or titanium, so it may be useful to select cytokines known to be responsive to those two metals in particular for future studies. When comparing metal levels between the two corrosion levels seen in the heads, there were no statistically significant differences in any of the metals between implants with a corrosion score of one and those with a corrosion score of two.
Discussion. This study was limited by the fact that the sample size for this study was very low. With only nineteen total implants, it was difficult to draw meaningful conclusions. Additional implants are being recruited in order to increase this sample size for future studies. Additionally, it was difficult for meaningful correlations to be seen when comparing any factor to the inflammatory cytokine concentrations, as these values were clustered around the lower limit of detection. However, this was expected with well-functioning implants. While it is difficult to draw meaningful conclusions when used as a correlation, this data will be useful when comparing cytokine concentrations of a group of failed implants. This group is able to serve as a baseline value for each type of testing performed, and will help to make sense of the same testing of failed implants in the future
Why is Family Medicine Different?
Get PDFAn understanding of the role and function of Family Medicine in the healthcare system can provide important insights for Enterprise Architecture. It is often stated that the thought process utilized by Family Medicine physicians is different from that of specialty physicians, but heretofore there has been little or no analysis of what that difference is. This article examines that difference from the perspective of the complex adaptive system that is healthcare today, and shows how it is that Family Medicine physicians perform the vital function of decreasing the entropy or disorder in the patient care system via decision loops, as opposed to the decision trees of linear or classical logic. The generalist function of Family Medicine physicians results in the integration and coordination of the various specialty functions in healthcare
Discovery of Novel Tubulin Inhibitors and Selective Survivin Inhibitors for Advanced Melanoma and Total Synthesis of Bioactive 20S-hydroxyvitamin D3
Get PDFAccording to the statistics from American Cancer Society, the 5-year survival rate for patients with advanced melanoma is as low as 5%. Treatment of advanced melanoma, therefore, represents an unmet medical need. In this dissertation, I will show the effort to develop new generations of bioavailable tubulin inhibitors targeting the colchicine binding site and selective small-molecule survivin inhibitors for treating advanced melanoma. Extensive structure-activity relationship (SAR) studies of lead molecules ABI-231 and UC-112 have been performed.
Chapter 1 will introduce the current situation of advanced or metastatic melanoma, its clinical drug treatments, as well as problems in current drug treatments. Microtubule dynamics and survivin will be discussed as promising therapeutic targets for developing anticancer drugs. 20S-hydroxyvitamin D3 (20S-OH-D3) will be introduced as a promising anti-inflammatory scaffold.
Chapter 2 will disclose the SAR study of ABI-231, a previously reported potent tubulin inhibitor from our lab. In this chapter, a new synthetic method was developed to enable the synthesis of ABI-231 analogues modifying the indole moiety. The novel synthetic method involved the synthesis of a key diamine intermediate and imidazoline formation. From the new synthetic method, thirty ABI-231 analogues were synthesized and tested for activities. Among all analogues, 10ab with a 4-methyl-3-indole moiety and 10bb with a 4-indole moiety showed the most potent antiproliferative activities against a panel of melanoma cell lines. 10ab and 10bb had IC50s of 2.2 and 3.0 nM, respectively. The SAR result revealed that modification of the indole moiety in ABI-231 was beneficial to activity.
In Chapter 3, we will describe our effort to develop the SAR study of ABI-231 focusing on modification of the 3,4,5-TMP moiety. This is selected since it is one of the most common moieties in current tubulin inhibitors targeting the colchicine binding site. To circumvent the use of potentially explosive azide reported in Chapter 2, an alternative was established to efficiently generate ABI-231 analogues. This new synthetic method involved Suzuki coupling and Grignard reactions to modify the 3,4,5-TMP moiety and to produce target compounds in gram-scale. Among the eight analogues synthesized, the one containing an unique 3-methoxybenzo[4,5]-dioxene moiety had the strongest antiproliferative activity against a panel of melanoma cell lines with an average IC50 of 1.9 nM. To our best knowledge, it represents the most successful instance of isosterically modifying the 3,4,5-TMP moiety in CA-4 derivatives.
Chapter 4 will highlight our effort to synthesize reverse ABI (RABI) analogues for SAR study. In this chapter, a novel and concise synthetic route was established toaccess RABI scaffold. RABI scaffold was constructed through a Grignard reaction/Suzuki-Miyaura coupling reaction strategy. From this new synthetic method, twelve novel RABI analogues were synthesized. Compared to MX-RABI (the previously reported most potent RABI), several new RABI analogues showed significantly improved cytotoxicities. In particular, analogue 15i with a 4-indazole moiety showed the most potent antiproliferative activity against a panel of melanoma cell lines and had an average IC50 of 0.8 nM. This is the first sub-nM anti-tubulin compound in the related scaffolds.
Chapter 5 will reveal our latest SAR study of UC-112, a previously reported selective survivin inhibitor. Fourteen UC-112 analogues modifying the benzyloxy moiety of UC-112 were synthesized. Their corresponding SAR result demonstrated that indole moiety was the most favorable (analogue 12a). Subsequent structural optimization of 12a by introducing either mono-substituent or di-substituent to the indole moiety led to the synthesis of another twenty-four new UC-112 analogues. Several substituted indole analogues showed equipotency to that of UC-112 and MX-106. Importantly, new indole analogues exhibited significant abilities to overcome multidrug-resistance mediated by Pgp overexpression.
Chapter 6 is characterized by the establishment of a total synthetic method of 20SOH-D3 which showed comparable antiproliferative activity to 1,25α-dihydroxyvitamin D3 without hypercalcemic toxic effect upto a concentration of 60 μg/kg in vivo. The total synthesis of 20S-OH-D3 involved parallel generation of key intermediates from ergocalciferol. The vitamin D3 core structure was constructed through Wittig-Horner coupling reaction. Deprotection of SEM and TBS was achieved in one step. 20S-OH-D3 was furnished in sixteen steps with an overall yield of 0.4%
Cannabinoid Receptor 2 (CB2) Ligands Downregulate Pro-Inflammatory Markers in Stimulated Primary Human Periodontal Ligament Fibroblasts (hPDLFs)
Get PDFThere are approximately 743 million individuals suffering from chronic periodontitis (PD) making it the sixth most prevalent condition worldwide. The affected adult population in the U.S. are nearly 64.7 million and the healthcare costs exceeds $14 billion. Recently, host response to pathogenic infection has been seen critical to the progression of PD and exhibit increase in various inflammatory markers. Marijuana is well known for its recreational usage and is a risk factor for periodontal disease, which is seen as a concern in society for its negative health consequences. However, many medical conditions can benefit from the pharmacological effects of cannabinoids. Plethora of evidence is available for the use of cannabinoids for the anti-inflammatory and immune modulating activity. The targets for therapeutic intervention include the cannabinoid type 1 and 2 receptors (CB1R and CB2R). The CB2 receptor is an attractive target in the endocannabinoid system (ECS), due to widespread expression in peripheral tissue, upregulated expression during inflammation and lacks adverse psychotropic effects associated with CB1 receptor. The expression of cannabinoid receptors are present in periodontal tissues, and play a role in the physiological protection of tissues against excessive inflammation. Our lead compound SMM-189 is a CB2 inverse agonist, which exhibited anti-inflammatory properties in various primary cell lines. We aimed to address the anti-inflammatory properties seen with cannabinoid synthetic compounds SMM-189 (CB2 selective inverse agonist), HU308 (CB2 selective agonist), and the endocannabinoid anandamide (AEA) in periodontal disease. Our primary investigation led us to evaluate the most potent stimuli for the study, from which IL-1β emerged as the most robust inducer of cytokine and chemokine responses. The study was further expanded to include the most prominent biomarkers associated with infection, inflammation and disease. We addressed the hypothesis that effective inhibition of IL-1β-stimulated primary human periodontal ligament fibroblasts (hPDLF’s) will be seen with SMM-189 and HU308 and AEA. The synthetic cannabinoids exhibit excellent profile as an anti-inflammatory agent, while the endocannabinoid AEA exhibits dualistic effects that exerted both anti-inflammatory and pro-inflammatory effects. Our study revealed that the most potent anti-inflammatory effects were seen with SMM-189, an inverse agonist. Suggesting that targeting the endocannabinoid system in chronic periodontitis by an inverse agonist may lead to the development of novel drug for periodontal therapy. The outcome from this study has provided hopes for the development of drugs that will aid in strategies that will improve public oral health
Age-Associated Expression Patterns of OATP 1B1 and OATP 1B3 and Their Effect on the Disposition of Fexofenadine
Get PDFAs part of the drug disposition process (absorption, distribution, metabolism, excretion), an often overlooked aspect is transport. In order for drugs to be metabolized and excreted from the body they go through the liver or other drug removal organs. For drugs that are polar or are large they must rely upon transport mechanisms to transport them across the biomembranes of the drug removal organs. OATP1B1 and OATP1B3 are transporters on the sinusoidal membrane of the liver which work in concert with the drug metabolizing enzymes as part of the drug removal process. It is known that the development of each drug metabolizing enzyme follows a unique pattern that is determined in part by age. This is not unique to drug metabolizing enzymes or even drug disposition processes. All aspects of physiologic development are governed in part by age from gastric pH, and kidney function, to skin thickness. With the knowledge that age is a major determinant of expression of drug metabolizing enzymes it was hypothesized that age would also impact the expression of drug transporters. The developmental protein expression pattern of the closely related hepatic uptake transporters, OATP1B1 and OATP1B3, was examined in human liver tissue samples. Samples were obtained from donors aged 14 days to 12 years. Western blot analysis revealed a very intriguing pattern. OATP1B3 showed very high expression at birth but tapered down to minimal expression by 6 months of age. The intriguing aspect of this data was that the protein expression level of OATP1B3 started to increase during the pre-adolescent period. When comparing the protein expression levels in infants less than 3 months to children aged 6-12 years, protein expression was 237% higher in the infants. The expression decreased to 33% in the 3 months to 2 years group as compared to the 6-12 years group with the 2-6 years group starting to increase expression to 50%. OATP1B1 showed large inter-individual variability but failed to show a significant difference in protein expression amongst the age groups examined. While ontogeny is a very important part of overall development, genetics is also very important. The term developmental pharmacogenetics describes the inter-play between age and genetics, as phenotypes can only be expressed if physiological development is at a point where it is possible to be seen. In order to learn more about the expression of OATP1B1 and OATP1B3 several common SNPs were examined, OATP1B1 388 A→G and 521 T→C as well as OATP1B3 334T→G and 699G→A. All four SNPs were found to be in Hardy-Weinberg equilibrium. The allele frequencies for each SNP were in agreement with previously published reports. OATP1B3 334T→G and 699G→A were in complete linkage disequilibrium. The linkage disequilibrium that was found between the SNPs in OATP1B3 and OATP1B1 388 A→G was a novel finding that deserves further exploration. Examination of protein expression and each SNP failed to show any association between the two which also deserves further exploration with a larger number of samples to confirm. Lastly, the functional consequences of the loss of OATP1B activity was explored using the Oatp1b2-/- mouse model. Oatp1b2-/- is the rodent member of the OATP1B family and shares greater than 65% homology to the human members. Using fexofenadine as a probe for Oatp1b2 activity, it was found that Oatp1b2-/- mice had 2.1 fold higher exposure as compared to the wild-type mice. The Oatp1b2-/- also had 55% of the fexofenadine clearance and 63% the volume of distribution as compared to wild-type mice. These findings underscore the importance that Oatp1b activity has in determining exposure, and thus efficacy and potential toxicity, to substrates. Drug transport is a very important part of drug disposition that has yet to be extensively studied especially in terms of drug uptake. The following studies examined the developmental expression patterns of hepatic uptake transporters OATP1B1 and OATP1B3, common genetic variations of the transporters, as well as the consequences the loss of OATP1B activity has on substrates. Understanding the developmental expression pattern of OATP1B will allow for better dosing strategies for patients, particularly infants and children who rarely participate in clinical trials. Better dosing strategies ultimately lead to improved outcomes and reduced toxicity in the pediatric population
THE ROLE OF HSF1 PROTEIN REGULATION ON NEURODEGENERATION
Get PDFCellular protein homeostasis is achieved by a delicate network of molecular chaperones and various proteolytic processes such as ubiquitin–proteasome system (UPS) to avoid a build-up of misfolded protein aggregates. The latter is a common denominator of neurodegeneration. Neurons are found to be particularly vulnerable to toxic stress from aggregation-prone proteins such as α-synuclein. Induction of heat-shock proteins (HSPs), such as through activated heat shock transcription factor 1 (HSF1) via Hsp90 inhibition, is being investigated as a therapeutic option for proteinopathic diseases. HSF1 is a master stress-protective transcription factor which activates genes encoding protein chaperones (e.g. iHsp70) and anti-apoptotic proteins. However, whether and how HSF1 is dysregulated during neurodegeneration has not been studied. Here, we discover aberrant HSF1 degradation by aggregated α-synuclein (or α-synuclein-induced proteotoxic stress) in transfected neuroblastoma cells. HSF1 dysregulation via α-synuclein was confirmed by in vivo assessment of mouse and in situ studies of human specimens with α-synucleinopathy. We demonstrate that elevated NEDD4 is implicated as the responsible ubiquitin E3 ligase for HSF1 degradation through UPS. Furthermore, pharmacologically induced SIRT1-mediated deacetylation can attenuate aberrant NEDD4-mediated HSF1 degradation. Indeed, we define the acetylation status of the Lys 80 residue located in the DNA-binding domain of HSF1 as a critical factor in modulating HSF1 protein stability in addition to its previously identified role in the transcriptional activity. Together with the finding that preserving HSF1 can alleviate α-synuclein toxicity, the first part of the study strongly suggests that aberrant HSF1 degradation is a key neurodegenerative mechanism underlying α-synucleinopathy. Chronic activation of another cellular stress response, unfolded protein response in ER, has been implicated in tauopathy including Alzheimer’s disease (AD). The unfolded protein response (UPR) in the endoplasmic reticulum (ER) and the cytoplasmic heat stress response are two major stress response systems necessary for maintaining proteostasis for cellular health. Failure of either of these systems, such as in sustained UPR activation or in insufficient heat shock response activation, can lead to the development of neurodegeneration. Alleviation of ER stress and enhancement of heat shock response through heat shock factor 1 (HSF1) activation have previously been considered as attractive potential therapeutic targets for AD—a prevalent and devastating tauopathy. The second part of the study concentrates on our attempts to understand the interplay of the two aforementioned systems and their cooperative role in AD. We provide compelling in vitro and in vivo evidence that strongly suggests an auto-propagating interplay of UPR activation and HSF1 degradation being a common pathogenic feature in both human AD and tau transgenic mouse AD models. We identify aging-associated AD-like neuropathological changes in the hippocampus of HSF1 heterozygous knock-out mice. We speculate that HSF1 loss as an early (earliest) event which constitutes a mechanistic connection between ER stress and tau hyperphosphorylation in tau pathology. Finally, we demonstrate that aged mice lacking HSF1 gene exhibited deficits in hippocampal-dependent functions including short-term working memory, spatial learning and long-term memory. All together, our work supports a previously underappreciated importance of this master stress regulator HSF1 in neuronal functions and in maintaining brain homeostasis
A Novel Distractive and Mobility-Enabling Lumbar Spinal Orthosis for Treating Mechanical Low Back Pain
Get PDFIntroduction: Lumbar spinal orthoses (LSOs) are often used as non-surgical treatment and serve to support the spine and alleviate low back pain. More recently, dynamic orthoses claiming to decompress the spine have been introduced. Currently, there is an unserved population of people that suffer from mechanical low back pain (LBP) conditions, such as degenerative disc disease or lumbar foraminal stenosis, that would benefit from spinal decompression and mobility. A previously-developed prototype of dynamic mobility orthosis (DMO1) was designed that provided a distractive load across the lumbar spine but required higher sagittal bending moments and was unable to maintain spinal off-loading throughout extended ranges of movement. The research objectives were to a) Design a new orthosis that reduced bending moment build up and sustained spinal off-loading throughout daily living ranges of flexion and extension movement, b) Test the new orthosis prototype in a controlled laboratory environment, and c) Organize and carry out a clinical pilot study with patients suffering from mechanical LBP to determine the immediate and short-term effects of the new orthosis prototype on LBP and overall patient quality of life. Methods: A mechanical analog upper torso model and programmable robotic testing platform were used to design features of the new prototype (DMO2): a mobility-enabling component (MEC) and a distractive force component (DFC). The DMO2 prototype was tested in a robotic testing platform (RTP) under a 300 N applied vertical torso load over a range of 25° flexion to 10° of extension utilizing a previously-developed protocol. For DMO2, loads carried by the brace were determined throughout flexion and extension. Applied moments to upper torso model and transferred moments to spine were measured. The difference in applied and transferred moments represented brace moment effects. It was determined that DMO2 had limitations, primarily with providing a distractive force to actual human subjects. Because of this, a new orthosis prototype (DMO3) was developed that improved upon the design of the DMO2 MEC and DFC. The DMO3 prototype was designed to provide a constant distractive force with minimal resistance to bending while effectively providing a distractive force to the wearer that could be felt. Also, the MEC of DMO3 included both flexion and extension as well as axial rotation. The DMO3 prototype was tested in the RTP under a 150 N applied vertical load over a range of 25° flexion to 10° of extension utilizing a previously-developed protocol. Also, the DMO3 prototype was tested in the RTP under simulated axial rotation without an applied vertical torso load. For this test, a measurement was made that determined how much axial rotation DMO3 allowed. A clinical study was organized in which two patients with LBP wore the DMO3 prototype during six physical therapy (PT) treatment sessions. Before the treatment sessions began, the patient had a radiograph (x-ray 1) taken and the patient completed a Modified Oswestry Disability Questionnaire (mODI). During each treatment session, a pain score was taken before wearing DMO3 and after completing exercises for 30-40 minutes while wearing the DMO. Additionally, on the last treatment session, a radiograph (x-ray 2) was taken. The lumbar disc height at the diseased and adjacent lumbar levels were measured on x-ray 1 and x-ray 2 and compared. Also, the mODI scores were compared before and after treatment. Results: The DMO2 prototype improved spinal off-loading capacity from 172 N to 290 N at end range flexion and from 247 N to 293 N at end range extension compared to the original DMO1 prototype. End range applied moments (flexion-DMO1: 32.4 Nm / DMO2: 21.7 Nm; extension-DMO1: 15.0 Nm / DMO2: 10.9 Nm) and brace moments (flexion DMO1: 18.6 Nm / DMO2: 6.6 Nm; extension-DMO1: 15.0 Nm / DMO2: 4.4 Nm) were also reduced. The DMO3 prototype was able to support 100% (at vertical stance), 104% (at end range flexion), and 97% (at end range extension) of the applied vertical torso load during simulated flexion and extension. Also, the DMO3 prototype contributed 0 of the 4.1 Nm (at end range flexion), and 4.4 of the 6.6 Nm (at end range extension) of the total bending moment. During simulated axial rotation, the DMO3 prototype was able to achieve 10 degrees of axial rotation in both the clockwise and counterclockwise directions. The DMO3 prototype immediately reduced pain for both patient 1 (p \u3c 0.001) and patient 2 (p = 0.005) during all PT treatment sessions that they participated in. Patient 1 showed an of 2 mm (resolution 0.14 mm) of lumbar disc height at the L5-S1 level between the start and end of the treatments with DMO3. Similarly, patient 2 showed an increase of 0.5 mm at both the L4-L5 and L5-S1 levels. Patient 1 and patient 2 showed a decrease in mODI scores from 70 % to 62% and from 46% to 22%, respectfully. Discussion: An advanced testing assembly was used in this study to carry out the design of two prototypes of a novel dynamic spinal orthosis having the unique design goal of providing spinal offloading while enabling mobility. Two prototype models (DMO2 and DMO3) were built and tested under simulated DLAs in a controlled laboratory environment. Throughout the development of the DMO, each prototype demonstrated significant improvements compared to its predecessor. The means by which the DMO3 prototype produced a distractive force allowed the force output of the DMO to remain constant even with minor fluctuations in vertical displacement as might be experienced in normal flexion and extension motions. Preliminary testing found DMO3 prototype completely removed all pain from patient 1 at five of her six PT treatment visits. Conclusion: A novel dynamic spinal orthosis was designed that maintained spinal off-loading throughout extended ranges of flexion and extension movement without buildup of adverse bending moments. Additionally, the novel orthosis was able to achieve enough axial rotation required for most daily living activities (DLAs). Preliminary testing found this orthosis was also effective on human subjects as pain was reduced during all PT treatment sessions for both patients. An orthosis that provides distraction and mobility has not been designed and the proposed spinal orthosis provided the first evidence of its clinical effectiveness on human subjects. Because of the efficacy of this orthosis, a larger clinical study is warranted to see if the DMO can have short-term and long-term effects on mechanical LBP
Therapeutic Effects of Ormeloxifene in Cervical Cancer Carcinogenesis
Get PDFCervical cancer (CxCa) remains the fourth leading cause of cancer related deaths among women worldwide. Cervical cancer is mainly (~ 99.7%) derived from high risk Human papillomavirus (HR HPV). HPV E6/E7 are the two main oncoproteins that interfere with p53 and pRb (retinoblastoma) cell cycle regulatory proteins and hinder their efficacy of controlling cell growth. Additionally, PI3K-Akt is a cell survival pathway that is aberrantly expressed in cervical cancer cells. This pathway has a profound role in inhibiting mitochondrial intrinsic apoptotic signaling pathway. Advanced stage cervical cancer is difficult to treat and patients diagnosed with metastatic disease have a poor survival rate. Therefore, there is an urgent need to develop newer treatment modalities. Ormeloxifene (ORM) is a non-hormonal, anti-estrogen, oral contraceptive for human use. Growing evidences also suggest that ormeloxifene has anti-cancerous properties in a variety of cancers. Developing nanoformulation of drugs has received much attention lately as nanoparticles have site specific targeted drug delivery. Nanoparticles have a specific size range that makes them capable of being entrapped and accumulated at the tumor site due to its leaky vasculature. As a result of it, drug is released from the particle core at a sustained rate; therefore, nanoparticulates offer enhanced bioavailability and better therapeutic efficacy. Considering these benefits, we engineered ormeloxifene loaded PLGA based novel nanoformulation (PLGA-ORM). In this work we validated anti-cancer properties of free ORM and its PLGA based nanoformulation. Our set of data showed that ormeloxifene significantly decreased the cellular proliferation and clonogenic potential of cervical cancer cells. Ormeloxifene also reduced the cellular motility and induced the apoptosis via targeting PI3K-Akt signaling in these cells. Furthermore, ormeloxifene modulated the HPV induced oncogenesis in Caski cells. Ormeloxifene also showed additive inhibitory effects on cellular proliferation and growth when used with radiation. Moreover, our novel PLGA-ORM had a particle size range of 100 – 280 nm and also exhibited excellent encapsulation of ormeloxifene in to PLGA core. PLGA-ORM was labeled with Coumarin 6 (green fluorescent) dye for its uptake studies, where PLGA-ORM internalized in cervical cancer cells in dose, time and energy dependent manner via endocytosis pathway. PLGA-ORM showed improved anti-proliferative/growth properties than free ormeloxifene in cervical cancer cells. When utilized in animals (an orthotopic mouse model) both ormeloxifene and PLGA-ORM showed great anti-tumorous properties, however PLGA-ORM had improved inhibitory effects on tumor growth than free ormeloxifene. To conclude, ormeloxifene and its nanoformulation have the potential to be a novel treatment modality for cervical cancer which can reduce the overall disease burden and improve patients’ life expectancy