Implementation of primary cells for mechanistic investigations of inflammatory and metabolic diseases

Cell based in vitro experiments consist an essential tool in many research fields as they offer a far less complex and easier to manipulate system compared to in vivo models. In most cases, investigators can either use established continuous cell lines or opt for primary cells directly isolated from the tissue of interest. While cell lines are cost effective and easy to obtain in high numbers, continuous growth is facilitated by their cancer background or by genetic manipulation, both of which often limit their ability to simulate the physiology of the tissue of origin. Primary cells on the other hand require labor-intensive isolation procedures but therefore much closer resemble the in vivo situation in terms of sensitivity, transporter expression and physiological behavior. To choose the appropriate tool for each experiment a detailed knowledge of the abilities and limitations of both systems is essential, whereas it is always recommendable to repeat at least some key experiments in a primary cell system. The present thesis aims to assess the implementation of bone marrow-derived macrophages (BMDMs), a 3D model of rat brain as well as primary proximal tubular cells (PTCs) for mechanistic investigations of inflammatory and metabolic diseases in different research projects. The first project implements BMDMs in the assessment of hexose 6 phosphate dehydrogenase (H6PD) function in macrophage differentiation and metabolism. Macrophages are phagocytic cells present in essentially all tissues fulfilling various functions vital for tissue repair, homeostasis and immunity. During pathological and homeostatic inflammatory reactions they arise from the bone marrow under the influence of macrophage-colony stimulating factor, patrol the body and eventually enter compromised tissue. Presence of lipopolysaccharide and interferon gamma will differentiate them into a pro-inflammatory M1 phenotype producing toxic effector molecules and inflammatory cytokines whereas interleukin 4 induces an anti-inflammatory M2 phenotype involved in resolution of inflammation and promotion of tissue remodeling. For the first study, BMDMs were derived from bone marrow progenitor cells flushed from the femurs of wild-type (WT) and H6pd knockout (KO) mice. The H6PD produces the cofactor NADPH for the activation of glucocorticoids by oxo-reduction activity of 11β hydroxysteroid dehydrogenase type 1 (11β HSD1) in the endoplasmic reticulum (ER). In macrophages this could influence the phenotypic and functional differentiation and therefore also their metabolism. Absence of H6pd was reported to cause a switch in the bidirectional 11β HSD1 towards an inactivation of glucocorticoids. Comparing BMDMs of WT and H6pd KO mice we found no such switch but only a decrease in 11β HSD1 oxo-reduction activity by 40 50 %, indicating an alternative source of NADPH. Furthermore, H6pd KO did not cause a major disturbance in macrophage phenotypic differentiation although it caused a slightly exaggerated M1 phenotype as well as an overall reduced glucose consumption. This study showed the suitability of BMDMs to study macrophage differentiation and to perform a variety of assays to assess characteristic macrophage parameters like phagocytosis, nitric oxide production or metabolism. Most importantly, by using animal derived cells, we could use a H6pd KO mouse which circumvents an incomplete gene knockdown by siRNA. In the second project, BMDMs were implemented to investigate the contribution of secondary calciprotein particles (CPPs) to the process of vascular calcification frequently observed in chronic kidney disease (CKD) patients. The formation of primary CPPs is a physiological process in which serum proteins prevent the precipitation of calcium and phosphate as hydroxyapatite by forming spherical complexes instead. In CKD patients, primary transform into secondary CPPs, which were shown to activate macrophages. Nuclear factor erythroid 2 related factor 2 (NRF2), a master regulator of oxidative cell defense, was reported to play an important role in CPP-induced inflammation in CKD. Using BMDMs derived from WT and Nrf2 KO mice we showed the induction of macrophages by secondary CPPs at concentrations measured in CKD patients. Mechanistic studies suggested a TLR4-mediated CPP response, which could be reduced or exacerbated by induction or knockdown of Nrf2. Whereas the use of Nrf2 KO mice facilitated complete absence of the target gene, the relevance of the study would benefit from the use of a human model like human PBMCs. A primary cell system that includes a specific type of macrophage, the microglia found in brain, can be obtained as part of an in vitro 3D brain model derived from rat embryonic brain tissue. These 3D cultures contain all cell types of the brain, including neurons, oligodendrocytes, astrocytes and microglia cells. The latter two are involved in neuroinflammation, which can be caused by heavy metals such as trimethyltin. The project investigates the combination of three risk factors of neurodegenerative diseases, namely the metabolic syndrome characterized by low brain insulin and high glucocorticoid levels as well as trimethyltin exposure. Therefore, an approach consisting of the described 3D rat brain model, the murine microglia cell line BV 2 as well as a mouse model of diabetes was used to report the absence of an additive effect of the risk factors to neurodegeneration but an increased neuroinflammatory response. The implementation of models with various levels of complexity allowed to address mechanistic questions using the BV 2 cell line but also to draw more in vivo relevant conclusions by using a primary 3D model and in vivo mouse experiments. In a fourth project, the implementation of primary PTCs in the investigation of metabolic acidosis was assessed. Within the functional unit of a kidney, the nephron, the proximal tubule is responsible for 65 % of the total sodium reabsorption as well as most solutes, amino acids and low molecular weight proteins. In addition, the proximal tubule plays an important role in counteracting metabolic acidosis via a process that involves the uptake of glutamine by the glutamine transporter SNAT3 which is mainly expressed in the second and third proximal tubule segment. To test the involvement of Nrf2 in the upregulation of SNAT3 in response to metabolic acidosis, we exposed primary PTCs isolated from mouse kidneys to acidified medium. We could observe an upregulation of Snat3 mRNA, which was prevented by siRNA knockdown of Nrf2. Studies in Nrf2 KO mice fed with high acid diet confirmed the in vitro findings but also revealed a compensatory adaption of other transporters not detected in the primary cell model. Overall, the four projects revealed many abilities but also some disabilities of the implemented primary cell systems. As the availability of human material is very limited, inducible pluripotent stem cell technology will grow more and more important as it increases translational relevance of in vitro experiments. However, the described primary cell models will probably remain of great use, especially in basic research

Hydrogeologic characterization of fractured rock: Site 32, Portsmouth, New Hampshire

Natural underground settings exhibit small and large scale spatial variations, making them difficult to characterize. This complexity is particularly difficult to overcome when delineating a heterogeneous fractured bedrock system. The following study involves the characterization of a fractured bedrock site. Hydraulically conductive fractures provide migratory pathways within a fractured rock. Interpreting their orientation in space is important in understanding contaminant movement. The data used include borehole geophysical, lithologic and hydraulic test data. The first method applied was a geostatistical analysis. This technique incorporates statistical characteristics of the geophysical and hydraulic data to develop fracture patterns. The second method is a deterministic study developed by analyzing the fracture orientations as they cross-cut a borehole. Hydraulic connections were inferred between two sets of wells through slug test analyses. Multiple response anomalies were identified within the data. Uncertainty and model reliability are a component of any model created to depict the real world.*

Epidemiology and costs of gastroesophageal reflux disease in Switzerland: a population-based study

Summary: Objectives: Assessment of the prevalence, health care resource use and cost of gastroesophageal reflux disease in Switzerland. Methods: A population-based telephone survey was conducted in German and French speaking Switzerland. Reflux cases were defined using a questionnaire proposed by the German Gastro League and answered additional questions on their personal characteristics and resource use. Results: 1274 out of 7222 participants were positively screened. The prevalence of reflux disease in Swiss adults was estimated at 17.6% (95% CI: 15.6%-19.7%) or 993000 individuals. Regular treatment with medication was reported by 38.0% of the reflux positive sample. Reflux-induced general practitioner consultations during the last year were reported by 25.9%. On average, there were 0.84 general practitioner consultations, 0.19 specialist consultations, 0.08 gastroscopies and 0.01 hospitalisations annually. Mean direct medical costs, dominated by medication costs, were CHF 185 per patient-year (95 % CI: CHF 140-230) or 0.5% of Switzerland's total health care expenditures. Total costs were CHF 234 (95% CI: CHF 185-284) per patient-year. Conclusions: The prevalence of reflux disease in Switzerland is similar to that in other industrialised countries. Reflux disease causes considerable costs, in the medical system and at the societal leve

Anticardiolipin antibodies and coronary heart disease

Arterial or venous thrombotic events have been described as complications in patients with positive anticardiolipin antibodies (aCL), affecting various organs including the heart. In order to see whether aCL could be, among others, a predisposing factor for coronary artery occlusions and whether it could serve as a prognostic marker for coronary heart disease, 232patients enrolled in the European Concerted Action on Thrombosis Angina Pectoris Study were studied. aCL and various other haemostatic parameters were determined at time of admittance in order to see whether a relationship existed between haemostasis at baseline and extent or prognosis of the cardiovascular disease. A follow-up at 12 and 24 months after angiography included information about relapsing coronary or other thrombotic events, treatment and outcome of the disease. aCL were not found to be a marker of either progressive cardiovascular disease or recurrent thrombotic events. No correlation was found, either in aCL positive or in aCL negative patients, between high levels of haemostasis activation markers, such as fi-thromboglobulin, platelet factor 4 or fibrinopeptide A and recurrent cardiovascular diseas

Colorectal cancer surveillance by colonoscopy in a prospective, population-based long-term Swiss screening study – outcomes, adherence, and costs

BACKGROUND: The success of colorectal cancer (CRC) screening depends mainly on screening quality, patient adherence to surveillance, and costs. Consequently, it is essential to assess the performance over time. METHODS: In 2000, a closed cohort study on CRC screening in individuals aged 50 to 80 was initiated in Uri, Switzerland. Participants who chose to undergo colonoscopy were followed over 18 years. We investigated the adherence to recommended surveillance and collected baseline characteristics and colonoscopy data. Risk factors at screening for the development of advanced adenomas were analyzed. Costs for screening and follow-up were evaluated retrospectively. RESULTS: 1278 subjects with a screening colonoscopy were included, of which 272 (21.3%; 69.5% men) had adenomas, and 83 (6.5%) had advanced adenomas. Only 59.8% participated in a follow-up colonoscopy, half of them within the recommended time interval. Individuals with advanced adenomas at screening had nearly five times the risk of developing advanced adenomas compared to individuals without adenomas (24.3% vs. 5.0%, OR 4.79 CI 2.30-9.95). Individuals without adenomas developed advanced adenomas in 4.9%, including four cases of CRC; three of them without control colonoscopy. The villous component in adenomas smaller than 10 mm was not an independent risk factor. Costs for screening and follow-up added up to CHF 1'934'521 per 1'000 persons screened, almost half of them for follow-up examinations; 60% of these costs accounted for low-risk individuals. CONCLUSION: Our findings suggest that follow-up of screening colonoscopy should be reconsidered in Switzerland; in particular, long-term adherence is critical. Costs for follow-up could be substantially reduced by adopting less expensive long-term screening methods for low-risk individuals

Hydrology of five forest roads in the Oregon Coast Range

Understanding the impact of low volume road networks on forested watersheds is important for future forest management and watershed restoration. This study characterized the hydrology of five segments of forest road in the Oregon Coast Range. Rainfall, infiltration, road surface runoff, and intercepted subsurface flow were measured at each road segment. Results indicate that these individual segments of forest road differ hydrologically, depending on how much subsurface flow they intercept from the hillslope. The first objective of this study was to compare and contrast hydrologic behavior of ditch flow resulting from infiltration-excess overland flow on the road surface with ditch flow that was intercepted subsurface flow from the hillslope. Overland flow and intercepted subsurface flow were physically separated in the ditch by a divider and routed through two trapezoidal flumes at the bottom of each road study segment. Runoff derived from infiltration-excess overland flow on the road surface was ephemeral, responding to high intensity rainfall, and it ceased within minutes to hours after rainfall. This was the only type of flow observed in road ditches at four of the five study sites. Subsurface flow intercepted from the hillslope was intermittent, occurring continuously during the rainy season with a more gradual, muted response to storms. This type of flow occurred, along with ephemeral ditch flow, at one of the five study sites. Ephemeral flow in the ditch of this site produced minimal runoff volume, no more than 4.5 m³ (16 mm / m² of road), for storms up to 140 mm in depth. In contrast, intermittent ditch flow intercepted from the hillslope produced up to 801 m³ (2800 mm / m² of road) for similar storms, 20 times more flow than all of the rainfall that had occurred on the road surface. Any ephemeral ditch flow derived from subsurface flow on the hillslope was not observed in this study, though it may exist. The second objective of this thesis was to quantify the relationship between rainfall intensity, infiltration capacity and road surface runoff at the study road segments. A rainfall simulator was used to measure road infiltration capacities. Estimates of infiltration capacity from the rainfall simulation averaged 4 mm/hr and ranged from 0 to 11 mm/hr. Despite the low infiltration capacities, runoff volumes from the road surface were on average only 5 percent of natural rainfall, because rainfall intensity remained lower than infiltration capacity during most of the duration of storms. Infrequent pulses of high intensity rainfall overwhelmed the infiltration capacity of the road and produced surface runoff. Median lag time from peak rainfall intensity to peak discharge of road-derived ditch flow was 10 minutes on road segments with up to 250 m² of surface area. Two other estimates of the infiltration capacity of a given road were 1) the maximum rainfall intensity that did not produce runoff, and 2) the minimum rainfall intensity that did produce runoff. These intensities ranged from 0.5 to 11 mm/hr, similar to the infiltration capacity estimates from rainfall simulation. Infiltration capacity and ephemeral road surface runoff were similar for all road segments in this study. Intermittent flow, intercepted from the hillslope, differed between roads and was two orders of magnitude greater than ephemeral runoff from the road surface. Intercepted subsurface flow has greater potential to cause erosive damage than ephemeral runoff from the road surface, because of its large peak discharges and flow volumes

Absence of nitroglycerin-induced heparin resistance in healthy volunteers

A previously described nitroglycerin-induced heparin resistance could not be verified by in-vitro experiments or in a randomized, double-blind, crossover trial in healthy volunteers. A clinically relevant attenuation of the anticoagulant effect of a heparin bolus (40 U.kg−1) by a concomitant infusion of nitroglycerin (100 µg.min−1) was absent. Activated partial thromboplastin time was not significantly different under nitroglycerin infusion as compared to placebo after heparin injection. Concentrations and activities of antithrombin III and heparin cofactor II remained unchanged during nitroglycerin infusion. An interaction of these two frequently combined drugs in patients with active thromboembolic disease or after a prolonged concomitant intravenous administration cannot be ruled out. Since this is of clinical importance, furt her studies must clarify a possible nitroglycerin-induced heparin resistanc

Role of Gastric Colonization in Nosocomial Infections and Endotoxemia: A Prospective Study in Neurosurgical Patients on Mechanical Ventilation

The role of gastric microbial colonization in nosocomial infections and endotoxemia was investigated prospectively in 40 neurosurgical patients requiring mechanical ventilation for >48 h. Each was studied up to 7 d. Swabs from the nose and oropharynx were cultured at admission, and aspirates from the stomach and trachea were cultured daily until enteral alimentation was started. Patients were evaluated every second day for endotoxemia and coagulation activation. Of 153 gastric aspirates, 66.7% contained microorganisms at a mean quantity of 107 cfu/ml, Nosocomial pneumonia occurred in 15 patients, septicemia in 5, and meningitis in 1. The stomach was the evident source of infection in only 1 patient with pneumonia. Of 140 plasma samples, 12 (8.6%) from 10 patients showed detectable endotoxin levels, but there was no association between endotoxemia or coagulation activation and the presence of microorganisms in the stomach. The stomach was not an important source for nosocomial infections or endotoxemia, even in patients with high gastric p

Cumplimiento del protocolo de manejo de Infecciones de Vías Urinarias en embarazadas atendidas en la sala de Alto Riesgo Obstétrico y Ginecología del Hospital Escuela Regional Santiago, Jinotepe durante el año 2017 -2018

Objetivo: Determinar el cumplimiento del protocolo de manejo de infecciones de vías urinarias en embarazadas atendidas en la sala de ARO y ginecología del servicio de Ginecoobstetricia del Hospital Escuela Regional Santiago, Jinotepe durante el año 2017 -2018. Diseño metodológico: Se realizó un estudio, descriptivo, de corte transversal, donde se revisaron 68 expedientes clínicos de pacientes con IVU. Resultados: El promedio de edad en las pacientes estudiadas, fue la edad mínima de 15 años y la edad máxima fue de 35 años, la edad que con mayor frecuencia se repitió (moda) fue de 19 años, siendo el intervalo más frecuente el comprendido de edad menor de 20 años fue predominante en el 47,1%(n=32), el 67,6%(n=46) se encontraba acompañada, un 63,2%(n=43) procedía dela rural, el 67,6%(n=46) era ama de casa con un grado de escolaridad secundaria aprobada en el 51,1%(n=35).La infección de vías urinarias severa en un 58,8% fue la clasificación más frecuente presentada en las pacientes. Estas se diagnosticaron mediante Clínica y uso de cinta reactiva en un 59,9% El protocolo MINSA para el manejo de la IVU en embarazadas no se cumple al 100%, se evidenció que un 14,7% de los expedientes no cumplían con los criterios estipulados en la norma. Conclusiones: La mayoría de las pacientes diagnosticadas con IVU son pacientes jóvenes en la segunda década de la vida, procedentes del aérea rural, acompañadas, amas de casa, con secundaria aprobada, en la cuales se diagnosticó la IVU Severa mediante clínica y cinta reactiva según los criterios estipulados en la normativa MINSA, se porta un cumplimiento del 85,3% de los expedientes clínicos revisados y un 14,7% que no cumple los criterios clínicos establecidos en la norma. Palabras clave: Cumplimiento Norma IVU; Protocolo IVU MINSA; IVU y Norma MINSA

Absence of hexose-6-phosphate dehydrogenase results in reduced overall glucose consumption but does not prevent 11β-hydroxysteroid dehydrogenase-1-dependent glucocorticoid activation

Hexose-6-phosphate dehydrogenase (H6PD) is thought to be the major source of NADPH within the endoplasmic reticulum (ER), determining 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) reaction direction to convert inert 11-oxo- to potent 11β-hydroxyglucocorticoids. Here, we tested the hypothesis whether H6pd knock-out (KO) in primary murine bone marrow-derived macrophages results in a switch from 11β-HSD1 oxoreduction to dehydrogenation, thereby inactivating glucocorticoids (GC) and affecting macrophage phenotypic activation as well as causing a more aggressive M1 macrophage phenotype. H6pdKO did not lead to major disturbances of macrophage activation state, although a slightly more pronounced M1 phenotype was observed with enhanced proinflammatory cytokine release, an effect explained by the decreased 11β-HSD1-dependent GC activation. Unexpectedly, ablation of H6pd did not switch 11β-HSD1 reaction direction. A moderately decreased 11β-HSD1 oxoreduction activity by 40-50% was observed in H6pdKO M1 macrophages but dehydrogenation activity was undetectable, providing strong evidence for the existence of an alternative source of NADPH in the ER. H6pdKO M1 activated macrophages showed decreased phagocytic activity, most likely a result of the reduced 11β-HSD1-dependent GC activation. Other general macrophage functions reported to be influenced by GC, such as nitrite production and cholesterol efflux, were altered negligibly or not at all. Importantly, assessment of energy metabolism using an extracellular flux analyzer and lactate measurements revealed reduced overall glucose consumption in H6pdKO M1 activated macrophages, an effect that was GC independent. The GC-independent influence of H6PD on energy metabolism and the characterization of the alternative source of NADPH in the ER warrant further investigations. ENZYMES: 11β-HSD1, EC 1.1.1.146; H6PD, EC 1.1.1.47