Browsing by Author "Robertson, John L."

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    7.0-T Magnetic Resonance Imaging Characterization of Acute Blood-Brain-Barrier Disruption Achieved with Intracranial Irreversible Electroporation
    Garcia, Paulo A.; Rossmeisl, John H. Jr.; Robertson, John L.; Olson, JohnD.; Johnson, Annette J.; Ellis, Thomas L.; Davalos, Rafael V. (PLOS, 2012-11-30)
    The blood-brain-barrier (BBB) presents a significant obstacle to the delivery of systemically administered chemotherapeutics for the treatment of brain cancer. Irreversible electroporation (IRE) is an emerging technology that uses pulsed electric fields for the non-thermal ablation of tumors. We hypothesized that there is a minimal electric field at which BBB disruption occurs surrounding an IRE-induced zone of ablation and that this transient response can be measured using gadolinium (Gd) uptake as a surrogate marker for BBB disruption. The study was performed in a Good Laboratory Practices (GLP) compliant facility and had Institutional Animal Care and Use Committee (IACUC) approval. IRE ablations were performed in vivo in normal rat brain (n = 21) with 1-mm electrodes (0.45 mm diameter) separated by an edge-to-edge distance of 4 mm. We used an ECM830 pulse generator to deliver ninety 50-ms pulse treatments (0, 200, 400, 600, 800, and 1000 V/cm) at 1 Hz. The effects of applied electric fields and timing of Gd administration (25, +5, +15, and +30 min) was assessed by systematically characterizing IRE-induced regions of cell death and BBB disruption with 7.0-T magnetic resonance imaging (MRI) and histopathologic evaluations. Statistical analysis on the effect of applied electric field and Gd timing was conducted via Fit of Least Squares with a = 0.05 and linear regression analysis. The focal nature of IRE treatment was confirmed with 3D MRI reconstructions with linear correlations between volume of ablation and electric field. Our results also demonstrated that IRE is an ablation technique that kills brain tissue in a focal manner depicted by MRI (n = 16) and transiently disrupts the BBB adjacent to the ablated area in a voltage-dependent manner as seen with Evan’s Blue (n = 5) and Gd administration.
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    Advancements in Irreversible Electroporation for the Treatment of Cancer
    Arena, Christopher Brian (Virginia Tech, 2013-05-03)
    Irreversible electroporation has recently emerged as an effective focal ablation technique. When performed clinically, the procedure involves placing electrodes into, or around, a target tissue and applying a series of short, but intense, pulsed electric fields. Oftentimes, patient specific treatment plans are employed to guide procedures by merging medical imaging with algorithms for determining the electric field distribution in the tissue. The electric field dictates treatment outcomes by increasing a cell's transmembrane potential to levels where it becomes energetically favorable for the membrane to shift to a state of enhanced permeability. If the membrane remains permeabilized long enough to disrupt homeostasis, cells eventually die. By utilizing this phenomenon, irreversible electroporation has had success in killing cancer cells and treating localized tumors. Additionally, if the pulse parameters are chosen to limit Joule heating, irreversible electroporation can be performed safely on surgically inoperable tumors located next to major blood vessels and nerves. As with all technologies, there is room for improvement. One drawback associated with therapeutic irreversible electroporation is that patients must be temporarily paralyzed and maintained under general anesthesia to prevent intense muscle contractions occurring in response to pulsing. The muscle contractions may be painful and can dislodge the electrodes. To overcome this limitation, we have developed a system capable of achieving non-thermal irreversible electroporation without causing muscle contractions. This progress is the main focus of this dissertation. We describe the theoretical basis for how this new system utilizes alterations in pulse polarity and duration to induce electroporation with little associated excitation of muscle and nerves. Additionally, the system is shown to have the theoretical potential to improve lesion predictability, especially in regions containing multiple tissue types. We perform experiments on three-dimensional in vitro tumor constructs and in vivo on healthy rat brain tissue and implanted tumors in mice. The tumor constructs offer a new way to rapidly characterize the cellular response and optimize pulse parameters, and the tests conducted on live tissue confirm the ability of this new ablation system to be used without general anesthesia and a neuromuscular blockade. Situations can arise in which it is challenging to design an electroporation protocol that simultaneously covers the targeted tissue with a sufficient electric field and avoids unwanted thermal effects. For instance, thermal damage can occur unintentionally if the applied voltage or number of pulses are raised to ablate a large volume in a single treatment. Additionally, the new system for inducing ablation without muscle contractions actually requires an elevated electric field. To ensure that these procedures can continue to be performed safely next to major blood vessels and nerves, we have developed new electrode devices that absorb heat out of the tissue during treatment. These devices incorporate phase change materials that, in the past, have been reserved for industrial applications. We describe an experimentally validated numerical model of tissue electroporation with phase change electrodes that illustrates their ability to reduce the probability for thermal damage. Additionally, a parametric study is conducted on various electrode properties to narrow in on the ideal design.
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    Advancements in the Treatment of Malignant Gliomas and Other Intracranial Disorders With Electroporation-Based Therapies
    Lorenzo, Melvin Florencio (Virginia Tech, 2021-04-19)
    The most common and aggressive malignant brain tumor, glioblastoma (GBM), demonstrates on average a 5-year survival rate of only 6.8%. Difficulties arising in the treatment of GBM include the inability of large molecular agents to permeate through the blood-brain barrier (BBB); migration of highly invasive GBM cells beyond the solid tumor margin; and gross, macroscopic intratumor heterogeneity. These characteristics complicate treatment of GBM with standard of care, resulting in abysmal prognosis. Electroporation-based therapies have emerged as attractive alternates to standard of care, demonstrating favorable outcomes in a variety of tumors. Notably, irreversible electroporation (IRE) has been used for BBB disruption and nonthermal ablation of intracranial tumor tissues. Despite promising results, IRE can cause unintended muscle contractions and is susceptible to electrical heterogeneities. Second generation High-frequency IRE (H-FIRE) utilizes bursts of bipolar pulsed electric fields on the order of the cell charging time constant (~1 μs) to ablate tissue while reducing nerve excitation, muscle contraction, and is far less prone to differences in electrical heterogeneities. Throughout my dissertation, I discuss investigations of H-FIRE for the treatment of malignant gliomas and other intracranial disorders. To advance the versatility, usability, and understanding of H-FIRE for intracranial applications, my PhD thesis focuses on: (1) characterizing H-FIRE-mediated BBB disruption effects in an in vivo healthy rodent model; (2) the creation of a novel, real-time impedance spectroscopy technique (Fourier Analysis SpecTroscopy, FAST) using waveforms compatible with existing H-FIRE pulse generators; (3) development of FAST as an in situ technique to monitor ablation growth and to determine patient-specific ablation endpoints; (4) conducting a preliminary efficacy study of H-FIRE ablation in an orthotopic F98 rodent glioma model; and (5) establishing the feasibility of MRI-guided H-FIRE for the ablation malignant gliomas in a spontaneous canine glioma model. The culmination of this thesis advances our understanding of H-FIRE in intracranial tissues, as well as develops a novel, intraoperative impedance spectroscopy technique towards determining patient-specific ablation endpoints for intracranial H-FIRE procedures.
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    Alterations in the molecular composition of COVID-19 patient urine, detected using Raman spectroscopic/computational analysis
    Robertson, John L.; Senger, Ryan S.; Talty, Janine; Du, Pang; Sayed-Issa, Amr; Avellar, Maggie L.; Ngo, Lacy T.; Gomez de la Espriella, Mariana; Fazili, Tasaduq N.; Jackson-Akers, Jasmine Y.; Guruli, Georgi; Orlando, Giuseppe (PLOS, 2022-07-01)
    We developed and tested a method to detect COVID-19 disease, using urine specimens. The technology is based on Raman spectroscopy and computational analysis. It does not detect SARS-CoV-2 virus or viral components, but rather a urine ‘molecular fingerprint’, representing systemic metabolic, inflammatory, and immunologic reactions to infection. We analyzed voided urine specimens from 46 symptomatic COVID-19 patients with positive real time-polymerase chain reaction (RT-PCR) tests for infection or household contact with test-positive patients. We compared their urine Raman spectra with urine Raman spectra from healthy individuals (n = 185), peritoneal dialysis patients (n = 20), and patients with active bladder cancer (n = 17), collected between 2016–2018 (i.e., pre-COVID-19). We also compared all urine Raman spectra with urine specimens collected from healthy, fully vaccinated volunteers (n = 19) from July to September 2021. Disease severity (primarily respiratory) ranged among mild (n = 25), moderate (n = 14), and severe (n = 7). Seventy percent of patients sought evaluation within 14 days of onset. One severely affected patient was hospitalized, the remainder being managed with home/ambulatory care. Twenty patients had clinical pathology profiling. Seven of 20 patients had mildly elevated serum creatinine values (>0.9 mg/dl; range 0.9–1.34 mg/dl) and 6/7 of these patients also had estimated glomerular filtration rates (eGFR) <90 mL/min/1.73m2 (range 59–84 mL/min/1.73m2). We could not determine if any of these patients had antecedent clinical pathology abnormalities. Our technology (Raman Chemometric Urinalysis—Rametrix®) had an overall prediction accuracy of 97.6% for detecting complex, multimolecular fingerprints in urine associated with COVID-19 disease. The sensitivity of this model for detecting COVID-19 was 90.9%. The specificity was 98.8%, the positive predictive value was 93.0%, and the negative predictive value was 98.4%. In assessing severity, the method showed to be accurate in identifying symptoms as mild, moderate, or severe (random chance = 33%) based on the urine multimolecular fingerprint. Finally, a fingerprint of ‘Long COVID-19’ symptoms (defined as lasting longer than 30 days) was located in urine. Our methods were able to locate the presence of this fingerprint with 70.0% sensitivity and 98.7% specificity in leave-one-out cross-validation analysis. Further validation testing will include sampling more patients, examining correlations of disease severity and/or duration, and employing metabolomic analysis (Gas Chromatography–Mass Spectrometry [GC-MS], High Performance Liquid Chromatography [HPLC]) to identify individual components contributing to COVID-19 molecular fingerprints.
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    The Application of BioHeat Perfusion Sensors to Analyze Preservation Temperature and Quantify Pressure Ischemia of Explanted Organs
    O'Brien, Timothy J. (Virginia Tech, 2015-03-09)
    The development of an organ preservation system (primarily kidneys and livers, but could be adapted to fit hearts, lungs, and even limbs in the future) that can provide surgeons and doctors with real-time quantitative feedback on the health of the organ would be a significant improvement on current transplant practices. This organ transport system will provide surgeons and doctors the opportunity to make more educated decisions towards whether or not to proceed with organ transplantation. Here, we discuss the use Smart Perfusion's organ preservation system as a platform for determining the optimal perfusion temperature of an organ. Porcine kidneys were procured and perfused with a modified PBS solution on the Vasowave™. While on this organ preservation system, a heart emulating pressure waveform (90/50 mmHg) was generated and sent to the specimen. The pressure response, flow rate, temperature, pH, dissolved oxygen content, and conductivity of the fluid stream were all monitored throughout the duration of experimentation. In addition to inline sensors, IR imaging captured the surface temperature of the organ while on the system. Lastly, the use of a combined heat flux-temperature (CHFT) sensor, previously developed at Virginia Tech, was applied for the first time to monitor and measure local tissue perfusion of an explanted organ. A total of 12 experiments were performed (6 at a set fluid temperature of 15°C, and 6 at 20°C). All system data was collected, statistically evaluated and finally compared against blind histological readings (taken at the termination of each experiment at the hilum and pole) to investigate the effects of temperature on organ vasculature. The results of this experiment indicated that the effects of temperature on explanted kidneys can be affectively measured using a non-invasive bioheat perfusion sensor. Specifically, the lower temperature group of kidneys was measured to have lower perfusion. Furthermore, an enhancement to the CHFT sensor technology (CHFT+) was developed and tested for compliance. A controllable thin filmed heat resistor was added to the CHFT assembly to replace the current convective thermal event. This enhancement improved the measured heat flux and temperature signals and enables autonomy. Also, the thin and semi-flexible nature of the new CHFT+ sensor allows for perfusion measurements to be taken from the underside of the organ, permitting a quantitative measure of pressure ischemia. Results from a live tissue test illustrated, for the first time, the effects of pressure ischemia on an explanted porcine kidney.
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    The Applications of Raman Spectroscopy Assisted Urinalysis: Hematuria and Bladder Cancer Detection
    Carswell, William Forrester (Virginia Tech, 2020-10-29)
    Early detection and screening for urinary tract illnesses is a complex and widespread process which has implications for both preventative care, diagnosis, and treatment monitoring. In this paper, we investigate the use of Raman spectroscopy (RS) for the analysis of urine, a complex biological solution, for the detection of bladder cancer (BCa) and hematuria. Raman spectroscopy is a rapid, low cost, non-destructive analysis method with wide-ranging applicability due to the holistic data capturing nature of the scanning technique. Each Raman scan can be considered a 'snapshot' of the molecular makeup of the sample, and, through proper applications of algorithmic transformation and statistical analysis, many types of assessments can be performed on each sample. In this paper we address creating and utilizing a data pipeline for the purposes of analyzing and characterizing potential samples with hematuria and BCa. The algorithmic transformations utilized include baselining using either the Goldindec or ISREA methods, and intensity normalization. The statistical analysis methods utilized include principal component analysis (PCA), discriminant analysis of principal components (DAPC), analysis of variance (ANOVA), pairwise ANOVA, leave-one-out cross-validation (LOOCV), and partial least squares regression (PLSR). These components of the data pipeline serve to output qualitative or quantitative data, depending on the application. The Rametrix toolbox encompasses the tools required to transform and assess Raman spectra with PCA and DAPC. Using the Rametrix toolbox as well as ANOVA, pairwise ANOVA, and LOOCV, we were able to significantly detect the presence of bladder cancer in a specimen with 80% accuracy. Using the Rametrix toolbox, ANOVA, pairwise ANOVA, LOOCV, and PLSR, we were able to classify samples as pure urine, micro-, or macrohematuria with a greater than 91% accuracy, and quantify the amount of blood in the sample with a high correlation (R-squared value of 0.92). In combination, this style of data pipeline is shown to rapidly and accurately test for multiple symptoms or diseases using similar methodologies.
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    Atrial natriuretic peptide and streptozotocin-induced diabetes in rats
    Black, Leslie Seale (Virginia Tech, 1991-04-17)
    This study was undertaken to determine whether immunoreactive atrial natriuretic peptide (irANP) concentrations in plasma and atrial tissue are altered in experimental diabetes mellitus (DM), and to compare the response of the DM and normal groups to exogenous administration of ANP. OM was induced by intraperitoneal injection of 45 mg/kg streptozotocin in male Sprague-Dawley rats. After three weeks of established OM (glucosuria and blood glucose> 250 mg/dl), plasma irANP levels were 149.6 ± 19.4 pg/ml in the OM group (n = 18) and 86.3 + 12.9 pg/ml in the normal group en = 12, P <0.01). Atrial tissue irANP levels were significantly lower in the OM group (38.1 ± 7.8 ng/mg, n = 7) than in the normal group (60.1 ± 1.3 ng/mg, n = 4, P < 0.02). In response to intravenous infusion of ANP (2.5 ug/kg prime, followed by 0.1 ug/kg/min for 30 minutes), urine flow rate and urine sodium and potassium excretion rates increased significantly in the normal group (n = 6, P < 0.05), while no significant responses were found in the OM group (n = 6). It is concluded that plasma levels of ANP are significantly elevated in streptozotocin-induced diabetes in rats, and that atrial tissue stores are significantly depleted in this diabetic model. In addition, the renal response to exogenously administered ANP appears to be diminished in streptozotocin-induced OM.
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    Bacterial Kidney Disease and Its effect on the Salmonid Immune response
    Densmore, Christine L. (Virginia Tech, 1997-11-04)
    Renibacterium salmoninarum, the etiological agent of bacterial kidney disease (BKD) of salmonid fish, is a pathogen of great concern among fisheries and the aquaculture industry worldwide. Previous investigations have indicated the pathogenesis of BKD is complex. It is a chronic, multisystemic, granulomatous disease with a number of potential immunomodulatory effects on the host. Given the current limitations for treatment and control of BKD, it is imperative that the pursuit of development of methods of prevention, namely management strategies and vaccination, be continued. To do so, the immunology of BKD must be elucidated in order to better understand and manipulate the associated immune responses to our advantage. This dissertation is composed of four chapters which relate to BKD and the associated immune responses of three species of susceptible salmonid fish as follows: Exogenous stress factors, through stress-induced immunosuppression, have been shown to influence BKD development in cultured salmonids. Chapter 1 examines the effects of two environmental stressors common to fish culture, overcrowding and overfeeding, as they affect BKD development and R. salmoninarumantigen prevalence among juvenile chinook salmon (Oncorhynchus tshawytscha) Immunomodulatory interaction between pathogen and host in BKD is widely reported and merits further investigation. Particularly, the immunological parameters affected and the role of the extracellular protein (ECP) of R. salmoninarum are of interest. Chapter 2 examines the in vivo immune response of rainbow trout (Oncorhynchus mykiss) following exposure to the ECP in terms of both humoral and cell-mediated immunological parameters, including the immune response against another bacterial pathogen. Chapter 3 addresses the in vitro effects of the ECP upon specific splenic immunocyte functions, phagocytosis and respiratory burst activity, in brook trout (Salvelinus fontinalis). The immune-complex mediated hypersensitivity reported to occur with BKD has considerable ramifications for control measures involving immunostimulation via antigen exposure. Further investigation is warranted to discern the significance and consistency of immunological hypersensivity in BKD pathogenesis. Chapter 4 examines the renal lesions, including immunopathologic changes and indications of immune-mediated disease, of brook trout exposed to R. salmoninarum.
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    Biocompatibility of the fiberoptic microneedle device chronically implanted in the rat brain
    Kani, Yukitaka; Hinckley, Jonathan; Robertson, John L.; Mehta, Jason M.; Rylander, Christopher G.; Rossmeisl, John H. Jr. (Elsevier, 2022-03)
    The fiberoptic microneedle device (FMD) is a fused-silica microcatheter capable of co-delivery of fluids and light that has been developed for convection-enhanced delivery and photothermal treatments of glioblastoma. Here we investigate the biocompatibility of FMD fragments chronically implanted in the rat brain in the context of evaluating potential mechanical device failure. Fischer rats underwent craniectomy procedures for sham control (n & nbsp;= 16) or FMD implantation (n = 16) within the brain. Rats were examined daily after implantation, and at 14, 30, 90, and 180 days after implantation were evaluated via computed tomography of the head, hematologic and blood biochemical profiling, and necropsy examinations. Clinical signs of illness and distant implant migration were not observed, and blood analyses were not different between control and FMD implanted groups at any time. Mild inflammatory and astrogliotic reactions localized to the treatment sites within the brain were observed in all groups, more robust in FMD implanted groups compared to controls at days 30 and 90, and decreased in severity over days 90-180 of the study. One rat developed a chronic, superficial surgical site pyogranuloma attributed to the FMD silica implant. Chronically implanted FMD fragments were well tolerated clinically and resulted in anticipated mild, localized brain tissue responses that were comparable with other implanted biomaterials in the brain.
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    Biomanufacturing of Bacteria-Mediated Drug Delivery Systems and Investigation of Their Interaction with the Tumor Microenvironment
    Zhan, Ying (Virginia Tech, 2024-05-14)
    The limited transport of conventional chemotherapy within the tumor microenvironment (TME) is due to irregular vascularization, increased tumor interstitial pressure, and a dense extracellular matrix (ECM). The lack of selectivity of anticancer drugs often leads to systemic toxicity and damage to healthy tissues. Bacteria-based cancer therapy (BBCT) is a promising alternative, as tumor-targeting bacteria have been shown to preferentially colonize primary and metastatic tumors and induce anti-tumor effects. In this dissertation, we focus on several aspects of bacteria-nanoparticle conjugates, wherein BBCT is synergistically combined with nanomedicine to augment the efficacy of both treatment modalities. We explore biofabrication of our bacteria-nanoparticle conjugates called NanoBEADS (Nanoscale Bacteria Enabled Autonomous Drug Delivery Systems) and their interaction with the TME. Specifically, (1) we investigate the effects of two bacteria-NP conjugation chemistry and assembly process parameters of mixing method, volume, and duration, on NP attachment density and repeatability. We evaluate the influence of linkage chemistry and NP size on NP attachment density, viability, growth rate, and motility of NanoBEADS. (2) We investigate the effect of dense stroma and ECM production on the intratumoral penetration of bacteria with a mathematical model of bacterial intratumoral transport and growth. (3) We develop a microfluidic device with multicellular tumor spheroids to study the transport of tumor-targeting bacteria and support real-time imaging and long-term experiments. (4) We develop a new type of bacteria-based bio-hybrid drug delivery system using engineered cell surface display for enhancing the attachment of nanoparticles.
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    Bursts of Bipolar Microsecond Pulses Inhibit Tumor Growth
    Sano, Michael B.; Arena, Christopher B.; Bittleman, Katelyn Rose; DeWitt, Matthew R.; Cho, Hyung J.; Szot, Cchristopher S.; Saur, Dieter; Cissell, James M.; Robertson, John L.; Lee, Yong Woo; Davalos, Rafael V. (Nature Publishing Group, 2015-10-13)
    Irreversible electroporation (IRE) is an emerging focal therapy which is demonstrating utility in the treatment of unresectable tumors where thermal ablation techniques are contraindicated. IRE uses ultra-short duration, high-intensity monopolar pulsed electric fields to permanently disrupt cell membranes within a well-defined volume. Though preliminary clinical results for IRE are promising, implementing IRE can be challenging due to the heterogeneous nature of tumor tissue and the unintended induction of muscle contractions. High-frequency IRE (H-FIRE), a new treatment modality which replaces the monopolar IRE pulses with a burst of bipolar pulses, has the potential to resolve these clinical challenges. We explored the pulse-duration space between 250 ns and 100 μs and determined the lethal electric field intensity for specific H-FIRE protocols using a 3D tumor mimic. Murine tumors were exposed to 120 bursts, each energized for 100 μs, containing individual pulses 1, 2, or 5 μs in duration. Tumor growth was significantly inhibited and all protocols were able to achieve complete regressions. The H-FIRE protocol substantially reduces muscle contractions and the therapy can be delivered without the need for a neuromuscular blockade. This work shows the potential for H-FIRE to be used as a focal therapy and merits its investigation in larger pre-clinical models.
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    Canine Butterfly Glioblastomas: a Neuroradiological Review
    Rossmeisl, John H. Jr.; Clapp, Kemba; Pancotto, Theresa E.; Emch, Samantha; Robertson, John L.; Debinski, Waldemar (Frontiers, 2016-05-19)
    In humans, high-grade gliomas may infiltrate across the corpus callosum resulting in bihemispheric lesions that may have symmetrical, winged-like appearances. This particular tumor manifestation has been coined a “butterfly” glioma (BG). While canine and human gliomas share many neuroradiological and pathological features, the BG morphology has not been previously reported in dogs. Here, we describe the magnetic resonance imaging (MRI) characteristics of BG in three dogs and review the potential differential diagnoses based on neuroimaging findings. All dogs presented for generalized seizures and interictal neurological deficits referable to multifocal or diffuse forebrain disease. MRI examinations revealed asymmetrical (2/3) or symmetrical (1/3), bihemispheric intra-axial mass lesions that predominantly affected the frontoparietal lobes that were associated with extensive perilesional edema, and involvement of the corpus callosum. The masses displayed heterogeneous T1, T2, and fluid-attenuated inversion recovery signal intensities, variable contrast enhancement (2/3), and mass effect. All tumors demonstrated classical histopathological features of glioblastoma multiforme (GBM), including glial cell pseudopalisading, serpentine necrosis, microvascular proliferation as well as invasion of the corpus callosum by neoplastic astrocytes. Although rare, GBM should be considered a differential diagnosis in dogs with an MRI evidence of asymmetric or symmetric bilateral, intra-axial cerebral mass lesions with signal characteristics compatible with glioma.
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    Canine Cancer Screening Via Ultraviolet Absorbance And Fluorescence Spectroscopy Of Serum Proteins
    Dickerson, Bryan Douglas; Geist, Brian L.; Spillman, William B. Jr.; Robertson, John L. (Optical Society of America, 2007-01-01)
    A cost-effective optical cancer screening and monitoring technique was demonstrated in a pilot study of canine serum samples and was patented for commercialization. Compared to conventional blood chemistry analysis methods, more accurate estimations of the concentrations of albumin, globulins, and hemoglobin in serum were obtained by fitting the near UV absorbance and photoluminescence spectra of diluted serum as a linear combination of component reference spectra. Tracking these serum proteins over the course of treatment helped to monitor patient immune response to carcinoma and therapy. For cancer screening, 70% of dogs with clinical presentation of cancer displayed suppressed serum hemoglobin levels (below 20 mg/dL) in combination with atypical serum protein compositions, that is, albumin levels outside of a safe range (from 4 to 8 g/dL) and globulin levels above or below a more normal range (from 1.7 to 3.7 g/dL). Of the dogs that met these criteria, only 20% were given a false positive label by this cancer screening test. (C) 2007 Optical Society of America.
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    Cell Death Characterization In Tumor Constructs Using Irreversible Electroporation
    Prokop, Katherine Jane (Virginia Tech, 2013-10-04)
    Pancreatic and prostate cancer are both prevalent cancers in the United States with pancreatic being one of the most aggressive of all cancers and prostate cancer being one of the most common, ranking as the number one cancer in men. Treatment of both cancers can be quite challenging as the anatomy of the pancreas and prostate, as well as the development and diagnosis of the disease can greatly limit treatment options. Therefore, it is necessary to develop new cancer treatments to help manage and prevent these cancers. Irreversible electroporation is a new non-thermal focal ablation therapy utilizing short, pulsed electric fields to damage cell membranes leading to cell death. The therapy is minimally invasive, involving the insertion of needle electrodes into the region of interest and lasts less than two minutes. Heat sink effects that thermal therapies experience near large blood vessels do not affect irreversible electroporation. This allows the treatment to be used on tumors near vasculature as well as critical structures without harming these vital regions. While irreversible electroporation is a promising new cancer therapy, further developments are necessary to improve treatment planning models. This work aims to further understand the electric field thresholds necessary to kill different types of cancer cells with a focus on pancreatic and prostate cancer. The work is done using an in vitro tumor (hydrogel) model as this model is better than traditional cell suspension studies, with added benefits over the immediate use of tissue and animal models.
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    Characterization of Apoptotic Cells in Equine Proximal Suspensory Desmitis
    Hewes, Christina Andrea (Virginia Tech, 2006-05-30)
    Suspensory desmitis is a common problem and affects a broad cross section of equine athletes in various disciplines. For this study, the proximal portion of the suspensory ligament was collected from 6 horses without suspensory ligament injury (16 ligaments) and 4 horses with degeneration of the suspensory ligament (11 ligaments). Specimens were collected immediately after euthanasia and placed in neutral-buffered 10% formalin. The tissue was fixed, sectioned, and stained with hematoxylin and eosin (H&E), Masson's trichrome, and for apoptosis by the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique. Histological changes in the abnormal ligaments included mineralization, fibroplasias, neovascularization, collagen degeneration, and significant architecture disruption in 2 ligaments. There was a trend for increased apoptosis in the injured ligaments compared to the normal ligaments.
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    Characterization of the Beta-2 Adrenergic Receptor Mechanism in Bovine Neutrophils, and Some Effects of Inflammatory Stimuli on its Function
    LaBranche, Timothy Paul (Virginia Tech, 2005-04-12)
    The bovine polymorphonuclear leukocyte (neutrophil) is a central component of the acute inflammatory response, and is capable of reacting to a myriad of pro-inflammatory chemical signals that have been characterized in the context of bovine respiratory disease (BRD). Human neutrophils and bovine macrophages are known to react to pro-inflammatory signals as well; however, they are also capable of responding to anti-inflammatory signals from the autonomic nervous system. In particular, activation of the beta2-adrenergic receptor on these cells decreases several aspects of inflammatory activity, including reactive oxygen species production, chemotaxis, degranulation, and inflammatory mediator production. Dysfunction of beta-adrenergic receptors is known to contribute to the pathophysiology of numerous diseases in both people and animals. For example, congestive heart failure, asthma, cystic fibrosis, atopic dermatitis, pheochromocytoma, myasthenia gravis, hypertension, and sepsis have all been linked to decreased beta1- / beta2-adrenergic receptor density (depending on the cell type) and / or uncoupling of the respective receptor from its effector enzyme, adenylyl cyclase. Dysfunction of the beta2-adrenergic receptor mechanism has also been described in pulmonary airway and vascular smooth muscle tissue from cattle, sheep, and rats exposed to Manheimia haemolytica, which provides insight into the pathophysiology of BRD. Despite the prominent role of the bovine neutrophil in the acute inflammatory stage of BRD, and despite the potential for dysfunction following excessive exposure to inflammatory stimuli, there are no reports that describe the presence of the beta2-adrenergic receptor on bovine neutrophils, nor function of the components responsible for its signal transduction cascade. Without complimentary work with bovine neutrophils, using data from human neutrophils to examine treatment options for the acute inflammatory stage of BRD is unrealistic. For this reason, the present dissertation proposed that 1) bovine neutrophils possess the beta2-adrenergic receptor mechanism, 2) components of the beta2-adrenergic receptor mechanism work in concert to increase bovine neutrophil adenosine 3,5-cyclic monophosphate (cAMP) levels and suppress superoxide anion production, and 3) the beta2-adrenergic receptor mechanism is dysfunctional following exposure to inflammatory stimuli. Using the nonselective beta1- / beta2-adrenergic receptor antagonist [3H]CGP-12177 we observed a maximum specific binding density (Bmax) value of 0.19 fmol per 100,000 bovine neutrophils. Although this value is approximately equal to what we observed with dairy cow neutrophils, human neutrophil Bmax values with this radioligand are anywhere from five to ten-fold greater, which suggests a significant species difference. We further defined the adrenergic receptor population on bovine neutrophils to be dominated by the beta2-subtype. Next, we characterized the function of beta2-adrenergic receptors by stimulating cAMP production with the beta2-adrenergic receptor agonist, terbutaline. The role of the beta2-subtype was confirmed when the terbutaline-mediated effect was negated by ICI-118,551, a beta2-adrenergic receptor antagonist. Also, the role of the phosphodiesterase enzyme in cAMP recycling in bovine neutrophils was illustrated, as the terbutaline-mediated rise in cAMP concentration was dependent upon phosphodiesterase inhibition by 3-isobutyl-1-methylxanthine (IBMX). This study confirms the anti-inflammatory nature of the beta2-adrenergic receptor on bovine neutrophils by demonstrating the ability of terbutaline and IBMX to decrease superoxide anion production in a dose-dependent manner. The synthetic cAMP analog, 8-bromo-cAMP also decreased superoxide anion production, but the effect was time-dependent because of its need to diffuse across the cell membrane. Moreover, IBMX exaggerated the terbutaline-mediated effect on superoxide anion production, while cAMP exaggerated the IBMX-mediated effect on superoxide anion, demonstrating that the beta2-adrenergic receptor acts in concert with adenylyl cyclase, while the phosphodiesterase enzyme functions to decrease their signal. By increasing the dose of the inflammatory stimulant opsonized zymosan eight-fold, we were able to eliminate the ability of various concentrations of terbutaline and IBMX to reduce superoxide anion production. We sought to provide a more specific demonstration of this phenomenon by activating protein kinase C (PKC) via phorbol 12-myristate 13-acetate (PMA) administration. However, preincubation with PMA actually increased terbutaline-mediated cAMP production, in a dose and time-dependent manner. At this time, we cannot explain why increasing the dose of opsonized zymosan and PMA had opposite effects on beta2-adrenergic receptor mechanism function. The answer may reside in the many reported functions of PKC isoforms. Additional studies that identify the PKC isoform repertoire in bovine neutrophils may illustrate the potential for selective inhibition, and may lead to more specific identification and treatment of beta2-adrenergic receptor mechanism dysfunction. Also, it remains to be seen how the various components of the bovine neutrophil beta2-adrenergic receptor mechanism function in-vivo during the acute inflammatory stage of BRD.
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    A Combined In Vivo and In Vitro Approach to the Study of Endotoxemia in Swine
    Smedley, Jeremy Vance (Virginia Tech, 2000-06-06)
    The cardiopulmonary effects of endotoxin administration (1 microgram/kg) were evaluated in 8-10 week old SPF-derived Yorkshire pigs, both because endotoxemia is a common and important swine problem, and because the pig is a good model for human adult respiratory distress syndrome. Physiological changes included sustained increases in mean pulmonary artery pressure, pulmonary vascular resistance, pulmonary arterial wedge pressure, heart rate, hematocrit, and the arterial partial pressure of carbon dioxide. Transient increases were also observed in central venous pressure and airway pressure. Transient increases, followed by decreases, were observed in mean systemic arterial pressures and systemic vascular resistance. Decreases were seen in cardiac output, cardiac index, arterial partial pressure of oxygen and oxygen saturation. The number of circulating leukocytes, lymphocytes and segmented neutrophils decreased with endotoxin infusion. To investigate the role of airway smooth muscle, bronchial rings were isolated and exposed to contractile agents in tissue baths. A hyperresponsiveness of the third generation bronchi to substance P, carbachol, bradykinin and electric field stimulation was observed. However the increase in response to bradykinin and electric field stimulation were not statistically significant. Histopathology of the lungs demonstrated congestion, hemorrhage and neutrophilic infiltration.
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    Comparison of linear and volumetric criteria for the determination of therapeutic response in dogs with intracranial gliomas
    Garcia Mora, Josefa Karina; Robertson, John L.; Hsu, Fang-Chi; Shinn, Richard Levon; Larson, Martha M.; Rylander, Christopher G.; Whitlow, Christopher T.; Debinski, Waldemar; Davalos, Rafael V.; Daniel, Gregory B.; Rossmeisl, John H. Jr. (Wiley, 2022-05)
    Background: Brain tumor therapeutic responses can be quantified from magnetic resonance images (MRI) using 1- (1D) and 2-dimensional (2D) linear and volumetric methods, but few studies in dogs compare these techniques. Hypotheses: Linear methods will be obtained faster, but have less agreement than volumetric measurements. Therapeutic response agreement will be highest with the total T2W tumor volumetric (TTV) method. Therapeutic response at 6-weeks will correlate with overall survival (OS). Animals: Forty-six dogs with intracranial gliomas. Methods: Prospective study. Three raters measured tumors using 1D and 2D linear, TTV, and contrast-enhancing volumetric (CEV) techniques on 143 brain MRI to determine agreement between methods, define therapeutic responses, and assess relations with OS. Results: Raters performed 1D the fastest (2.9 ± 0.57 minutes) and CEV slowest (17.8 ± 6.2 minutes). Inter- and intraobserver agreements were excellent (intraclass correlations ≥.91) across methods. Correlations between linear (1D vs 2D; ρ >.91) and volumetric (TTV vs CEV; ρ >.73) methods were stronger than linear to volumetric comparisons (ρ range,.26-.59). Incorporating clinical and imaging data resulted in fewer discordant therapeutic responses across methods. Dogs having partial tumor responses at 6 weeks had a lower death hazard than dogs with stable or progressive disease when assessed using 2D, CEV, and TTV (hazard ration 2.1; 95% confidence interval, 1.22-3.63; P =.008). Conclusions and Clinical Importance: One-dimensional, 2D, CEV, and TTV are comparable for determining therapeutic response. Given the simplicity, universal applicability, and superior performance of the TTV, we recommend its use to standardize glioma therapeutic response criteria.
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    Complexity, fractals, disease time, and cancer
    Spillman, William B. Jr.; Robertson, John L.; Huckle, William R.; Govindan, B. S.; Meissner, Kenith E. (American Physical Society, 2004-12)
    Despite many years of research, a method to precisely and quantitatively determine cancer disease state remains elusive. Current practice for characterizing solid tumors involves the use of varying systems of tumor grading and staging and thus leaves diagnosis and clinical staging dependent on the experience and skill of the physicians involved. Although numerous disease markers have been identified, no combination of them has yet been found that produces a quantifiable and reliable measure of disease state. Newly developed genomic markers and other measures based on the developing sciences of complexity offer promise that this situation may soon be changed for the better. In this paper, we examine the potential of two measures of complexity, fractal dimension and percolation, for use as components of a yet to be determined "disease time" vector that more accurately quantifies disease state. The measures are applied to a set of micrographs of progressive rat hepatoma and analyzed in terms of their correlation with cell differentiation, ratio of tumor weight to rat body weight and tumor growth time. The results provide some support for the idea that measures of complexity could be important elements of any future cancer "disease time" vector.
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    Conformal Additive Manufacturing for Organ Interface
    Singh, Manjot (Virginia Tech, 2017-06-08)
    The inability to monitor the molecular trajectories of whole organs throughout the clinically relevant ischemic interval is a critical problem underlying the organ shortage crisis. Here, we report a novel technique for fabricating manufacturing conformal microfluidic devices for organ interface. 3D conformal printing was leveraged to engineer and fabricate novel organ-conforming microfluidic devices that endow the interface between microfluidic channels and the organ cortex. Large animal studies reveal microfluidic biopsy samples contain rich diagnostic information, including clinically relevant biomarkers of ischemic pathophysiology. Overall, these results suggest microfluidic biopsy via 3D printed organ-conforming microfluidic devices could shift the paradigm for whole organ preservation and assessment, thereby relieving the organ shortage crisis through increased availability and quality of donor organs.