In vivo clearance of Japanese encephalitis virus by adoptively transferred virus specific cytotoxic T lymphocytes

Japanese encephalitis virus (JEV) is a positive stranded RNA virus that belongs to the flavivirus group, JEV infection damages the central nervous system (CNS) and is one of the main causative agents of acute encephalitis, H-2 restricted virus-specific cytotoxic T lymphocytes (CTL) have been generated specifically against JEV in our laboratory and these CTL have been shown to protect mice against lethal challenge with JEV, Virus replication was found to be inhibited in the brains of animals that mere adoptively transferred with JEV specific CTL as revealed by immunohistological staining as,veil as viral plaque assays. We further show that virus specific CTL could be recovered from such protected mice as long as 45 days after adoptive transfer.

Angelica archengelica extract induced perturbation of rat skin and tight junctional protein (ZO-1) of HaCaT cells

Background and purpose of the study: Herbal enhancers compared to the synthetic ones have shown less toxis effects. Coumarins have been shown at concentrations inhibiting phospoliphase C-Y (Phc-Y) are able to enhance tight junction (TJ) permeability due to hyperpoalation of Zonolous Occludense-1 (ZO-1) proteins. The purpose of this study was to evaluate the influence of ethanolic extract of Angelica archengelica (AA-E) which contain coumarin on permeation of repaglinide across rat epidermis and on the tight junction plaque protein ZO-1 in HaCaT cells. Methods: Transepidermal water loss (TEWL) from the rat skin treated with different concentrations of AA-E was assessed by Tewameter. Scanning and Transmission Electron Microscopy (TEM) on were performed on AA-E treated rat skin portions. The possibility of AA-E influence on the architecture of tight junctions by adverse effect on the cytoplasmic ZO-1 in HaCaT cells was investigated. Finally, the systemic delivery of repaglinide from the optimized transdermal formulation was investigated in rats. Results: The permeation of repaglinide across excised rat epidermis was 7-fold higher in the presence of AA-E (5% w/v) as compared to propylene glycol:ethanol (7:3) mixture. The extract was found to perturb the lipid microconstituents in both excised and viable rat skin, although, the effect was less intense in the later. The enhanced permeation of repaglinide across rat epidermis excised after treatment with AA-E (5% w/v) for different periods was in concordance with the high TEWL values of similarly treated viable rat skin. Further, the observed increase in intercellular space, disordering of lipid structure and corneocyte detachment indicated considerable effect on the ultrastructure of rat epidermis. Treatment of HaCaT cell line with AA-E (0.16% w/v) for 6 hrs influenced ZO-1 as evidenced by reduced immunofluorescence of anti-TJP1 (ZO-1) antibody in Confocal Laser Scanning Microscopy studies (CLSM) studies. The plasma concentration of repaglinide from transdermal formulation was maintained higher and for longer time as compared to oral administration of repaglinide. Major conclusion: Results suggest the overwhelming influence of Angelica archengelica in enhancing the percutaneous permeation of repaglinide to be mediated through perturbation of skin lipids and tight junction protein (ZO-1).

Macromolecular Transport Through Porous Arterial Walls

Arteries are heterogeneous, composite structures that undergo large cyclic deformations during blood transport. Presence, build-up and consequent rupture of blockages in blood vessels, called atherosclerotic plaques, lead to disruption in the blood flow that can eventually be fatal. Abnormal lipid profile and hypertension are the main risk factors for plaque progression. Treatments span from pharmacological methods, to minimally invasive balloon angioplasty and stent procedures, and finally to surgical alternatives. There is a need to understand arterial disease progression and devise methods to detect, control, treat and manage arterial disease through early intervention. Local delivery through drug eluting stents also provide an attractive option for maintaining vessel integrity and restoring blood flow while releasing controlled amount of drug to reduce and alleviate symptoms. Development of drug eluting stents is hence interesting albeit challenging because it requires an integration of knowledge of mechanical properties with material transport of drug through the arterial wall to produce a desired biochemical effect. Although experimental models are useful in studying such complex multivariate phenomena, numerical models of mass transport in the vessel have proved immensely useful to understand and delineate complex interactions between chemical species, physical parameters and biological variables. The goals of this review are to summarize literature based on studies of mass transport involving low density lipoproteins in the arterial wall. We also discuss numerical models of drug elution from stents in layered and porous arterial walls that provide a unique platform that can be exploited for the design of novel drug eluting stents.

Withania somnifera reverses Alzheimer's disease pathology by enhancing low-density lipoprotein receptor-related protein in liver

A 30-d course of oral administration of a semipurified extract of the root of Withania somnifera consisting predominantly of withanolides and withanosides reversed behavioral deficits, plaque pathology, accumulation of beta-amyloid peptides (A beta) and oligomers in the brains of middle-aged and old APP/PS1 Alzheimer's disease transgenic mice. It was similarly effective in reversing behavioral deficits and plaque load in APPSwInd mice (line J20). The temporal sequence involved an increase in plasma A beta and a decrease in brain A beta monomer after 7 d, indicating increased transport of A beta from the brain to the periphery. Enhanced expression of low-density lipoprotein receptor-related protein (LRP) in brain microvessels and the A beta-degrading protease neprilysin (NEP) occurred 14-21 d after a substantial decrease in brain A beta levels. However, significant increase in liver LRP and NEP occurred much earlier, at 7 d, and were accompanied by a rise in plasma sLRP, a peripheral sink for brain A beta. In WT mice, the extract induced liver, but not brain, LRP and NEP and decreased plasma and brain A beta, indicating that increase in liver LRP and sLRP occurring independent of A beta concentration could result in clearance of A beta. Selective down-regulation of liver LRP, but not NEP, abrogated the therapeutic effects of the extract. The remarkable therapeutic effect of W. somnifera mediated through up-regulation of liver LRP indicates that targeting the periphery offers a unique mechanism for A beta clearance and reverses the behavioral deficits and pathology seen in Alzheimer's disease models.

Does aluminium bind to histidine? an NMR investigation of Amyloid beta 12 and Amyloid beta 16 fragments

Aluminium and zinc are known to be the major triggering agents for aggregation of amyloid peptides leading to plaque formation in Alzheimer's disease. While zinc binding to histidine in A (amyloid ) fragments has been implicated as responsible for aggregation, not much information is available on the interaction of aluminium with histidine. In the NMR study of the N-terminal A fragments, DAEFRHDSGYEV (A12) and DAEFRHDSGYEVHHQK (A16) presented here, the interactions of the fragments with aluminium have been investigated. Significant chemical shifts were observed for few residues near the C-terminus when aluminium chloride was titrated with A12 and A16 peptides. Surprisingly, it is nonhistidine residues which seem to be involved in aluminium binding. Based on NMR constrained structure obtained by molecular modelling, aluminium-binding pockets in A12 were around charged residues such as Asp, Glu. The results are discussed in terms of native structure propagation, and the relevance of histidine residues in the sequences for metal-binding interactions. We expect that the study of such short amyloid peptide fragments will not only provide clues for plaque formation in aggregated conditions but also facilitate design of potential drugs for these targets.

The Modified Selenenyl Amide, M-hydroxy Ebselen, Attenuates Diabetic Nephropathy and Diabetes-Associated Atherosclerosis in ApoE/GPx1 Double Knockout Mice

Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in in vitro studies for their ability to scavenge reactive oxygen and nitrogen species, including hydrogen peroxide and peroxynitrite. In this study, we investigated the efficacies of two Eb analogues, m-hydroxy ebselen (ME) and ethanol-ebselen (EtE) and compared these with Eb in cell based assays. We found that ME is superior in attenuating the activation of hydrogen peroxide-induced pro-inflammatory mediators, ERK and P38 in human aortic endothelial cells. Consequently, we investigated the effects of ME in an in vivo model of diabetes, the ApoE/GPx1 double knockout (dKO) mouse. We found that ME attenuates plaque formation in the aorta and lesion deposition within the aortic sinus of diabetic dKO mice. Oxidative stress as assessed by 8-OHdG in urine and nitrotyrosine immunostaining in the aortic sinus and kidney tubules, was reduced by ME in diabetic dKO mice. ME also attenuated diabetes-associated renal injury which included tubulointerstitial fibrosis and glomerulosclerosis. Furthermore, the bioactivity of the pro-fibrotic cytokine transforming growth factor-beta (TGF-beta) as assessed by phospho-Smad2/3 immunostaining was attenuated after treatment with ME. TGF-beta-stimulated increases in collagen I and IV gene expression and protein levels were attenuated by ME in rat kidney tubular cells. However, in contrast to the superior activity of ME in in vitro and cell based assays, ME did not further augment the attenuation of diabetes-associated atherosclerosis and renal injury in our in vivo model when compared with Eb. In conclusion, this study strengthens the notion that bolstering GPx-like activity using synthetic mimetics may be a useful therapeutic strategy in lessening the burden of diabetic complications. However, these studies highlight the importance of in vivo analyses to test the efficacies of novel Eb analogues, as in vitro and cell based assays are only partly predictive of the in vivo situation.

Coral diseases are major contributors to coral mortality in Shingle Island, Gulf of Mannar, southeastern India

The present study reports coral mortality, driven primarily by coral diseases, around Shingle Island, Gulf of Mannar (GOM), Indian Ocean. In total, 2910 colonies were permanently monitored to assess the incidence of coral diseases and consequent mortality for 2 yr. Four types of lesions consistent with white band disease (WBD), black disease (BD), white plaque disease (WPD), and pink spot disease (PSD) were recorded from 4 coral genera: Montipora, Pocillopora, Acropora, and Porites. Porites were affected by 2 disease types, while the other 3 genera were affected by only 1 disease type. Overall disease prevalence increased from 8% (n = 233 colonies) to 41.9% (n = 1219) over the 2 yr study period. BD caused an unprecedented 100% mortality in Pocillopora, followed by 20.4 and 13.1% mortality from WBD in Montipora and Acropora, respectively. Mean disease progression rates of 0.8 +/- 1.0 and 0.6 +/- 0.5 cm mo(-1) over live coral colonies were observed for BD and WBD. Significant correlations between temperature and disease progression were observed for BD (r = 0.86, R-2 = 0.75, p < 0.001) and WBD (R-2 = 0.76, p < 0.001). This study revealed the increasing trend of disease prevalence and progression of disease over live coral in a relatively limited study area; further study should investigate the status of the entire coral reef in the GOM and the role of diseases in reef dynamics.

An enzyme-linked immuno focus assay for rapid detection and enumeration, and a newborn mouse model for human non-polio enteroviruses associated with acute diarrhea

We have recently reported significant association of non-polio enteroviruses (NPEVs) with acute and persistent diarrhea (18-21% of total diarrheal cases), and non-diarrheal Increased Frequency of Bowel Movements (IFoBM-ND) (about 29% of the NPEV infections) in children and that the NPEV-associated diarrhea was as significant as rotavirus diarrhea. However, their diarrhea-causing potential is yet to be demonstrated in an animal model system. Since the determination of virus titers by the traditional plaque assay takes 4-7 days, there is a need for development of a rapid method for virus titer determination to facilitate active clinical research on enterovirus-associated diarrhea. The goal of this study is to develop a cell-based rapid detection and enumeration method and to demonstrate the diarrhea-inducing potential of purified and characterized non-polio enteroviruses, which were isolated from diarrheic children. Here we describe generation of monoclonal and polyclonal antibodies against purified strains belonging to different serotypes, and development of an enzyme-linked immuno focus assay (ELIFA) for detection and enumeration of live NPEV particles in clinical and purified virus samples, and a newborn mouse model for NPEV diarrhea. Plaque-purified NPVEs, belonging to different serotypes, isolated from children with diarrhea, were grown in cell culture and purified by isopycnic CsCl density gradient centrifugation. By ELIFA, NPEVs could be detected and enumerated within 12 h post-infection. Our results demonstrated that Coxsackievirus B1 (CVB1) and CVB5 strains, isolated from diarrheic children, induced severe diarrhea in orally-inoculated 9-12 day-old mouse pups, fulfilling Koch's postulates. The methods described here would facilitate studies on NPEV-associated gastrointestinal disease. (C) 2015 Elsevier B.V. All rights reserved.

The hemodynamic effects of in-tandem carotid artery stenosis: implications for carotid endarterectomy.

OBJECTIVES: It remains controversial whether patients with severe disease of the internal carotid artery and a coexisting stenotic lesion downstream would benefit from a carotid endarterectomy (CEA) of the proximal lesion. The aim of this study was to simulate the hemodynamic and wall shear effects of in-tandem internal carotid artery stenosis using a computational fluid dynamic (CFD) idealized model to give insight into the possible consequences of CEA on these lesions. METHODS: A CFD model of steady viscous flow in a rigid tube with two asymmetric stenoses was introduced to simulate blood flow in arteries with multiple constrictions. The effect of varying the distance between the two stenoses, and the severity of the upstream stenosis on the pressure and wall shear stress (WSS) distributions on the second plaque, was investigated. The influence of the relative positions of the two stenoses was also assessed. RESULTS: The distance between the plaques was found to have minimal influence on the overall hemodynamic effect except for the presence of a zone of low WSS (range -20 to 30 dyne/cm2) adjacent to both lesions when the two stenoses were sufficiently close (<4 times the arterial diameter). The upstream stenosis was protective if it was larger than the downstream stenosis. The relative positions of the stenoses were found to influence the WSS but not the pressure distribution. CONCLUSIONS: The geometry and positions of the lesions need to be considered when considering the hemodynamic effects of an in-tandem stenosis. Low WSS is thought to cause endothelial dysfunction and initiate atheroma formation. The fact that there was a flow recirculation zone with low WSS in between the two stenoses may demonstrate how two closely positioned plaques may merge into one larger lesion. Decision making for CEA may need to take into account the hemodynamic situation when an in-tandem stenosis is found. CFD may aid in the risk stratification of patients with this problem.

Age-dependent decrease in glutamine synthetase expression in the hippocampal astroglia of the triple transgenic Alzheimer's disease mouse model : mechanism for deficient glutamatergic transmission?

Astrocytes are fundamental for brain homeostasis and the progression and outcome of many neuropathologies including Alzheimer's disease (AD). In the triple transgenic mouse model of AD (3xTg-AD) generalised hippocampal astroglia atrophy precedes a restricted and specific beta-amyloid (A beta) plaque-related astrogliosis. Astrocytes are critical for CNS glutamatergic transmission being the principal elements of glutamate homeostasis through maintaining its synthesis, uptake and turnover via glutamate-glutamine shuttle. Glutamine synthetase (GS), which is specifically expressed in astrocytes, forms glutamine by an ATP-dependent amination of glutamate. Here, we report changes in GS astrocytic expression in two major cognitive areas of the hippocampus (the dentate gyrus, DG and the CA1) in 3xTg-AD animals aged between 9 and 18 months. We found a significant reduction in Nv (number of cell/mm(3)) of GS immunoreactive (GS-IR) astrocytes starting from 12 months (28.59%) of age in the DG, and sustained at 18 months (31.65%). CA1 decrease of GS-positive astrocytes Nv (33.26%) occurs at 18 months. This Nv reduction of GSIR astrocytes is paralleled by a decrease in overall GS expression (determined by its optical density) that becomes significant at 18 months (21.61% and 19.68% in DG and CA1, respectively). GS-IR Nv changes are directly associated with the presence of A beta deposits showing a decrease of 47.92% as opposed to 23.47% in areas free of A beta. These changes in GS containing astrocytes and GS-immunoreactivity indicate AD-related impairments of glutamate homeostatic system, at the advanced and late stages of the disease, which may affect the efficacy of glutamatergic transmission in the diseased brain that may contribute to the cognitive deficiency.

GFAP Isoforms in Adult Mouse Brain with a Focus on Neurogenic Astrocytes and Reactive Astrogliosis in Mouse Models of Alzheimer Disease

24 p.

细胞黏附的力学建模及其力敏感性的机理研究

细胞黏附在机体的生理和病理过程中起着重要的作用。作为细胞内、外信息交流和传递的通道，细胞黏附斑具有独特的力敏感性。实验表明，在力的作用下，黏附斑不仅可以生长、成熟和破坏，而且还能感知外部环境的力学性质，如基底硬度、硬度梯度和形貌等等。细胞黏附如何响应不同的力学刺激，物理机理是什么，如何定量描述这些物理机理？这些问题是细胞生物学和细胞力学中的重要问题。本论文通过在分子和亚细胞尺度上的力学建模研究了黏附斑的力敏感性机理，主要包括以下几方面的内容： (1) 发展了一个非线性的撕裂模型，研究了细胞黏附的稳定性和边缘依赖性。通过引入黏附分子键的非线性本构关系，并考虑黏附分子键的多种分布形式，我们发现黏附分子键的非线性效应对维持细胞黏附的稳定性起着至关重要的作用。黏附分子键的非线性力学性质使黏附分子键可以同时承载，降低了细胞对黏附分子键分布的依赖性，大大提高了细胞的黏附强度。本文的预测结果与实验结果一致。 (2) 建立了细胞黏附的细观力学模型，研究了在力作用下黏附斑生长和失稳的分子机理。在细观力学模型中，引入了“整联蛋白的聚集”和“整联蛋白-配体的反应”两个分子作用机理，并用两个化学反应来描述。通过基于Monte Carlo思想的Gillespie算法模拟了细胞黏附在不同载荷下的响应。我们发现黏附斑只能在一定范围的张力下生长，在这个范围内整联蛋白的聚集机制占主导。而当张力大于某个临界值时，黏附斑将失稳并导致破坏，这时整联蛋白-配体分子键的解离机制占主导。因此，黏附斑对作用力的不同响应，是不同分子作用机制在力作用下相互消长的结果。同时我们还建立了一个唯象的热力学模型中，验证了我们的细观力学模型。 (3) 基于细胞黏附的细观力学模型，研究了周期性载荷下细胞的重排和转向机理。在细观力学模型中，通过黏附块(adhesion plaque)，将整联蛋白-配体分子键和细胞骨架联系起来。基于Monte Calro模拟，我们发现存在一个载荷临界值，当外载大于临界值时，细胞将进行重排。细胞重排的原因是在周期性载荷下黏附斑的失稳。通过引入整联蛋白-配体成键的化学反应动力学和应力纤维的粘弹性性质，解释了细胞黏附稳定性的频率依赖性。本文预测的细胞转向临界载荷和重排方向，与实验结果一致。

Flexible microimplants for in vivo sensing

The work in this thesis develops two types of microimplants for the application of cardiovascular in vivo biomedical sensing, one for short-term diagnosis and the other for long-term monitoring.

Despite advances in diagnosis and therapy, atherosclerotic cardiovascular disease remains the leading cause of morbidity and mortality in the Western world. Predicting metabolically active atherosclerotic plaques has remained an unmet clinical need. A stretchable impedance sensor manifested as a pair of quasi-concentric microelectrodes was developed to detect unstable intravascular. By integrating the impedance sensor with a cardiac catheter, high-resolution Electrochemical Impedance Spectroscopy (EIS) measurements can be conducted during cardiac catheterization. An inflatable silicone balloon is added to the sensor to secure a well-controlled contact with the plaque under test in vivo. By deploying the device to the explants of NZW rabbit aorta and live animals, distinct EIS measurements were observed for unstable atherosclerotic plaques that harbored active lipids and inflammatory cells.

On the other hand, zebrafish (Danio rerio) is an emerging genetic model for heart regenerative medicine. In humans, myocardial infarction results in the irreversible loss of cardiomyocytes. Zebrafish hearts can fully regenerate after two months with 20% ventricular resection. Long-term electrocardiogram (ECG) recording can characterize the heart regeneration in a functional dimension. A flexible microelectrode membrane was developed to be percutaneously implanted onto a zebrafish heart and record epicardial ECG signals from specific regions on it. Region-specific aberrant cardiac signals were obtained from injured and regenerated hearts. Following that, in order to achieve continuous and wireless recording from non-sedated and non-restricted small animal models, a wireless ECG recording system was designed for the microelectrode membrane, prototyped on a printed circuit board and demonstrated on a one-day-old neonatal mouse. Furthermore, a flexible and compact parylene C printed circuit membrane was used as the integration platform for the wireless ECG recording electronics. A substantially miniature wireless ECG recording system was achieved.

Avaliação da eficácia da terapia fotodinâmica como adjunto ao tratamento periodontal de pacientes com periodontite agressiva

A periodontite agressiva é um processo inflamatório de origem bacteriana mediado pelo sistema imunológico do hospedeiro e é provavelmente a forma mais grave de doença periodontal, apresentando destruição das estruturas tanto de proteção quanto de suporte dentário, num período relativamente rápido, normalmente levando a perda prematura dos elementos dentários e, em alguns casos, terapia antimicrobiana adjunta é necessária em adição à terapia mecânica. O objetivo do presente ensaio clínico randomizado controlado foi avaliar o efeito clínico da terapia fotodinâmica como adjunto ao tratamento periodontal não cirúrgico no tratamento da periodontite agressiva Os seguintes parâmetros clínicos foram avaliados: índice de placa visível, sangramento à sondagem, profundidade de bolsa à sondagem, nível de inserção clínica relativo, envolvimento de furca e mobilidade. Foram selecionados dez pacientes com periodontite agressiva, os quais foram examinados no dia zero e após três meses.O desenho do estudo consistiu em um modelo de boca dividida, onde um hemiarco foi tratado com raspagem e alisamento radicular e terapia fotodinâmica (laser diodo) e o outro apenas com raspagem e alisamento radicular. Três meses após o término do tratamento, os grupos terapêuticos apresentaram resultados semelhantes para todos os parâmetros clínicos avaliados: ambas as terapias tiveram sucesso, como redução de profundidade de bolsa, ganho de nível de inserção clínica relativo, redução de índice de placa visível, redução de sangramento à sondagem, diminuição de envolvimento de furca e diminuição de mobilidade, porém sem diferenças estatisticamente significantes entre elas. Dentro das limitações do presente estudo, os resultados sugerem que a terapia fotodinâmica adjuntamente ao tratamento periodontal não cirúrgico mecânico foi tão eficaz quanto o tratamento periodontal não cirúrgico mecânico sozinho.

Wide Field-of-view Microscopes and Endoscopes for Time-lapse Imaging and High-throughput Screening

Wide field-of-view (FOV) microscopy is of high importance to biological research and clinical diagnosis where a high-throughput screening of samples is needed. This thesis presents the development of several novel wide FOV imaging technologies and demonstrates their capabilities in longitudinal imaging of living organisms, on the scale of viral plaques to live cells and tissues.

The ePetri Dish is a wide FOV on-chip bright-field microscope. Here we applied an ePetri platform for plaque analysis of murine norovirus 1 (MNV-1). The ePetri offers the ability to dynamically track plaques at the individual cell death event level over a wide FOV of 6 mm × 4 mm at 30 min intervals. A density-based clustering algorithm is used to analyze the spatial-temporal distribution of cell death events to identify plaques at their earliest stages. We also demonstrate the capabilities of the ePetri in viral titer count and dynamically monitoring plaque formation, growth, and the influence of antiviral drugs.

We developed another wide FOV imaging technique, the Talbot microscope, for the fluorescence imaging of live cells. The Talbot microscope takes advantage of the Talbot effect and can generate a focal spot array to scan the fluorescence samples directly on-chip. It has a resolution of 1.2 μm and a FOV of ~13 mm². We further upgraded the Talbot microscope for the long-term time-lapse fluorescence imaging of live cell cultures, and analyzed the cells’ dynamic response to an anticancer drug.

We present two wide FOV endoscopes for tissue imaging, named the AnCam and the PanCam. The AnCam is based on the contact image sensor (CIS) technology, and can scan the whole anal canal within 10 seconds with a resolution of 89 μm, a maximum FOV of 100 mm × 120 mm, and a depth-of-field (DOF) of 0.65 mm. We also demonstrate the performance of the AnCam in whole anal canal imaging in both animal models and real patients. In addition to this, the PanCam is based on a smartphone platform integrated with a panoramic annular lens (PAL), and can capture a FOV of 18 mm × 120 mm in a single shot with a resolution of 100─140 μm. In this work we demonstrate the PanCam’s performance in imaging a stained tissue sample.