An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes

Background  

Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. It is a mosquito-borne viral disease, caused by dengue (DEN) viruses, which are members of the Flaviviridae family. There are four closely related serotypes, DEN-1, DEN-2, DEN-3 and DEN-4, each of which is capable of causing disease. As immunity to any one serotype can potentially sensitize an individual to severe disease during exposure to a heterologous serotype, the general consensus is that an effective vaccine should be tetravalent, that is, it must be capable of affording protection against all four serotypes. The current strategy of creating tetravalent vaccine formulations by mixing together four monovalent live attenuated vaccine viruses has revealed the phenomenon of viral interference leading to the manifestation of immune responses biased towards a single serotype.     

Site-specific sonoporation of human melanoma cells at the cellular level using high lateral-resolution ultrasonic micro-transducer arrays

We developed a new instrumental method by which human melanoma cells (LU1205) are sonoporated via radiation pressures exerted by highly-confined ultrasonic waves produced by high lateral-resolution ultrasonic micro-transducer arrays (UMTAs). The method enables cellular-level site-specific sonoporation within the cell monolayer due to UMTAs and can be applicable in the delivery of drugs and gene products in cellular assays. In this method, cells are seeded on the biochip that employs UMTAs for high spatial resolution and specificity. UMTAs are driven by 30-MHz sinusoidal signals and the resulting radiation pressures induce sonoporation in the targeted cells. The sonoporation degree and the effective lateral resolution of UMTAs are determined by performing fluorescent microscopy and analysis of carboxylic-acid-derivatized CdSe/ZnS quantum dots passively transported into the cells. Models representing the transducer-generated ultrasound radiation pressure, the ultrasound-inflicted cell membrane wound, and the transmembrane transport through the wound are developed to determine the ultrasound-pressure-dependent wound size and enhanced cellular uptake of nanoparticles. Model-based calculations show that the effective wound size and cellular uptake of nanoparticles increase linearly with increasing ultrasound pressure (i.e., at applied radiation pressures of 0.21, 0.29, and 0.40 MPa, the ultrasound-induced initial effective wound radii are 150, 460, and 650 nm, respectively, and the post-sonoporation intracellular quantum-dot concentrations are 7.8, 22.8, and 29.9 nM, respectively) and the threshold pressure required to induce sonoporation in LU1205 cells is ～0.12 MPa.     

Augmentation of arginase 1 expression by exposure to air pollution exacerbates the airways hyperresponsiveness in murine models of asthma

Background

Arginase overexpression contributes to airways hyperresponsiveness (AHR) in asthma. Arginase expression is further augmented in cigarette smoking asthmatics, suggesting that it may be upregulated by environmental pollution. Thus, we hypothesize that arginase contributes to the exacerbation of respiratory symptoms following exposure to air pollution, and that pharmacologic inhibition of arginase would abrogate the pollution-induced AHR.

Methods

To investigate the role of arginase in the air pollution-induced exacerbation of airways responsiveness, we employed two murine models of allergic airways inflammation. Mice were sensitized to ovalbumin (OVA) and challenged with nebulized PBS (OVA/PBS) or OVA (OVA/OVA) for three consecutive days (sub-acute model) or 12 weeks (chronic model), which exhibit inflammatory cell influx and remodeling/AHR, respectively. Twenty-four hours after the final challenge, mice were exposed to concentrated ambient fine particles plus ozone (CAP+O₃), or HEPA-filtered air (FA), for 4 hours. After the CAP+O₃ exposures, mice underwent tracheal cannulation and were treated with an aerosolized arginase inhibitor (S-boronoethyl-L-cysteine; BEC) or vehicle, immediately before determination of respiratory function and methacholine-responsiveness using the flexiVent^®. Lungs were then collected for comparison of arginase activity, protein expression, and immunohistochemical localization.

Results

Compared to FA, arginase activity was significantly augmented in the lungs of CAP+O₃-exposed OVA/OVA mice in both the sub-acute and chronic models. Western blotting and immunohistochemical staining revealed that the increased activity was due to arginase 1 expression in the area surrounding the airways in both models. Arginase inhibition significantly reduced the CAP+O₃-induced increase in AHR in both models.

Conclusions

This study demonstrates that arginase is upregulated following environmental exposures in murine models of asthma, and contributes to the pollution-induced exacerbation of airways responsiveness. Thus arginase may be a therapeutic target to protect susceptible populations against the adverse health effects of air pollution, such as fine particles and ozone, which are two of the major contributors to smog.     

Cerebrospinal Fluid Catecholamine Levels in Japanese Encephalitis Patients with Movement Disorders

Norepinephrine and dopamine have important role in movement disorders but their role in movement disorders associated with Japanese encephalitis (JE) has not been evaluated. Therefore, in the present study, cerebrospinal fluid (CSF) catecholamine levels and its metabolites in JE patients with movement disorders were compared with those without JE. CSF was collected by lumbar puncture and analyzed by HPLC-ED. Norepinephrine, dopamine and homovanillic acid concentrations were significantly (P<0.005) lower in JE patients compared to control groups. Low levels of catecholamines in JE associated movement disorders compared to idiopathic Parkinson’s disease and other extrapyramidal symptoms may be due to severe structural damage to thalamus, basal ganglia and brainstem in JE patients as revealed by MRI findings.     

Identification of domains of ataxia-telangiectasia mutated required for nuclear localization and chromatin association

Ataxia-telangiectasia mutated (ATM) is essential for rapid induction of cellular responses to DNA double strand breaks (DSBs). In this study, we mapped a nuclear localization signal (NLS), 385KRKK388, within the amino terminus of ATM and demonstrate its recognition by the conventional nuclear import receptor, the importin alpha1/beta1 heterodimer. Although mutation of this NLS resulted in green fluorescent protein (GFP) x ATM(NLSm) localizing predominantly within the cytoplasm, small amounts of nuclear GFP x ATM(NLSm) were still sufficient to elicit a DNA damage response. Insertion of an heterologous nuclear export signal between GFP and ATM(NLSm) resulted in complete cytoplasmic localization of ATM, concomitantly reducing the level of substrate phosphorylation and increasing radiosensitivity, which indicates a functional requirement for ATM nuclear localization. Interestingly, the carboxyl-terminal half of ATM, containing the kinase domain, which localizes to the cytoplasm, could not autophosphorylate itself or phosphorylate substrates, nor could it correct radiosensitivity in response to DSBs even when targeted to the nucleus by insertion of an exogenous NLS, demonstrating that the ATM amino terminus is required for optimal ATM function. Moreover, we have shown that the recruitment/retention of ATM at DSBs requires its kinase activity because a kinase-dead mutant of GFP x ATM failed to form damage-induced foci. Using deletion mutation analysis we mapped a domain in ATM (amino acids 5-224) required for its association with chromatin, which may target ATM to sites of DNA damage. Combined, these data indicate that the amino terminus of ATM is crucial not only for nuclear localization but also for chromatin association, thereby facilitating the kinase activity of ATM in vivo.