Background: Medical errors are a danger to patient safety and a significant cause of morbidity and mortality. Additionally, they increase expenditures in an already significantly indebted U.S. health care system. Much confusion exists about definitions of medical errors, which include medication errors and adverse drug events (ADEs). Several federal and international organizations have attempted to standardize definitions in order to streamline data collection, but until these standards are universally adopted, error reports and trends are still subject to questions of validity. Reporting errors, in general, has become a more socially acceptable practice in health care with the advent of several anonymous reporting databases. There have also been several initiatives aimed at reducing the incidence of errors, which range from national programs to intrafacility guidelines. Several pieces of health information technology (HIT) have made an impact on error incidence and data collection, although there is much room for improvement. Patient controlled analgesia (PCA) pumps for pain management have been in existence for decades, and "smart pump" software has improved their safety and ease of programming. PCA use in children presents challenges to clinicians, and the characteristics of providers who write PCA orders and those who program PCA pumps may play a role in the incidence of events related to PCA. This study seeks to elucidate trends in errors as they related to these different PCA providers in a pediatric hospital in the northeastern U.S. and provide recommendations for how PCA practice can be improved in this facility. Methods: Safety Event Reporting System (SERS) reports of PCA events (n = 117) during the period of 2004 - 2012 were analyzed retrospectively to determine several key variables for data analysis. The main focus of this analysis was those variable trends related to providers, including: proportion of events caused by human error, proportion of events related to subcategories of human error, proportion of types of prescribers involved in PCA events, proportion of errors in medical and surgical patients, proportion of errors occurring on day and night shifts for the nursing staff, and proportion of events that were dosing mistakes. Statistical analysis was performed for these results when possible to determine significance. Results: Human errors were implicated in 84.1% of events, whereas PCA pump mechanical errors and software errors were implicated in 7.1% and 7.9% of events, respectively. Statistically significant differences were found in all variables tested, including the proportion of nursing errors (60.9%) versus prescriber errors (28.7%) (p < 0.0002). For types of prescribers, the proportion of PCA events occurring when a M.D. wrote the PCA order (56.41%) was statistically different than when a N.P. wrote the PCA order (39.32%) (p = 0.0129). More surgical patients (61.5%) were affected by PCA events than medical patients (36.8%) (p < 0.0002). There were more events occurring on the nursing staff day shift (59.8%) than the night shift (36.8%) (p = 0.0004). Finally, dosing mistakes (66.7%) were implicated in significantly more PCA events than any other error type (33.3%) (p < 0.0002). Conclusion: Several recommendations for improving the safety of PCA in pediatric pain management are justified by the results of this data analysis. First, further education and simulation for entering PCA orders into the CPOE system is needed for all prescribers. Secondly, further education and simulation in PCA pump programming and system set-up is needed for all nursing staff members. In regard to prescriber credentials, it is recommended that Pain Treatment Service (PTS) staff members train M.D. residents in writing PCA orders and entering them into the CPOE system. Finally, it is recommended that the SERS management team publish standardized error report content and entry format in order to streamline data analysis for quality improvement (QI) purposes.

<p> Human ALS patient samples can be converted into motor neurons for the study of ALS pathogenesis <i>in vitro.</i> A major challenge, however is the lack of maturation of the stem cell-derived tissue, representing a major barrier to therapy development. The body of work herein details a series of studies to interrogate the relative maturation of induced pluripotent stem cell derived MNs and determine the effects of brain microvascular endothelial cells on their maturation. Finally, a novel microphysiological system for the modeling of ALS and the blood brain barrier is developed that includes the co-culture of brain microvascular endothelial cells under continuous flow of media with a focus on motor neuron function and vascular infiltration. </p>

BACKGROUND: Retinal capillary basement membrane thickening is closely associated with the development of vascular lesions in diabetic retinopathy. Thickened capillary basement membrane can compromise blood-retinal-barrier (BRB) characteristics and contribute to retinal vascular permeability, a significant clinical manifestation of diabetic retinopathy. We have previously shown that high glucose (HG) increases the expression and activity of lysyl oxidase (LOX), a crosslinking enzyme, in retinal endothelial cells. Additionally, concomitant with overexpression of LOX, increased vascular permeability was observed in diabetic rat retinas. However, it is unknown whether decreasing LOX overexpression may have protective effects against development of retinal vascular lesions associated with diabetic retinopathy. OBJECTIVE: To investigate whether reducing LOX level protects against diabetes-induced development of retinal vascular lesions characteristic of diabetic retinopathy METHODS: In Experiment 1, wild type (WT) control mice, streptozotocin (STZ)-induced diabetic mice, LOX heterozygous knockout (LOX +/-) mice, and STZ-induced diabetic LOX +/- mice were used in the study. In Experiment 2, WT rats, diabetic rats, and diabetic rats intravitreally injected with LOX short interfering RNA (siRNA), or scrambled siRNA as control, were used in the study. 1 month after the onset of diabetes, intravitreal injections were initiated at monthly intervals for up to 3 times. At the end of the study, retinas were assessed for LOX protein level by Western Blot (WB) analysis, and retinal capillary networks were assessed for the number of acellular capillaries (AC) and pericyte loss (PL). Vascular leakage was analyzed by measuring the extravasation of FITC-dextran in retinal capillaries after tail vein injection of FITC-Dextran. RESULTS: A significant increase in LOX expression was detected in the diabetic retinas compared to those of the WT control retinas while a significant decrease in LOX expression was observed in the diabetic LOX +/- retinas and in the diabetic retinas injected with LOX siRNA. Importantly, when diabetes-induced LOX overexpression was reduced, retinal vascular cell loss and vascular leakage were ameliorated. CONCLUSION: Decreasing diabetes-induced LOX overexpression may have protective effects against the development of vascular lesions characteristic of diabetic retinopathy. Therefore, LOX overexpression may be a potential target in preventing retinal vascular cell loss and excess permeability associated with diabetic retinopathy.

INTRODUCTION: Trauma to the musculoskeletal system can result in heterotopic ossification, a condition where aberrant bone tissue is synthesized and mineralized in the soft tissues of the body. Satellite cells expressing Pax7 are the predominant stem cell population found within adult skeletal muscle tissues. Once activated by trauma, satellite cells are primarily implicated in skeletal muscle regeneration by differentiating towards myocytes. Previous research in the lab has shown that no Pax7 derived cells were seen in either the fracture callus or periosteal ectopic bone induced by demineralized bone matrix (DBM). Questions however persist whether trauma can activate Pax7 cells to contribute to ectopic bone formation and whether muscle trauma will enhance the ability of DBM to induce ectopic bone in muscle. OBJECTIVES: Identify how muscle trauma effects DBM-induced ectopic bone formation and characterize the contribution of the Pax7 satellite cell population in DBM-induced ectopic bone formation after muscle trauma. METHODS: The tamoxifen inducible Pax7^tm1(cre/ER2)Gaka/J transgenic mice were crossed with B6.Cg-Gt(ROSA)26sor<tm14(CAG-tdTomato)Hze>/J to create Pax7/Ai14 reporter. These animals were subsequently crossed with B6,129S7-Rag1^tm1Mom/J mice. This created a transgenic reporter mouse that allows for the implantation of human DBM. Two tamoxifen doses (within 48 hours) were given to the animals approximately 31 days prior to surgery. Ectopic bone was induced by surgical implantation of DBM (50 mg) with 0.1 µg of bone morphogenic protein 2 (BMP-2) on the femoral periosteum or in the skeletal muscle tissue of the upper hind limb. Following implantation, mice received varying amounts of blunt force trauma to induce skeletal muscle trauma. Ectopic bone was then evaluated radiologically using plain film and micro-computed tomography, and histologically through fluorescence and brightfield microscopy. Micro-computed tomography allowed for the calculation of the ectopic bone volume, as well as the creation of 3D renderings of the ectopic bone. Fluorescence microscopy allowed for the visualization of recruited Pax7 positive satellite cells to the DBM-induced ectopic bone. Trichrome staining techniques allowed for the visualization and categorization of tissue types including skeletal muscle, un-mineralized and mineralized bone, and cartilage. RESULTS: Muscle trauma did not significantly change the volume of ectopic bone that was induced by BMP-2 supplemented DBM that was implanted on either the periosteum or in skeletal muscle. However, the amount of BMP2 that was needed within DBM to induce ectopic bone within muscle was greatly decreased with muscle trauma. Intriguingly, muscle trauma resulted in the activation and recruitment of Pax7 positive cells to the DBM-induced ectopic bone in both periosteal and skeletal muscle implants. CONCLUSIONS: Skeletal muscle trauma does not appear to impact the resulting bone volume of BMP2 supplemented DBM induced ectopic bone formation. However, the decreased dose of BMP-2 that was needed to induce ectopic bone formation within muscle suggests that trauma sensitized the stem cell populations that contribute to ectopic bone to BMP induction. The appearance of Pax7 within the newly formed ectopic bone with muscle trauma suggests that the muscle trauma effects the plasticity of Pax7 satellite enabling them to contribute to ectopic bone formation. Further research is needed to elucidate the molecular mechanism(s), which muscle trauma activates that favor ectopic bone formation and promotes the plasticity of Pax7 muscle satellite cells. These studies provide basis for the identification of novel therapeutic targets to treat heterotopic ossification.

Lung adenocarcinoma (LUAD) is a subtype of non small-cell lung cancer that has been characterized by late stage diagnosis and poor clinical outcomes. Recent advances in tumor genotyping and precision therapies have shifted the landscape of LUAD management, drastically improving outcomes for a large subset of patients. Specific tyrosine kinase inhibitors have been developed to target oncogenic aberrations harbored by different molecular subtypes of LUAD. Although many of these targeted therapies have proven to be more efficacious than traditional chemotherapy, the constant battle against acquired resistance has limited their success. This has warranted the development of second- and third-generation tyrosine kinase inhibitors, chronically directed at more and more specific targets. This review focuses on the eight most frequent LUAD oncogenic drivers and the effectiveness of their associated targeted therapies. The goal of this thesis is to evaluate each molecular target as a candidate for precision medicine, identify targeted therapies which hold the most promise, and characterize the current state of LUAD precision therapy. By comparing progression free survival rates, safety profiles, and resistance data for each genetic aberration, it was concluded that epidermal growth factor receptor (EGFR) and ROS proto-oncogene 1 (ROS1) directed therapies hold the most promise for improving clinical outcomes for EGFR mutant-positive and ROS1 fusion-positive patient populations. Though curative options will likely not be seen in the near future, the progress made by precision medicine is encouraging. By focusing further research on elucidating resistance mechanisms, identifying novel oncogenic drivers, and trialing combination therapies, patient outcomes can continue to improve.

Clostridium difficile infection (CDI) is the most common hospital-associated infection worldwide and is a significant threat in public health, causing morbidity, mortality, and healthcare costs. It is caused by C. difficile, ubiquitous, gram-positive, spore-forming bacteria that colonize in patients with a disruption in normal intestinal microflora. In the past, antibiotics were recommended as standard therapies. However, as treatments that involve the use of antibiotics have been associated with high rates of recurrence, recent advancements have been made to treat patients with CDI. One of these alternative therapies is fecal microbiota transplantation (FMT) that aims to restore colonic flora using samples collected from healthy donors. As the elderly population is at a higher risk for CDI, this study aims to review published literature to determine the efficacy and limitations of current treatment options available for elderly patients with CDI.

The kidneys are responsible for filtering out toxins, reabsorption of water, maintaining ion balance, as well as many other functions. Damage to the kidneys can be harmful to the body and even life threatening. Sudden damage to the kidney is categorized as acute kidney injury (AKI), while damage to the kidney over a long period of time, is known as chronic kidney disease (CKD). End-stage renal disease (ESRD) or kidney failure, is the last stage of CKD. At this stage, the kidneys are damaged to the point where they cannot function adequately for survival. Currently there is no cure for ESRD and kidneys at this stage require dialysis or a kidney transplant. Therefore, research on kidney diseases, development, and mechanisms will help reduce the number of patients with ESRD. Kidney development has been studied for decades but there are still many unanswered questions about nephrogenesis mechanisms, genetic factors, and biochemical pathways. Kidney organoids are a versatile tool used to study the functions of human kidneys. They originate from human pluripotent stem cells (hPSCs) which are derived from skin cells. After certain growth factors are added they function as miniature kidneys in a dish which acts as a platform for research in nephrogenesis, drug therapies, fibrosis, mutations and response to injury. Since these organoids are derived from hPSC, genome editing techniques like CRISPR/Cas9 are able to introduce modifications and mutations to replicate certain diseases or specific differentiations. The aim of this project is to develop tools to better understand kidney development and ultimately improve kidney organoid models to make advances against kidney diseases, injuries, and complications. In this project we make genetic modifications to hPSCs using the iCRISPR platform to provide new experimental tools in kidney organoids. To create the iCas9 hPSC line we utilize a knockin strategy into the AAVS1 locus. We also worked on generating a Nephron Progenitor Cell (NPC) – specific fluorescent reporter hPSC line corresponding with CITED1. Our goal is to provide tools for potential use in CRISPR screening experiments, create the ability to have live imaging of NPC behavior, and provide easy identification and isolation of NPCs for downstream analysis.

Acromegaly is a multi-system disorder whose etiology is most often traced back to a growth hormone-secreting pituitary adenoma (PA). Growth hormone (GH) secretion promotes insulin-like growth factor 1 (IGF-1) release from peripheral tissues, leading to the clinical manifestations of acromegaly. Current treatment methods for acromegaly include surgery, medical therapy, and radiation therapy. The goals of acromegaly treatment are to reduce GH levels and IGF-1 levels to age/sex-normalized levels, relieve comorbidities, normalize mortality rate, and to remove the pituitary mass causing high hormone levels. This study aims to provide a comprehensive review of current treatment methods and an analysis of novel therapies for treatment of acromegaly. The primary treatment method of acromegaly is surgery due to limited complications, relatively low cost, and remission in the majority of cases. However, surgery is not an effective treatment method for invasive macroadenomas with extension into the intracranial space. Medical therapies such as dopamine agonists (DAs) and somatostatin receptor ligands (SRLs) are effective at reducing GH and IGF-1 levels and may have anti-tumor effects. However, DAs are only effective at treating minor elevations in GH and IGF-1 levels and SRLs may cause hyperglycemia after prolonged treatment. In contrast to DAs and SRLs, Pegvisomant does not have anti-tumor effects, but it is more effective at reducing GH and IGF-1 levels. The disadvantages of Pegvisomant are the possibility of irreversible liver damage and the overwhelming cost of treatment. Stereotactic radiosurgery (SRS) is another mode of treatment for acromegaly, however, there are many disadvantages to SRS including prolonged latency period, hypopituitarism, radio-necrosis of normal brain tissue, and secondary tumor formation. Novel therapies for acromegaly include antisense drugs and modified botulin neurotoxins. Despite the success of antisense drugs and modified botulin neurotoxins in animal models, greater research is required prior to application in human clinical trials. Gene therapy is an emerging treatment method for acromegaly and proper manipulation of viral immunogenic effects could prove as a successful treatment for large macroadenomas, invasive PAs, and recurrent PAs. Despite the success of surgery in treating microadenomas and noninvasive macroadenomas, therapeutic alternatives must be explored to treat invasive PAs, macroadenomas, and recurrent PAs. Future research in immunotherapies and gene therapies may provide greater insight into the development of more effective and less invasive treatment methods for acromegaly.

OBJECTIVE: Radiotherapy (RT) elicits tumor cell death that leads to activation of dendritic cells (DC) and promotes a T-cell immune response. This type of cell death, termed immunogenic cell death (ICD), results in the release of immune signals (HMGB1 and ATP), and the cell surface translocation of calreticulin (CRT). TSA syngeneic murine model of mammary carcinoma were used in these experiments because of their ability to induce abscopal responses when treated with anti CTLA4 antibody, and therefore, a good “immunogenic” tumor model (Dewan et al. 2009). Experiments were done using wild-type cells, as well as TSA cells transfected with various plasmids that allow for easier recording. It has been shown that RT with X-rays is able to increase these three markers of ICD in a dose-dependent manner (Golden et al. 2014). Less is known about the effects of higher linear energy transfer (LET) radiation and how it may impact tumor ICD. This study investigates the impact of increasing LET particle radiation on the expressed levels of these established ICD markers. High LET particle irradiation offers several potential therapeutic advantages over X-rays: increased cell kill, more focused dose distribution and more densely generated double stranded DNA breaks. This experiment investigates the immunogenic effects of high LET particle radiation compared to X-rays. We have previously demonstrated that ICD markers (ATP, HMGB1 and CRT) are increased with high LET ions when compared to X-ray treatment. In addition to in vitro experiments, a tumor vaccination model has been created to test the differences between X-ray RT and charged particle RT in vivo. Tumor cells were treated with either X-ray RT or charged particle RT. These irradiated tumor cells are then injected subcutaneously into a mouse and serves as a systemic vaccine, protecting a mouse from a subsequent challenge of non-irradiated tumor cells. Cells treated with X-ray RT appear to have a vaccination effect that appears in a dose-dependent matter; with increasing radiation dose, we observed an increase in vaccination strength. The ability for irradiated TSA cells to serve as a vaccine appears to plateau at doses of 24 Gy. RESULTS: Preliminary data suggest that the immunogenicity of tumor cells as measured by immune cell death signals appear to increase with ion irradiation at LET greater than 60 keV/µm when compared to X-ray irradiation at the same dose. These marker levels appear to decrease at LET greater than 110 keV/µm. Preliminary in vivo experiments show that mice injected with irradiated TSA cells are able to provide a systemic vaccination effect that appears to be correlated with the original dose of radiation. As the dose is increased from 2 Gy to 50 Gy X-rays, there is a correlative increase in vaccination effect. For mice that grew tumors with lower doses of radiation, the size of tumor growth was significantly slower than control groups. CONCLUSIONS: Using wild-type TSA cells, irradiated cells showed an increase of ICD markers when compared to non-irradiated cells. There was no significant difference between charged particle RT and X-ray RT in their ability to increase markers of ICD. Using the TSA cell line with plasmid transfections, increasing LET leads to increased release of ICD markers. The degree of release appears to plateau after 100 keV/um. The TSA reporter cell line appears to show an increase in ICD marker expression when irradiated with high LET radiation. Our in vivo model shows that irradiated tumor cells injected subcutaneously into a mouse are able to induce an in situ vaccine effect so that subsequent challenge with non-irradiated tumor cells is able to reject tumor formation. Further studies are warranted to investigate the effects of LET and types of charged particle irradiation on the tumor immune response. / 2021-06-14T00:00:00Z

BACKGROUND: Significant and paramount discoveries in the past two centuries have propelled studying the gastrointestinal tract (GI) and brain connection to the forefront of all research. As the second most ranked common inflammatory disease, inflammatory bowel disease (IBD) is a chronic disorder of the GI tract marked by periods of relapse and remission with symptoms of abdominal pain, weight loss, GI bleeding, and diarrhea. It affects as many as 3.1 million people in the US (1.3% of the total population) and 2.2 million people in Europe. In addition to contributing significantly to the morbidity, mortality, and disability demands on the health care system, IBD also considerably diminishes the quality of life due to its frequent reoccurrences, low remission rate and debilitating symptomology. Due to its chronic intestinal inflammatory state, pain (both inflammatory and referred visceral pain to the abdomen) is a defining symptom of the disease activity. However, due to its idiopathic nature, treatment of the inflammation and pain pathologies distinct to IBD has been unsatisfactory. Among the many treatments of IBD, sulfasalazine (SSZ) is an sepiapterin reductase (SPR) inhibitor of the tetrahydrobiopterin (BH4) pathway. However, SSZ has a more modest effect and is a less potent inhibitor when compared to other known SPR inhibitors, such as SPRi3, of which have proven to not be ideal in humans do to lack of solubility. Because SSZ is used as IBD treatment and because enterochromaffin (EC) cells, located mainly in the colon, produce over 90% of the body’s serotonin (5-HT), a neurotransmitter that requires BH4 for its synthesis, we questioned whether BH4 plays a critical role in the pathogenesis of IBD. Many studies have characterized EC cells as gate keepers to pain signaling, where they bridge the connection between intestinal contents and the somatosensory system located underneath that then connects back to the central nervous system. Our lab has further characterized BH4 as an essential key regulator for T-cell proliferation, a key mediator in IBD, by using a novel SPR inhibitor, QM385, to not only block its proliferation, but to also alleviate chronic pain. QM385 has the same inhibitory effects as SPRi3, but at a lower concentration with minimal side effects, longer half-life and more favorable central nervous system penetration kinetics in rodents. These groundbreaking findings allow us to hypothesize that BH4 plays a significant dual role in IBD inflammation and the resultant pain, by acting on: 1) Macrophage and T-cell activation and function and 2) 5-HT signaling from EC cells. Moreover, we hypothesize that the inhibition of BH4 with our newly created pro-drug, QM385, will reduce the sensory input from EC cells and the inflammatory response in IBD simultaneously. METHODS: The widely accepted DSS animal model was used to better understand the pathologies of pain and inflammation in IBD and to a deeper extent examine the effects of the QM385 treatment. Six-day 2% DSS treated mice and control were all were assessed for inflammation and pain using the following: disease activity index (DAI), abdominal hypersensitivity with von Frey filaments, colon microscopic lesions, levels of neopterin (BH4 pathway synthesis biomarker) and sepiapterin (SPR inhibition biomarker) and gene expression for GCH1 (BH4 pathway enzyme), TNF-α (inflammatory mediator), CD68 (macrophage marker), CD4 (T-cell marker), SPR (BH4 pathway enzyme), and TrpA1 (nociceptive ion channel expressed on EC cells). RESULTS/CONCLUSIONS: In this study, the DSS induced IBD model in mice was successfully implemented evidenced by the DAI data, morphological changes, and mechanical hypersensitivity. DSS treatment increased mechanical hypersensitivity, clinical and morphological inflammatory symptoms, BH4 activity (neopterin, GCH1, SPR), inflammatory mediators (CD68, CD4, and TNF-α), and nociceptive receptor transcription, TRPVA1 production. Sepiapterin was observed after QM385 treatment, suggesting BH4 inhibition of DSS-induced IBD. QM385’s role in alleviating the immune response in DSS-induced IBD was evident with the improvements in the macroscopic clinical symptoms of inflammation (DAI) and recovery in colon length. QM385 also demonstrated a significant improvement in referred visceral abdominal pain at 1 and 3 mg/kg. Moreover, QM385 appears to resolve the upregulation of 5-HT and GCH1 in the distal colon after DSS treatment; therefore, we show that QM385 suppresses the BH4 pathway in EC cells (i.e. suppress EC cell activation), by causing less 5-HT production and therefore, less activation of the gut sensory neurons. In summary, QM385 treatment alleviated the degree of colitis caused by DSS and the degree of inflammatory and referred visceral pain, suggesting that QM385 might be a novel agent for the treatment of chronic colitis as seen in IBD. / 2021-06-17T00:00:00Z