Analysis of the spatial distribution of amyotrophic lateral sclerosis in Virginia.

Objective: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that is usually fatal. Environmental exposures have been posited in the etiology of ALS, but few studies have modeled the spatial risk of ALS over large geographic areas. In this paper, our goal was to analyze the spatial distribution of ALS in Virginia and identify any areas with significantly elevated risk using Virginia ALS Association administrative data. Methods: We used Bayesian hierarchical spatial regression models to estimate the relative risk for ALS in Virginia census tracts, adjusting for several covariates posited to be associated with the disease. We used an intrinsic conditional autoregressive prior to allow for spatial correlation in the risk estimates and stabilize estimates over space. Results: Considerable variation in ALS risk existed across Virginia, with greater relative risk found in the central and western parts of the state. We identified significantly elevated relative risk in a number of census tracts. In particular, Henrico, Albemarle, and Botetourt counties all contained at least four census tracts with significantly elevated risk. Conclusions: We identified several areas with significantly elevated ALS risk across Virginia census tracts. These results can inform future studies of potential environmental triggers for the disease, whose etiology is still being understood.

Trends in gastrostomy tube placement with concomitant Nissen fundoplication for infants and young children at Pediatric Tertiary Centers.

PURPOSE: In infants and toddlers, gastrostomy tube placement (GT) is typically accompanied by consideration of concomitant Nissen fundoplication (NF). Historically, rates of NF have varied across providers and institutions. This study examines practice variation and longitudinal trends in NF at pediatric tertiary centers. METHODS: Patients ≤ 2 years who underwent GT between 2008 and 2018 were identified in the Pediatric Health Information System database. Patient demographics and rates of NF were examined. Descriptive statistics were used to evaluate the variation in the proportion of GT with NF at each hospital, by volume and over time. RESULTS: 40,348 patients were identified across 40 hospitals. Most patients were male (53.8%), non-Hispanic white (49.5%) and publicly-insured (60.4%). Rates of NF by hospital varied significantly from 4.2 to 75.2% (p < 0.001), though were not associated with geographic region (p = 0.088). Rates of NF decreased from 42.8% in 2008 to 14.2% in 2018, with a mean annual rate of change of - 3.07% (95% CI - 3.53, - 2.61). This trend remained when stratifying hospitals into volume quartiles. CONCLUSION: There is significant practice variation in performing NF. Regardless of volume, the rate of NF is also decreasing. Objective NF outcome measurements are needed to standardize the management of long-term enteral access in this population.

Role of FOXM1 in vascular smooth muscle cell survival and neointima formation following vascular injury.

BACKGROUND: Accelerated smooth muscle cell (SMC) proliferation is the primary cause of intimal hyperplasia (IH) following vascular interventions. Forkhead Box M1 (FOXM1) is considered a proliferation-associated transcription factor. However, the presence and role of FOXM1 in IH following vascular injury have not been determined. OBJECTIVE: We examined the expression of FOXM1 in balloon-injured rat carotid arteries and investigated the effect of FOXM1 inhibition in SMCs and on the development of IH. METHODS AND RESULTS: FOXM1 was detected by immunofluorescent staining in balloon-injured rat carotid arteries where we observed an upregulation at day 7, 14, and 28 compared to uninjured controls. Immunofluorescence staining revealed FOXM1 coincided with proliferating cell nuclear antigen (PCNA). FOXM1 was also detectable in human carotid plaque samples. Western blot showed an upregulation of FOXM1 protein in serum-stimulated SMCs. Inhibition of FOXM1 using siRNA or chemical inhibition led to the induction of apoptosis as measured by flow cytometry and western blot for cleaved caspase 3. Perturbations in survival signaling were measured by western blot following FOXM1 inhibition, which showed a decrease in phosphorylated AKT and ß-catenin. The chemical inhibitor thiostrepton was delivered by intraperitoneal injection in rats that underwent balloon injury and led to reduced intimal thickening compared to DMSO controls. CONCLUSIONS: FOXM1 is an important molecular mediator of IH that contributes to the proliferation and survival of SMCs following vascular injury.

Cross-sectional study of hepatitis B antibody status in health care workers immunized as children at an academic medical center in Wisconsin.

Individuals who received the hepatitis B vaccine series as young children are entering the healthcare workforce. Our study measured the persistence of antibody to the hepatitis B surface antigen (anti-HBs) at time of employment. Among 986 individuals born in 1991 or more recently with documentation of completion of the hepatitis B vaccine series, 51% had anti-HBs < 10mIU/ml. Of these 507 healthcare workers, 446 (88%) received documented fourth dose of hepatitis B vaccine followed by another anti-HBs ≥ 28 days post vaccination; 11% (50/446 or 5% of the total population) did not mount an anamnestic response. The non-responders were more likely to be male or complete the vaccine series prior to age 7 months. Measuring anti-HBs at the time of hire in this population of healthcare workers who had documentation of hepatitis B series completion as young children may be unnecessary because of the high rate of hepatitis B vaccine protection.

MANAGEMENT OF THYROTOXICOSIS AMONG GENERAL PRACTITIONERS IN TRINIDAD COMPARED WITH 2016 AMERICAN THYROID ASSOCIATION GUIDELINES FOR HYPERTHYROIDISM.

Objective: A previous Trinidadian survey highlighted the investigative and therapeutic approaches selected by general practitioners (GPs) in managing thyrotoxicosis. The main objective of this study was to compare practice with existing guidelines. Methods: In this cross-sectional study a pretested de novo questionnaire was self-administered to GPs throughout Trinidad. The survey evaluated GPs' choices in management of thyrotoxicosis cases and compared their responses to the 2016 American Thyroid Association guidelines as well as with those previously reported locally. Results: A total of 159 completed questionnaires were analyzed (59% response rate). Thyroid stimulating hormone was the preferred (94%) biochemical test to confirm thyrotoxicosis etiology. A combination of ultra-sound and thyroid scintigraphy, thyroid ultrasound alone, and scintigraphy only were the testing options selected by 41%, 38%, and 12%, respectively. Generally medical therapy with antithyroid drugs was the preferred treatment option with 86% of respondents selecting this option for the index case of newly diagnosed female Graves disease. The greatest proportion of respondents that selected radioactive iodine (RAI) was 35% for both the index case as well as the male equivalent. Surgery was the most popular option at 25% for patients with a toxic multinodular goiter. Having access to RAI and scintigraphy was reported by 32% and 28%, respectively. Conclusion: GPs appear to be constrained to making rational choices based upon availability rather than what the guidelines recommend. In the absence of formal continuing medical education for GPs on thyrotoxicosis, dissemination of guidelines at the primary care level may reduce this gap. Abbreviations: ATA = American Thyroid Association; ATD = antithyroid drugs; CME = continued medical education; GP = general practitioner; RAI = radioactive iodine; SURG = surgery; T4 = thyroxine; TSH = thyroid-stimulating hormone.

Inhibition of Receptor-Interacting Protein Kinase 1 with Necrostatin-1s ameliorates disease progression in elastase-induced mouse abdominal aortic aneurysm model.

Abdominal aortic aneurysm (AAA) is a common aortic disease with a progressive nature. There is no approved pharmacological treatment to effectively slow aneurysm growth or prevent rupture. Necroptosis is a form of programmed necrosis that is regulated by receptor-interacting protein kinases (RIPs). We have recently demonstrated that the lack of RIP3 in mice prevented aneurysm formation. The goal of the current study is to test whether perturbing necroptosis affects progression of existing aneurysm using the RIP1 inhibitors Necrostatin-1 (Nec-1) and an optimized form of Nec-1, 7-Cl-O-Nec-1 (Nec-1s). Seven days after aneurysm induction by elastase perfusion, mice were randomly administered DMSO, Nec-1 (3.2 mg/kg/day) and Nec-1s (1.6 mg/kg/day) via intraperitoneal injection. Upon sacrifice on day 14 postaneurysm induction, the aortic expansion in the Nec-1s group (64.12 ± 4.80%) was significantly smaller than that of the DMSO group (172.80 ± 13.68%) (P < 0.05). The mean aortic diameter of Nec-1 treated mice appeared to be smaller (121.60 ± 10.40%) than the DMSO group, though the difference was not statistically significant (P = 0.1). Histologically, the aortic structure of Nec-1s-treated mice appeared normal, with continuous and organized elastin laminae and abundant αActin-expressing SMCs. Moreover, Nect-1s treatment diminished macrophage infiltration and MMP9 accumulation and increased aortic levels of tropoelastin and lysyl oxidase. Together, our data suggest that pharmacological inhibition of necroptosis with Nec-1s stabilizes pre-existing aneurysms by diminishing inflammation and promoting connective tissue repair.

The aging disc: using an ovine model to examine age-related differences in the biomechanical properties of the intralamellar matrix of single lamellae.

PURPOSE: To determine the effect of age on the biomechanical properties of the intralamellar matrix of single annulus fibrosus (AF) lamellae. METHODS: One intervertebral disc (IVD) was excised from five young (<12 months), five middle-aged (2-4 years) and five older (5-7 years) ovine lumbar spines. From each IVD, a maximum of four single AF lamellae samples were harvested: two from the anterior region and two from the posterior region. Tissues were mounted in a tensile testing apparatus such that tension was applied perpendicular to the orientation of the collagen fibers to isolate the intralamellar matrix. Variables of interest from the stress-strain relationship were: end of toe-region strain and corresponding stress, initial failure stress and strain, and elastic stiffness. RESULTS: When compared to the middle-aged and old samples, the intralamellar matrix of young AF samples displayed significantly higher stress values at the end of the end of toe-region (p = 0.008) and at initial failure (p = 0.002). Further, the young samples were stiffer than both middle-aged and old samples (p = 0.04). CONCLUSIONS: This study was the first to show that the intralamellar matrix of single AF lamellae is weaker and more compliant in middle-aged and old ovine IVDs compared to young IVDs. These findings are likely a result of the remarkable age-related changes that occur that ultimately weaken the IVD as a whole.

The use of intermittent trunk flexion to alleviate low back pain during prolonged standing.

The current study examined of the effect of intermittent, short-term periods of full trunk flexion on the development of low back pain (LBP) during two hours of standing. Sixteen participants completed two 2-h standing protocols, separated by one week. On one day, participants stood statically for 2h (control day); on the other day participants bent forward to full spine flexion (termed flexion trials) to elicit the flexion relaxation (FR) phenomenon for 5s every 15min (experimental day). The order of the control and experimental day was randomized. During both protocols, participants reported LBP using a 100mm visual analogue scale every 15min. During the flexion trials, lumbar spine posture, erector spinae and gluteus medius muscle activation was monitored. Ultimately, intermittent trunk flexion reduced LBP by 36% (10mm) at the end of a 2-h period of standing. Further, erector spinae and gluteus medius muscle quietening during FR was observed in 91% and 65% of the flexion trials respectively, indicating that periods of rest did occurred possibly contributing to the reduction in LBP observed. Since flexion periods do not require any aids, they can be performed in most workplaces thereby increasing applicability.

Complex changes in von Willebrand factor-associated parameters are acquired during uncomplicated pregnancy.

BACKGROUND: The coagulation protein von Willebrand Factor (VWF) is known to be elevated in pregnancy. However, the timing and nature of changes in VWF and associated parameters throughout pregnancy are not well understood. OBJECTIVES: To better understand the changes in VWF provoked by pregnancy, we studied VWF-associated parameters in samples collected over the course of healthy pregnancies. METHODS: We measured VWF antigen (VWF:Ag), VWF propeptide (VWFpp), Factor VIII (FVIII), and ADAMTS13 activity in samples collected from 46 women during pregnancy and at non-pregnant baseline. We also characterized pregnant vs. non-pregnant VWF multimer structure in 21 pregnancies, and performed isoelectric focusing (IEF) of VWF in two pregnancies which had samples from multiple trimesters. RESULTS: VWF:Ag and FVIII levels were significantly increased during pregnancy. ADAMTS13 activity was unchanged. VWFpp levels increased much later in pregnancy than VWF:Ag, resulting in a progressive decrease in VWFpp:Ag ratios. FVIII:VWF ratios also decreased in pregnancy. Most pregnancies exhibited a clear loss of larger VWF multimers and altered VWF triplet structure. Further evidence of acquired VWF qualitative changes in pregnancy was found in progressive, reversible shifts in VWF IEF patterns over gestation. CONCLUSIONS: These data support a new view of pregnancy in which VWF can acquire qualitative changes associated with advancing gestational age. Modeling supports a scenario in which both increased VWF production and doubling of the VWF half-life would account for the data observed. We propose that gestation induces a prolongation in VWF survival, which likely contributes to increased total VWF levels and altered VWF structure.

Highly efficient differentiation of neural precursors from human embryonic stem cells and benefits of transplantation after ischemic stroke in mice.

INTRODUCTION: Ischemic stroke is a leading cause of death and disability, but treatment options are severely limited. Cell therapy offers an attractive strategy for regenerating lost tissues and enhancing the endogenous healing process. In this study, we investigated the use of human embryonic stem cell-derived neural precursors as a cell therapy in a murine stroke model. METHODS: Neural precursors were derived from human embryonic stem cells by using a fully adherent SMAD inhibition protocol employing small molecules. The efficiency of neural induction and the ability of these cells to further differentiate into neurons were assessed by using immunocytochemistry. Whole-cell patch-clamp recording was used to demonstrate the electrophysiological activity of human embryonic stem cell-derived neurons. Neural precursors were transplanted into the core and penumbra regions of a focal ischemic stroke in the barrel cortex of mice. Animals received injections of bromodeoxyuridine to track regeneration. Neural differentiation of the transplanted cells and regenerative markers were measured by using immunohistochemistry. The adhesive removal test was used to determine functional improvement after stroke and intervention. RESULTS: After 11 days of neural induction by using the small-molecule protocol, over 95% of human embryonic stem-derived cells expressed at least one neural marker. Further in vitro differentiation yielded cells that stained for mature neuronal markers and exhibited high-amplitude, repetitive action potentials in response to depolarization. Neuronal differentiation also occurred after transplantation into the ischemic cortex. A greater level of bromodeoxyuridine co-localization with neurons was observed in the penumbra region of animals receiving cell transplantation. Transplantation also improved sensory recovery in transplant animals over that in control animals. CONCLUSIONS: Human embryonic stem cell-derived neural precursors derived by using a highly efficient small-molecule SMAD inhibition protocol can differentiate into electrophysiologically functional neurons in vitro. These cells also differentiate into neurons in vivo, enhance regenerative activities, and improve sensory recovery after ischemic stroke.

Vector-free and transgene-free human iPS cells differentiate into functional neurons and enhance functional recovery after ischemic stroke in mice.

Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited, stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently, the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition, the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However, the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation, we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice, hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling, increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice.

Inflammatory dendritic cells migrate in and out of transplanted chronic mycobacterial granulomas in mice.

An estimated one-third of the world's population is infected with Mycobacterium tuberculosis, although most affected individuals maintain a latent infection. This control is attributed to the formation of granulomas, cell masses largely comprising infected macrophages with T cells aggregated around them. Inflammatory DCs, characterized as CD11c+CD11b+Ly6C+, are also found in granulomas and are an essential component of the acute immune response to mycobacteria. However, their function during chronic infection is less well understood. Here, we report that CD11c+ cells dynamically traffic in and out of both acute and chronic granulomas induced by Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) in mice. By transplanting Mycobacterium-induced granulomas containing fluorescently labeled CD11c+ cells and bacteria into unlabeled mice, we were able to follow CD11c+ cell trafficking and T cell activation. We found that half of the CD11c+ cells in chronic granulomas were exchanged within 1 week. Compared with tissue-resident DC populations, CD11c+ cells migrating out of granuloma-containing tissue had an unexpected systemic dissemination pattern. Despite low antigen availability, systemic CD4+ T cell priming still occurred during chronic infection. These data demonstrate that surveillance of granulomatous tissue by CD11c+ cells is continuous and that these cells are distinct from tissue-resident DC populations and support T cell priming during both stages of Mycobacterium infection. This intense DC surveillance may also be a feature of Mycobacterium tuberculosis infection and other granuloma-associated diseases.

Small molecule promoted feeder free and adherent differentiation of functional neurons from human embryonic and induced pluripotent stem cells.

While human embryonic stem (hES) and induced pluripotent stem (hiPS) cells offer exciting prospects in the fields of regenerative medicine and developmental biology, efficient directed differentiation of these cells is still difficult. Neural induction protocols often include suspension culture or co-culture with other cell types, introducing heterogeneity and complicating analysis. In addition, expensive recombinant factors are often used over processes that take weeks to complete, making such experiments financially difficult. We have developed a fully adherent and feeder free neural differentiation protocol using small molecules such as dorsomorphin and common medium supplements. Using this protocol, we obtain >90% of cells developing into neural precursors, as measured by nestin staining. Neurons derived from these precursors are electrophysiologically active. After three weeks of terminal differentiation, we obtain functional neurons which fire high-amplitude action potentials upon depolarization. A subset of neurons also fires repetitive trains. This protocol offers a simpler and less expensive method for investigations involving the differentiation of neural precursors and neurons in culture.

Morphologic adaptation of arterial endothelial cells to longitudinal stretch in organ culture.

Arteries in vivo are subjected to large longitudinal stretch, which changes significantly due to vascular disease and surgery. However, little is known about the effect of longitudinal stretch on arterial endothelium. The aim of this study was to determine the morphologic adaptation of arterial endothelial cells (ECs) to elevated axial stretch. Porcine carotid arteries were stretched 20% more than their in vivo length while being maintained at physiological pressure and flow rate in an organ culture system. The ECs were elongated with the application of the axial stretch (aspect ratio 2.81+/-0.25 versus 3.65+/-0.38, n=8, p<0.001). The elongation was slightly decreased after three days and the ECs recovered their normal shape after seven days, as measured by the shape index and aspect ratio (0.55+/-0.03 versus 0.56+/-0.04, and 2.93+/-0.28 versus 2.88+/-0.20, respectively, n=5). Cell proliferation was increased in the intima of stretched arteries in three days as compared to control arteries but showed no difference after seven days in organ culture. These results demonstrate that the ECs adapt to axial stretch and maintain their normal shape.