ALTERNATING CURRENT DIELECTROPHORETIC MANIPULATION OF ERYTHROCYTES IN MEDICAL MICRODEVICE TECHNOLOGY

Medical microdevices have gained popularity in the past few decades because they allow the medical laboratory to be taken out into the field and for disease diagnostics to happen with a smaller sample volume, at a lower cost and much faster. Blood is the human body's most readily available and informative diagnostic fluid because of the wealth of information it provides about the body's general health including enzymatic, proteomic and immunological states. The purpose of this project is to optimize operating conditions and study ABO-Rh erythrocytes dielectrophoretic responses to alternating current electric signals. The end goal of this project is the creation of a relatively inexpensive microfluidic device, which can be used for the ABO-Rh typing of a blood sample. This dissertation presents results showing how blood samples of a known ABO- Rh blood type exhibit differing behavior to the same electrical stimulus based on their blood type. The first panel of donors and experiments, presented in Chapter 4 occurred when a sample of known blood type was injected into a microdevice with a T-shaped electrode configuration and the erythorcytes were found to rupture at a rate specific to their ABO-Rh blood type. The second set of experiments, presented in Chapter 5, were originally published in Electrophoresis in 20111. Novel in this work was the discovery that treatment of human erythrocytes with β-galactosidase successfully removed ABO surface antigens such that native A and B blood no longer agglutinated with the proper antibodies. This work was performed in a medium of conductivity 0.9S/m which is close to the measured conductivity of pooled plasma (~1.1S/m). The ability to perform dielectrophoresis experiments at physiological conductivities conditions is advantageous for future portable devices because the device/instrument would not need to store dilution buffers. The final results of this project, presented in Chapter 6, explore the entire dielectrophoretic spectra of the ABO-Rh erythrocytes including the cross-over frequency and the magnitudes of the positive or negative dielectrophoretic response. These were completed at lower medium conductivities of 0.1S/m and 0.01-0.04S/m. These results show that by using the sweep function built into the Agilent alternating current generator it is possible to explore how a single group of blood cells will react to rapid changes in frequency and will provide the user with curve that can be matched the theoretical dielectrophoretic response curves. As a whole this project shows that it is possible to distinguish human erythrocytes by their ABO-Rh blood type via three different dielectrophoretic methods. This work builds on the foundation of that it is possible to distinguish healthy from infected cells2-7, similar cell types1,7-14 and other work regarding the dielectrophoresis of human erythrocytes1,10,11. This work has implications in both medical diagnostics and future dielectrophoretic work because it has shown that ABO-Rh blood type is now a factor, which must be identified when working with a human blood sample. It also shows that the creation of a microfluidic device that subjects human erythrocytes to a dielectrophoretic impulse and then exports an ABO-Rh blood type is a near future possibility.

Different response to eccentric and concentric training in older men and women

Sarcopenia is the age-related loss of muscle mass and strength and has been associated with an increased risk of falling and the development of metabolic diseases. Various training protocols, nutritional and hormonal interventions have been proposed to prevent sarcopenia. This study explores the potential of continuous eccentric exercise to retard age-related loss of muscle mass and function. Elderly men and women (80.6 +/- 3.5 years) were randomized to one of three training interventions demanding a training effort of two sessions weekly for 12 weeks: cognitive training (CT; n = 16), conventional resistance training (RET; n = 23) and eccentric ergometer training (EET; n = 23). Subjects were tested for functional parameters and body composition. Biopsies were collected from M. vastus lateralis before and after the intervention for the assessment of fiber size and composition. Maximal isometric leg extension strength (MEL: +8.4 +/- 1.7%) and eccentric muscle coordination (COORD: -43 +/- 4%) were significantly improved with EET but not with RET (MEL: +2.3 +/- 2.0%; COORD: -13 +/- 3%) and CT (MEL: -2.3 +/- 2.5%; COORD: -12 +/- 5%), respectively. We observed a loss of body fat (-5.0 +/- 1.1%) and thigh fat (-6.9 +/- 1.5%) in EET subjects only. Relative thigh lean mass increased with EET (+2.5 +/- 0.6%) and RET (+2.0 +/- 0.3%) and correlated negatively with type IIX/type II muscle fiber ratios. It was concluded that both RET and EET are beneficial for the elderly with regard to muscle functional and structural improvements but differ in their spectrum of effects. A training frequency of only two sessions per week seems to be the lower limit for a training stimulus to reveal measurable benefits.

Heart rate response determines long term exercise capacity after heart transplantation

BACKGROUND: Exercise capacity after heart transplantation (HTx) remains limited despite normal left ventricular systolic function of the allograft. Various clinical and haemodynamic parameters are predictive of exercise capacity following HTx. However, the predictive significance of chronotropic competence has not been demonstrated unequivocally despite its immediate relevance for cardiac output. AIMS: This study assesses the predictive value of various clinical and haemodynamic parameters for exercise capacity in HTx recipients with complete chronotropic competence evolving within the first 6 postoperative months. METHODS: 51 patients were enrolled in this exercise study. Patients were included when at least >6 months after HTx and without negative chronotropic medication or factors limiting exercise capacity such as significant transplant vasculopathy or allograft rejection. Clinical parameters were obtained by chart review, haemodynamic parameters from current cardiac catheterisation, and exercise capacity was assessed by treadmill stress testing. A stepwise multiple regression model analysed the proportion of the variance explained by the predictive parameters. RESULTS: The mean age of these 51 HTx recipients was 55.4 +/- 13.2 yrs on inclusion, 42 pts were male and the mean time interval after cardiac transplantation was 5.1 +/- 2.8 yrs. Five independent predictors explained 47.5% of the variance observed for peak exercise capacity (adjusted R2 = 0.475). In detail, heart rate response explained 31.6%, male gender 5.2%, age 4.1%, pulmonary vascular resistance 3.7%, and body-mass index 2.9%. CONCLUSION: Heart rate response is one of the most important predictors of exercise capacity in HTx recipients with complete chronotropic competence and without relevant transplant vasculopathy or acute allograft rejection.

Diminished prefrontal brain function in adults with psychopathology in childhood related to attention deficit hyperactivity disorder

The aim of the present study was to investigate prefrontal brain function and cognitive response control in patients with personality disorders who either suffered or did not suffer from psychopathology related to attention deficit hyperactivity disorder (ADHD) during childhood. For this purpose, 36 psychiatric out-patients with personality disorders--24 of whom showed ADHD-related psychopathology during childhood assessed by the German short form of the Wender Utah Rating Scale--and 24 healthy controls were investigated electrophysiologically by means of a cued Go-NoGo task (Continuous Performance Test). Topographical analyses were conducted to individually quantify the NoGo anteriorisation (NGA), a neurophysiological correlate of prefrontal response control that has been suggested to reflect activation of the anterior cingulate cortex. ADHD patients exhibited a significantly reduced mean NGA and diminished amplitudes of the Global Field Power, as well as a reduced increase of fronto-central P300 amplitudes, in NoGo-trials compared with the healthy controls, whereas patients with personality disorders alone did not differ from the control group in any of the electrophysiological parameters. The results indicate that ADHD-related psychopathology is associated with prefrontal brain dysfunction, probably related to processes of response inhibition and/or cognitive response control.

Copy number variation of the Beta-defensin genes in europeans: no supporting evidence for association with lung function, chronic obstructive pulmonary disease or asthma

Lung function measures are heritable, predict mortality and are relevant in diagnosis of chronic obstructive pulmonary disease (COPD). COPD and asthma are diseases of the airways with major public health impacts and each have a heritable component. Genome-wide association studies of SNPs have revealed novel genetic associations with both diseases but only account for a small proportion of the heritability. Complex copy number variation may account for some of the missing heritability. A well-characterised genomic region of complex copy number variation contains beta-defensin genes (DEFB103, DEFB104 and DEFB4), which have a role in the innate immune response. Previous studies have implicated these and related genes as being associated with asthma or COPD. We hypothesised that copy number variation of these genes may play a role in lung function in the general population and in COPD and asthma risk. We undertook copy number typing of this locus in 1149 adult and 689 children using a paralogue ratio test and investigated association with COPD, asthma and lung function. Replication of findings was assessed in a larger independent sample of COPD cases and smoking controls. We found evidence for an association of beta-defensin copy number with COPD in the adult cohort (OR = 1.4, 95%CI:1.02-1.92, P = 0.039) but this finding, and findings from a previous study, were not replicated in a larger follow-up sample(OR = 0.89, 95%CI:0.72-1.07, P = 0.217). No robust evidence of association with asthma in children was observed. We found no evidence for association between beta-defensin copy number and lung function in the general populations. Our findings suggest that previous reports of association of beta-defensin copy number with COPD should be viewed with caution. Suboptimal measurement of copy number can lead to spurious associations. Further beta-defensin copy number measurement in larger sample sizes of COPD cases and children with asthma are needed.

Loss-of-function mutations in the KCNJ8-encoded Kir6.1 K(ATP) channel and sudden infant death syndrome.

BACKGROUND Approximately 10% of sudden infant death syndrome (SIDS) may stem from cardiac channelopathies. The KCNJ8-encoded Kir6.1 (K(ATP)) channel critically regulates vascular tone and cardiac adaptive response to systemic metabolic stressors, including sepsis. KCNJ8-deficient mice are prone to premature sudden death, particularly with infection. We determined the spectrum, prevalence, and function of KCNJ8 mutations in a large SIDS cohort. METHODS AND RESULTS Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, comprehensive open reading frame/splice-site mutational analysis of KCNJ8 was performed on genomic DNA isolated from necropsy tissue on 292 unrelated SIDS cases (178 males, 204 white; age, 2.9±1.9 months). KCNJ8 mutations were coexpressed heterologously with SUR2A in COS-1 cells and characterized using whole-cell patch-clamp. Two novel KCNJ8 mutations were identified. A 5-month-old white male had an in-frame deletion (E332del) and a 2-month-old black female had a missense mutation (V346I). Both mutations localized to Kir6.1's C-terminus, involved conserved residues and were absent in 400 and 200 ethnic-matched reference alleles respectively. Both cases were negative for mutations in established channelopathic genes. Compared with WT, the pinacidil-activated K(ATP) current was decreased 45% to 68% for Kir6.1-E332del and 40% to 57% for V346I between -20 mV and 40 mV. CONCLUSIONS Molecular and functional evidence implicated loss-of-function KCNJ8 mutations as a novel pathogenic mechanism in SIDS, possibly by predisposition of a maladaptive cardiac response to systemic metabolic stressors akin to the mouse models of KCNJ8 deficiency.

The Molecular Crosstalk between the MET Receptor Tyrosine Kinase and the DNA Damage Response - Biological and Clinical Aspects

Radiation therapy remains an imperative treatment modality for numerous malignancies. Enduring significant technical achievements both on the levels of treatment planning and radiation delivery have led to improvements in local control of tumor growth and reduction in healthy tissue toxicity. Nevertheless, resistance mechanisms, which presumably also involve activation of DNA damage response signaling pathways that eventually may account for loco-regional relapse and consequent tumor progression, still remain a critical problem. Accumulating data suggest that signaling via growth factor receptor tyrosine kinases, which are aberrantly expressed in many tumors, may interfere with the cytotoxic impact of ionizing radiation via the direct activation of the DNA damage response, leading eventually to so-called tumor radioresistance. The aim of this review is to overview the current known data that support a molecular crosstalk between the hepatocyte growth factor receptor tyrosine kinase MET and the DNA damage response. Apart of extending well established concepts over MET biology beyond its function as a growth factor receptor, these observations directly relate to the role of its aberrant activity in resistance to DNA damaging agents, such as ionizing radiation, which are routinely used in cancer therapy and advocate tumor sensitization towards DNA damaging agents in combination with MET targeting.

Oxidative-nitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia

BACKGROUND Acute exposure to high altitude stimulates free radical formation in lowlanders, yet whether this persists during chronic exposure in healthy, well-adapted and maladapted highlanders suffering from chronic mountain sickness (CMS) remains to be established. METHODS Oxidative-nitrosative stress (as determined by the presence of the biomarkers ascorbate radical [A •- ], via electron paramagnetic resonance spectroscopy, and nitrite [NO 2 2 ], via ozone-based chemiluminescence) was assessed in venous blood of 25 male highlanders in Bolivia living at 3,600 m with CMS (n 5 13, CMS 1 ) and without CMS (n 5 12, CMS 2 ). Twelve age- and activity-matched, healthy, male lowlanders were examined at sea level and during acute hypoxia. We also measured fl ow-mediated dilatation (FMD), arterial stiffness defined by augmentation index normalized for a heart rate of 75 beats/min (AIx-75), and carotid intima-media thickness (IMT). RESULTS Compared with normoxic lowlanders, oxidative-nitrosative stress was moderately increased in the CMS 2 group ( P , .05), as indicated by elevated A •- (3,191 457 arbitrary units [AU] vs 2,640 445 AU) and lower NO 2 2 (206 55 nM vs 420 128 nM), whereas vascular function remained preserved. This was comparable to that observed during acute hypoxia in lowlanders in whom vascular dysfunction is typically observed. In contrast, this response was markedly exaggerated in CMS 1 group (A •- , 3,765 429 AU; NO 2 2 , 148 50 nM) compared with both the CMS 2 group and lowlanders ( P , .05). This was associated with systemic vascular dysfunction as indicated by lower ( P , .05 vs CMS 2 ) FMD (4.2% 0.7% vs 7.6% 1.7%) and increased AIx-75 (23% 8% vs 12% 7%) and carotid IMT (714 127 m M vs 588 94 m M). CONCLUSIONS Healthy highlanders display a moderate, sustained elevation in oxidative-nitrosative stress that, unlike the equivalent increase evoked by acute hypoxia in healthy lowlanders, failed to affect vascular function. Its more marked elevation in patients with CMS may contribute to systemic vascular dysfunction.

Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8(+) memory T cells.

Protection against malaria can be achieved by induction of a strong CD8(+) T-cell response against the Plasmodium circumsporozoite protein (CSP), but most subunit vaccines suffer from insufficient memory responses. In the present study, we analyzed the impact of postimmunization sporozoite challenge on the development of long-lasting immunity. BALB/c mice were immunized by a heterologous prime/boost regimen against Plasmodium berghei CSP that induces a strong CD8(+) T-cell response and sterile protection, which is short-lived. Here, we show that protective immunity is prolonged by a sporozoite challenge after immunization. Repeated challenges induced sporozoite-specific antibodies that showed protective capacity. The numbers of CSP-specific CD8(+) T cells were not substantially enhanced by sporozoite infections; however, CSP-specific memory CD8(+) T cells of challenged mice displayed a higher cytotoxic activity than memory T cells of immunized-only mice. CD4(+) T cells contributed to protection as well; but CD8(+) memory T cells were found to be the central mediator of sterile protection. Based on these data, we suggest that prolonged protective immunity observed after immunization and infection is composed of different antiparasitic mechanisms including CD8(+) effector-memory T cells with increased cytotoxic activity as well as CD4(+) memory T cells and neutralizing antibodies.

Is the relation between early post-session reports and treatment outcome an epiphenomenon of intake distress and early response? A multi-predictor analysis in outpatient psychotherapy

The early phase of psychotherapy has been regarded as a sensitive period in the unfolding of psychotherapy leading to positive outcomes. However, there is disagreement about the degree to which early (especially relationship-related) session experiences predict outcome over and above initial levels of distress and early response to treatment. The goal of the present study was to simultaneously examine outcome at post treatment as a function of (a) intake symptom and interpersonal distress as well as early change in well-being and symptoms, (b) the patient's early session-experiences, (c) the therapist's early session-experiences/interventions, and (d) their interactions. The data of 430 psychotherapy completers treated by 151 therapists were analyzed using hierarchical linear models. Results indicate that early positive intra- and interpersonal session experiences as reported by patients and therapists after the sessions explained 58% of variance of a composite outcome measure, taking intake distress and early response into account. All predictors (other than problem-activating therapists' interventions) contributed to later treatment outcomes if entered as single predictors. However, the multi-predictor analyses indicated that interpersonal distress at intake as well as the early interpersonal session experiences by patients and therapists remained robust predictors of outcome. The findings underscore that early in therapy therapists (and their supervisors) need to understand and monitor multiple interconnected components simultaneously

Ventilatory response to nitrogen multiple-breath washout in infants

BACKGROUND Nitrogen multiple-breath washout (N2 MBW) using 100% oxygen (O2 ) has regained interest to assess efficiency of tracer gas clearance in, for example, children with Cystic Fibrosis (CF). However, the influence of hyperoxia on the infants' respiratory control is unclear. We assessed safety and impact on breathing pattern from hyperoxia, and if exposure to 40% O2 first induces tolerance to subsequent 100% O2 for N2 MBW. METHODS We prospectively enrolled 39 infants aged 3-57 weeks: 15 infants with CF (8 sedated for testing) and 24 healthy controls. Infants were consecutively allocated to the protocols comprising of 100% O2 or 40/100% O2 administered for 30 breaths. Lung function was measured using an ultrasonic flowmeter setup. Primary outcome was tidal volume (VT ). RESULTS None of the infants experienced apnea, desaturation, or bradycardia. Both protocols initially induced hypoventilation. VT temporarily declined in 33/39 infants across 10-25 breaths. Hypoventilation occurred independent of age, disease, and sedation. In the new 40/100% O2 protocol, VT returned to baseline during 40% O2 and remained stable during 100% O2 exposure. End-tidal carbon dioxide monitored online did not change. CONCLUSION The classical N2 MBW protocol with 100% O2 may change breathing patterns of the infants. The new protocol with 40% O2 induces hyperoxia-tolerance and does not lead to changes in breathing patterns during later N2 washout using 100% O2 . Both protocols are safe, the new protocol seems an attractive option for N2 MBW in infants. Pediatr Pulmonol. © 2013 Wiley Periodicals, Inc.

BVDV: a pestivirus inducing tolerance of the innate immune response

Animals persistently infected (PI) with bovine viral diarrhea virus (BVDV) retain a strain-specific B- and T-cell immunotolerance. Pestiviral RNA triggers interferon (IFN) synthesis, and the viral RNase E(rns) inhibits IFN expression induced by extracellular viral RNA. In addition, N(pro) promotes the degradation of the transcription factor IRF-3, which effectively blocks IFN expression in BVDV-infected cells. As not all the potential target cells are infected in PI animals, these are 'chimeric' with respect to BVDV. This suggests that N(pro) and E(rns) are non-redundant IFN antagonists that act in infected and non-infected cells, respectively. Moreover, E(rns) may take a paradoxical function, both as virulence as well as "attenuation" factor: The former by preventing the activation of the innate and, consequently, of the adaptive immune system, the latter by minimizing the detrimental effects of systemic IFN production. Thus, BVDV maintains "self-tolerance" by avoiding the induction of IFN while itself being largely resistant to it without, however, interfering with the IFN action against unrelated viruses ('nonself'). This unique extension of 'self' to a virus suggests that the host's own RNases may have evolved as a guard against inadvertent activation of the innate immune system by host RNA, thus establishing a state of "innate tolerance".

Measurement of the vascular input function in mice for DCE-MRI

DCE-MRI is an important technique in the study of small animal cancer models because its sensitivity to vascular changes opens the possibility of quantitative assessment of early therapeutic response. However, extraction of physiologically descriptive parameters from DCE-MRI data relies upon measurement of the vascular input function (VIF), which represents the contrast agent concentration time course in the blood plasma. This is difficult in small animal models due to artifacts associated with partial volume, inflow enhancement, and the limited temporal resolution achievable with MR imaging. In this work, the development of a suite of techniques for high temporal resolution, artifact resistant measurement of the VIF in mice is described. One obstacle in VIF measurement is inflow enhancement, which decreases the sensitivity of the MR signal to the presence of contrast agent. Because the traditional techniques used to suppress inflow enhancement degrade the achievable spatiotemporal resolution of the pulse sequence, improvements can be achieved by reducing the time required for the suppression. Thus, a novel RF pulse which provides spatial presaturation contemporaneously with the RF excitation was implemented and evaluated. This maximizes the achievable temporal resolution by removing the additional RF and gradient pulses typically required for suppression of inflow enhancement. A second challenge is achieving the temporal resolution required for accurate characterization of the VIF, which exceeds what can be achieved with conventional imaging techniques while maintaining adequate spatial resolution and tumor coverage. Thus, an anatomically constrained reconstruction strategy was developed that allows for sampling of the VIF at extremely high acceleration factors, permitting capture of the initial pass of the contrast agent in mice. Simulation, phantom, and in vivo validation of all components were performed. Finally, the two components were used to perform VIF measurement in the murine heart. An in vivo study of the VIF reproducibility was performed, and an improvement in the measured injection-to-injection variation was observed. This will lead to improvements in the reliability of quantitative DCE-MRI measurements and increase their sensitivity.

Differential dynamic properties of scleroderma fibroblasts in response to perturbation of environmental stimuli.

Diseases are believed to arise from dysregulation of biological systems (pathways) perturbed by environmental triggers. Biological systems as a whole are not just the sum of their components, rather ever-changing, complex and dynamic systems over time in response to internal and external perturbation. In the past, biologists have mainly focused on studying either functions of isolated genes or steady-states of small biological pathways. However, it is systems dynamics that play an essential role in giving rise to cellular function/dysfunction which cause diseases, such as growth, differentiation, division and apoptosis. Biological phenomena of the entire organism are not only determined by steady-state characteristics of the biological systems, but also by intrinsic dynamic properties of biological systems, including stability, transient-response, and controllability, which determine how the systems maintain their functions and performance under a broad range of random internal and external perturbations. As a proof of principle, we examine signal transduction pathways and genetic regulatory pathways as biological systems. We employ widely used state-space equations in systems science to model biological systems, and use expectation-maximization (EM) algorithms and Kalman filter to estimate the parameters in the models. We apply the developed state-space models to human fibroblasts obtained from the autoimmune fibrosing disease, scleroderma, and then perform dynamic analysis of partial TGF-beta pathway in both normal and scleroderma fibroblasts stimulated by silica. We find that TGF-beta pathway under perturbation of silica shows significant differences in dynamic properties between normal and scleroderma fibroblasts. Our findings may open a new avenue in exploring the functions of cells and mechanism operative in disease development.

Effects of ex vivo γ-tocopherol on airway macrophage function in healthy and mild allergic asthmatics

Elevated inflammation and altered immune responses are features found in atopic asthmatic airways. Recent studies indicate γ-tocopherol (GT) supplementation can suppress airway inflammation in allergic asthma. We studied the effects of in vitro GT supplementation on receptor-mediated phagocytosis and expression of cell surface molecules associated with innate and adaptive immunity on sputum-derived macrophages. Cells from nonsmoking healthy (n = 6) and mild house dust mite-sensitive allergic asthmatics (n = 6) were treated ex vivo with GT (300 µM) or saline (control). Phagocytosis of opsonized zymosan A bioparticles (Saccharomyces cerevisiae) and expression of surface molecules associated with innate and adaptive immunity were assessed using flow cytometry. GT caused significantly decreased (p < 0.05) internalization of attached zymosan bioparticles and decreased (p < 0.05) macrophage expression of CD206, CD36 and CD86 in allergic asthmatics but not in controls. Overall, GT caused downregulation of both innate and adaptive immune response elements, and atopic status appears to be an important factor.