A Novel Approach for Foreign Substances Detection in Injection Using Clustering and Frame Difference

This paper focuses on developing a novel technique based on machine vision for detection of foreign substances in injections. Mechanical control yields spin/stop movement of injections which helps to cause relative movement between foreign substances in liquid and an ampoule bottle. Foreign substances are classified into two categories: subsiding-slowly object and subsiding-fast object. A sequence of frames are captured by a camera and used to recognize foreign substances. After image preprocessing like noise reduction and motion detection, two different methods, Moving-object Clustering (MC) and Frame Difference, are proposed to detect the two categories respectively. MC is operated to cluster subsiding-slowly foreign substances, based on the invariant features of those objects. Frame Difference is defined to calculate the difference between two frames due to the change of subsiding-fast objects. 200 ampoule samples filled with injection are tested and the experimental result indicates that the approach can detect the visible foreign substances effectively

Verification of criterion-related validity of the evaluation method of postural stability using the frame subtraction method.

It is important to quantify the postural stability. The frame subtraction method can calculate the motions of a subject, and might be easier to implement, with lower costs. However, validity of the evaluation of postural stability using this method have not been validated yet. Therefore, the purpose of this study was to verify criterion-related validity of the frame subtraction scores and the center of pressure (COP) parameters during maintenance of single leg standing. Twenty two healthy young subjects participated in this study. Motion tasks comprised right leg standing with eyes open and closed. The total length of COP displacements (LNG), Root mean square (RMS) area, anterior - posterior (AP) range, medial - lateral (ML) range were recorded using the force plate. Simultaneously, the motion images were acquired with digital video cameras from the front and right sides. After the motion images were analyzed using the frame subtraction method, the frame subtraction scores (maximumsum of the frame subtraction score on each planethe frontal and sagittal planes) were measured. To confirm the validity, Spearman's rank correlation coefficient between the frame subtraction scores and the COP parameters was calculated. The sum of the frame subtraction score on the frontal plane was significantly correlated with all COP displacements in the single leg standing. The result of this study indicated that the frame subtraction method could be applied to the evaluation of balance task with postural sway such as maintenance of single leg standing. The frame subtraction method is low cost and easy owing to its marker-less systems

Labeling of immune cells for in vivo monitoring of cell migration using magnetic resonance imaging and near-infrared imaging

In addition to macrophage localization, the opening of the blood brain barrier (BBB) as well as demyelination processes have been measured by MRI mapping gadolinium (Gd) related signal enhancement and magnetic transfer (MT), respectively. By successively applying this protocol at different time points during the EAE model progression, we were able to analyse the interdependence of immuno-cellular processes leading to axonal damage as well as the longitudinal evolution of pathological hallmarks of EAE. Furthermore, these techniques have been used to validate and quantify the anti-inflammatory effect of EDG-1 inhibitor FTY720 on EAE symptoms. Repeated USPIO administrations and MRI measurements combining the analysis of MT ratios and Gd-enhancement have been performed on vehicle and FTY720 treated animals. This study demonstrates that FTY720 can prevent inflammatory events in EAE rats by sequestrating immune cells in lymphoid organs during acute inflammation episodes. The third goal, was to translate the iron-labeling protocol from macrophages to T lymphocytes. As T-cells are initiators of the immune cascade leading to the occurence of symptoms in the EAE model, it would be highly relevant to visualize T lymphocytes prior to the onset of symptoms. Yet, as lymphocytes have no natural phagocytotic activity, in vivo tagging with CA was not feasible. We decided to label them in vitro with ferumoxides (FeO) and then, transfer iron-presenting cells adoptively to EAE animals intravenously. Different techniques have been used to evaluate the efficiency of lymphocytes labeling combining iron oxide particles with commonly available transfection agents (TAs) and the feasibility of labeling T lymphocytes in vitro has been demonstrated. However, the adoptive transfer of iron-tagged T-cells to EAE rats did not lead to the detection of these cells by MRI. As MR detection of iron-tagged cell in vivo was unsuccessful probably due to the inherent lack of sensitivity of the MRI technique for molecular changes and the dilution of labeled cells in the blood, we decided to switch to a more sensitive technique. Thus, the goal of the last part of the thesis was to label primary cultured T lymphocytes with a fluorescent dye: cyanine 5.5 (Cy5.5). The Tat peptide from the HIV virus chemically has been bound to the Cy5.5 to cargo the dye across T-cells membrane. The ability of this probe to penetrate T-cells and its potential toxicity has been evaluated in vitro. Subsequently, Cy5.5-Tat labeled lymphocytes were transferred to EAE rats in order to monitor their bio-distribution during EAE. Prominent signals have been obtained from rat brain and histological experimentation using confocal microscopy analysis have been performed to confirm the localization of Cy5.5 within the brain parenchyma

A proteomic investigation of the immune response of the South African abalone, Haliotis midae

Includes bibliographical referencesHaliotis midae is a commercially important abalone in South Africa, previously harvested from a stable, quota-managed fishery. However, the combined effects of overharvesting, increased illegal catches and negative environmental factors led to a collapse in wild populations in the mid-90s. Consequently, land-based aquaculture of H. midae has grown significantly in South Africa, to satisfy the global demand for abalone and alleviate pressure on wild stocks. Unfortunately, disease outbreaks have had a severe impact on the abalone aquaculture industry internationally and remain one of the single biggest factors contributing to economic loss. Understanding the effects of pathogen infection of abalone is therefore crucial to mitigating and controlling infection outbreaks on farms. Despite this, the molecular mechanisms underlying the immune response of H. midae remain obscure. High-throughput proteomics, a powerful tool to analyse global protein expression changes, can provide an integrated view of the immune system. Thus, this study aimed to elucidate the haemocyte proteome of H. midae and gain insight into regulatory molecular pathways underlying innate immunity. In this study, a comparative shotgun proteomics approach using isobaric tagging for relative and absolute quantification (iTRAQ) coupled with LC-MS/MS was employed to investigate H. midae proteome changes in response to Vibrio anguillarum challenge. A preliminary iTRAQ challenge trial was conducted which identified a putative early (1 and 2 hours post-injection) and late (48 hours post-injection) proteome response to bacterial-challenge. Using these time points, four independent challenge trials were conducted and analysed by iTRAQ and the results combined to produce a high-confidence dataset with good quantitative reproducibility for statistical analysis. A parallel set of experiments was conducted using mock-infected samples

2013 Annual Research Symposium Abstract Book

2013 annual volume of abstracts for science research projects conducted by students at Trinity College

Interactions of entomopathogenic fungi and other control agents: mechanism and field potential against Hylobius abietis larvae

Biological control is the beneficial application of natural enemies such as pathogens, predators and parasites in managing pests and their damage. Entomopathogenic fungi (EPF) have a crucial role in natural ecosystems and are being developed as alternative control agents for insect pests. Both Beauvaria bassiana and Metarhizium anisopliae have been proven to be effective biological control agents against a range of pests and are commercially produced. Advantages of using EPF for biological pest control include their degree of specificity, absence of effects on mammals, reduced probability of insects developing resistance and they may persist for long periods in some environments which could provide long term control effects. Disadvantages include that it takes EPF longer to kill insects than their chemical counterparts, application needs to be timed for high relative humidity and low pest numbers, and efficacy varies among different insect species. If a combination of treatments resulted in a synergistic interaction then the efficacy of these biopesticides would be increased. A number of laboratory and field studies have used combinations of entomopathogenic nematodes (EPN) and EPF against insect pests with resulting interactions ranging from antagonistic to synergistic. In the case of synergism resulting from combined applications it is suggested that EPF may make the host more susceptible through suppressing its immune system. To understand how this putative synergistic interaction between control agents could occur mechanistically, the effect of EPF supernatant was tested on the immune response of the forestry pest, Hylobius abietis, to screen for species with immunomodulating properties. The potential of the commonly used model organism, the greater wax moth, Galleria mellonella, as a model for the study of the immune response of H. abietis to pathogens was also explored. Hylobius abietis, the large pine weevil, is a major pest of reforestation in Europe. It is estimated that H. abietis costs the forestry industry approximately €140 million/year. Current control measures rely heavily on the synthetic chemical cypermethrin. However, due to concerns over its environmental impact cypermethrin is being phased out across Europe. Therefore there is an interest in the use of entomopathogens as biological control agents in integrated pest management. Thus one objective of this work was to assess the efficacy of EPF, EPN and EPF-EPN combinations for H. abietis suppression in the field in order to ultimately determine if the treatments used exert synergistic control over H. abietis. The effect of EPF supernatant on the immune response of insects was assessed through a number of bioassays that investigated the effect of EPF on haemocyte densities and yeast proliferation in the insect haemocoel, as well as testing whether pre-treatment with EPF increases larval susceptibility to subsequent pathogens. The effect of EPF supernatant on the humoral immune response was investigated by subjecting larval haemolymph to label free quantitative (LFQ) proteomic analysis. To enable proteomic investigations into the effects of EPF on the immune response of H. abietis and to compensate for the lack of genomic information for H. abietis; a de novo transcriptome study of H. abietis larvae was performed with Beijing Genomics Institute (BGI, Hong Kong). Bioassays indicated that M. anisopliae, B. bassiana and Beauveria caledonica, demonstrated immunomodulating effects on H. abietis larvae, while M. anisopliae and B. caledonica modulated the immune response of G. mellonella making the insect more susceptible to subsequent pathogens. LFQ analysis on larval haemolymph showed that in response to EPF supernatant both insect species displayed altered abundance of proteins involved in antimicrobial defence, the prophenoloxidase cascade, detoxification and detection and sensing. These patterns of alteration may be integral to the modulation of the host immune response by EPF. A major difference observed between the proteomic profiles of H. abietis and G. mellonella haemolymph was that H. abietis injected with B. caledonica supernatant had a major alteration in metabolic proteins involved in cellulose cleavage, reflective of its wood based diet. It was concluded G. mellonella may have an application as a model for looking for secondary metabolites or natural products that display immunomodulating properties so that EPF isolates could be screened for production of these products. However G. mellonella are not a substitute for the target pest for proteomic analysis. Moreover laboratory bioassays with either G. mellonella or H. abietis are not predictive of whether synergy will occur in the field as there are many more factors involved than just the ability of EPF to modulate the immune response. To investigate the ability of EPF and EPN to suppress H. abietis populations in the field, three field studies were carried out over three consecutive years. Treatments were applied to tree stumps harbouring H. abietis developmental stages. The efficacy of EPF and EPN was investigated alone and in combination through emergence trapping and destructive sampling. Three EPF strains were utilised in these field studies, commercial strains of M. anisopliae and B. bassiana and a strain of B. caledonica native to Ireland. Two EPN species were utilised in these field studies, Steinernema carpocapsae and Heterorhabditis downesi, the latter is native to Ireland. In this work EPN were found to offer superior control over H. abietis in the field than EPF, with all treatments that caused significant reduction in adult emergence being EPN alone or EPN in combination with EPF. Ultimately EPF are not suited to control of H. abietis using this strategy. Synergy between EPF-EPN was not achieved in any of the three field studies with all combinations tested giving additive results. The final aim of this work was to investigate if the little-studied native fungus B. caledonica produces immunomodulating compounds active against H. abietis. Identification, large scale production and structural determination of an abundant secreted natural product of B. caledonica was carried out using High Performance Liquid Chromatography (HPLC) and Nuclear magnetic resonance (NMR) spectroscopy, and the metabolite of interest was found to be oosporein. Subsequently assessment of the potential insecticidal, anti-feedant and immunomodulating effects of oosporein was carried out. Oosporein was identified as an abundant metabolite in B. caledonica supernatant that displayed immunomodulating properties in H. abietis larvae as well as being a feeding stimulant to H. abietis adults. The work presented throughout this thesis offers the first report of the effect of EPF on the cellular and humoral immune system of H. abietis. It presents a critical assessment of G. mellonella as a model organism for immune studies of H. abietis. It highlights that EPN are a superior biocontrol agent for the control of H. abietis in the context of the current approach used. However the native B. caledonica isolate tested throughout this thesis presents an interesting avenue for future research into the utilisation of EPF as biocontrol agents in a wider sense than researched in this work

Analysis of contrast-enhanced medical images.

Early detection of human organ diseases is of great importance for the accurate diagnosis and institution of appropriate therapies. This can potentially prevent progression to end-stage disease by detecting precursors that evaluate organ functionality. In addition, it also assists the clinicians for therapy evaluation, tracking diseases progression, and surgery operations. Advances in functional and contrast-enhanced (CE) medical images enabled accurate noninvasive evaluation of organ functionality due to their ability to provide superior anatomical and functional information about the tissue-of-interest. The main objective of this dissertation is to develop a computer-aided diagnostic (CAD) system for analyzing complex data from CE magnetic resonance imaging (MRI). The developed CAD system has been tested in three case studies: (i) early detection of acute renal transplant rejection, (ii) evaluation of myocardial perfusion in patients with ischemic heart disease after heart attack; and (iii), early detection of prostate cancer. However, developing a noninvasive CAD system for the analysis of CE medical images is subject to multiple challenges, including, but are not limited to, image noise and inhomogeneity, nonlinear signal intensity changes of the images over the time course of data acquisition, appearances and shape changes (deformations) of the organ-of-interest during data acquisition, determination of the best features (indexes) that describe the perfusion of a contrast agent (CA) into the tissue. To address these challenges, this dissertation focuses on building new mathematical models and learning techniques that facilitate accurate analysis of CAs perfusion in living organs and include: (i) accurate mathematical models for the segmentation of the object-of-interest, which integrate object shape and appearance features in terms of pixel/voxel-wise image intensities and their spatial interactions; (ii) motion correction techniques that combine both global and local models, which exploit geometric features, rather than image intensities to avoid problems associated with nonlinear intensity variations of the CE images; (iii) fusion of multiple features using the genetic algorithm. The proposed techniques have been integrated into CAD systems that have been tested in, but not limited to, three clinical studies. First, a noninvasive CAD system is proposed for the early and accurate diagnosis of acute renal transplant rejection using dynamic contrast-enhanced MRI (DCE-MRI). Acute rejection–the immunological response of the human immune system to a foreign kidney–is the most sever cause of renal dysfunction among other diagnostic possibilities, including acute tubular necrosis and immune drug toxicity. In the U.S., approximately 17,736 renal transplants are performed annually, and given the limited number of donors, transplanted kidney salvage is an important medical concern. Thus far, biopsy remains the gold standard for the assessment of renal transplant dysfunction, but only as the last resort because of its invasive nature, high cost, and potential morbidity rates. The diagnostic results of the proposed CAD system, based on the analysis of 50 independent in-vivo cases were 96% with a 95% confidence interval. These results clearly demonstrate the promise of the proposed image-based diagnostic CAD system as a supplement to the current technologies, such as nuclear imaging and ultrasonography, to determine the type of kidney dysfunction. Second, a comprehensive CAD system is developed for the characterization of myocardial perfusion and clinical status in heart failure and novel myoregeneration therapy using cardiac first-pass MRI (FP-MRI). Heart failure is considered the most important cause of morbidity and mortality in cardiovascular disease, which affects approximately 6 million U.S. patients annually. Ischemic heart disease is considered the most common underlying cause of heart failure. Therefore, the detection of the heart failure in its earliest forms is essential to prevent its relentless progression to premature death. While current medical studies focus on detecting pathological tissue and assessing contractile function of the diseased heart, this dissertation address the key issue of the effects of the myoregeneration therapy on the associated blood nutrient supply. Quantitative and qualitative assessment in a cohort of 24 perfusion data sets demonstrated the ability of the proposed framework to reveal regional perfusion improvements with therapy, and transmural perfusion differences across the myocardial wall; thus, it can aid in follow-up on treatment for patients undergoing the myoregeneration therapy. Finally, an image-based CAD system for early detection of prostate cancer using DCE-MRI is introduced. Prostate cancer is the most frequently diagnosed malignancy among men and remains the second leading cause of cancer-related death in the USA with more than 238,000 new cases and a mortality rate of about 30,000 in 2013. Therefore, early diagnosis of prostate cancer can improve the effectiveness of treatment and increase the patient’s chance of survival. Currently, needle biopsy is the gold standard for the diagnosis of prostate cancer. However, it is an invasive procedure with high costs and potential morbidity rates. Additionally, it has a higher possibility of producing false positive diagnosis due to relatively small needle biopsy samples. Application of the proposed CAD yield promising results in a cohort of 30 patients that would, in the near future, represent a supplement of the current technologies to determine prostate cancer type. The developed techniques have been compared to the state-of-the-art methods and demonstrated higher accuracy as shown in this dissertation. The proposed models (higher-order spatial interaction models, shape models, motion correction models, and perfusion analysis models) can be used in many of today’s CAD applications for early detection of a variety of diseases and medical conditions, and are expected to notably amplify the accuracy of CAD decisions based on the automated analysis of CE images

Regulation of B cell fate by chronic activity of the IgE B cell receptor.

IgE can trigger potent allergic responses, yet the mechanisms regulating IgE production are poorly understood. Here we reveal that IgE+ B cells are constrained by chronic activity of the IgE B cell receptor (BCR). In the absence of cognate antigen, the IgE BCR promoted terminal differentiation of B cells into plasma cells (PCs) under cell culture conditions mimicking T cell help. This antigen-independent PC differentiation involved multiple IgE domains and Syk, CD19, BLNK, Btk, and IRF4. Disruption of BCR signaling in mice led to consistently exaggerated IgE+ germinal center (GC) B cell but variably increased PC responses. We were unable to confirm reports that the IgE BCR directly promoted intrinsic apoptosis. Instead, IgE+ GC B cells exhibited poor antigen presentation and prolonged cell cycles, suggesting reduced competition for T cell help. We propose that chronic BCR activity and access to T cell help play critical roles in regulating IgE responses