An activatable multimodal/multifunctional nanoprobe for direct imaging of intracellular drug delivery

Multifunctional nanoparticles integrated with imaging modalities (such as magnetic resonance and optical) and therapeutic drugs are promising candidates for future cancer diagnostics and therapy. While targeted drug delivery and imaging of tumor cells have been the major focus in engineering nanoparticle probes, no extensive efforts have been made towards developing sensing probes that can confirm and monitor intracellular drug release events. Here, we present quantum dot (Qdot)-iron oxide (IO) based multimodal/multifunctional nanocomposite probe that is optically and magnetically imageable, targetable and capable of reporting on intracellular drug release events. Specifically, the probe consists of a superparamagnetic iron oxide nanoparticle core (IONP) decorated with satellite CdS:Mn/ZnS Qdots where the Qdots themselves are further functionalized with STAT3 inhibitor (an anti-cancer agent), vitamin folate (as targeting motif) and m-polyethylene glycol (mPEG, a hydrophilic dispersing agent). The Qdot luminescence is quenched in this nanocomposite probe (“OFF” state) due to combined electron/energy transfer mediated quenching processes involving IONP, folate and STAT3 agents. Upon intracellular uptake, the probe is exposed to the cytosolic glutathione (GSH) containing environment resulting in restoration of the Qdot luminescence (“ON” state), which reports on uptake and drug release. Probe functionality was validated using fluorescence and MR measurements as well as in vitro studies using cancer cells that overexpress folate receptors.     

Inactivated Francisella tularensis live vaccine strain protects against respiratory tularemia by intranasal vaccination in an immunoglobulin A-dependent fashion

Francisella tularensis is a gram-negative intracellular bacterium that is considered to be a potential category A biological weapon due to its extreme virulence. Although vaccination with the attenuated live vaccine strain (LVS) of F. tularensis can protect against lethal challenge, use of inactivated or subunit forms as vaccine candidates for induction of protective antibody responses has not been fully evaluated. In the present study, we examined whether immune protection in the lung could be stimulated by intranasal administration of inactivated LVS together with interleukin-12 (IL-12) as an adjuvant. LVS was inactivated by heat, paraformaldehyde treatment, or exposure to UV, and inactivation of the preparations was confirmed by assessing bacterial growth and the survival of mice after direct inoculation. We found that mucosal vaccination with inactivated LVS provided 90 to 100% protection in mice after lethal intranasal challenge with 10(4) CFU of LVS, and this protection was dependent on inclusion of exogenous IL-12 during vaccine administration. Survival of vaccinated mice after live bacterial challenge was correlated with reduced bacterial burden, decreased pulmonary inflammation, increased serum antibody titers, and lower levels of gamma interferon (IFN-gamma), tumor necrosis factor alpha, and IL-6 in the lungs, livers, and spleens. Whereas NK cells were primarily responsible for the production of IFN-gamma in unvaccinated, challenged animals, vaccinated mice had increased levels of lung IFN-gamma+ CD4+ T cells after challenge. Significantly, mice genetically deficient in immunoglobulin A (IgA) expression were unable to survive lethal challenge after vaccination. These results are the first results to demonstrate that IgA-mediated protection against lethal respiratory tularemia occurs after mucosal vaccination with inactivated F. tularensis LVS.     

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays

AIMS: To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). METHODS: The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques-an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. RESULTS: All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (kappa > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (kappa = 0.63). CONCLUSION: The assay kit marketed as Varelisa allows accurate detection of LKM1.     

Localization and regulation of bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase by cyclic AMP-dependent protein kinase

Calmodulin-dependent cyclic nucleotide phosphodiesterase is one of the key enzymes involved in the complex interactions, which occur between the cyclic nucleotide and Ca2+ second-messenger systems. In eye, cAMP regulation is important in a variety of physiological processes such as aqueous humor regulation, photoreceptor signal transduction and retinal blood flow. Bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase was purified to apparent homogeneity and the isolated enzyme had a significantly higher affinity for calmodulin and Ca2+. Immunohistology revealed calmodulin-dependent cyclic nucleotide phospho-diesterase expression in corneal epithelium, retina and optic nerve of the eye. The cAMP-dependent protein kinase was found to catalyze the phosphorylation of bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase and the following observations were made. Firstly, the phosphorylation resulted in the incorporation of 1 mol of phosphate per mol of subunit, resulting in higher calmodulin and Ca2+ concentration requirement for calmodulin-dependent cyclic nucleotide phosphodiesterase activation. Secondly, Ca2+ and calmodulin prevented the phosphorylation. Thirdly, the phosphorylation of calmodulin-dependent cyclic nucleotide phosphodiesterase could be reversed by the calmodulin-dependent phosphatase, calcineurin. Analysis of the complex regulatory properties of the calmodulin-dependent cyclic nucleotide phosphodiesterase in the eye has led to the suggestion that fluxes of cAMP and Ca2+ during cell activation are closely coupled and that calmodulin-dependent cyclic nucleotide phosphodiesterase plays a key role in this signal coupling phenomenon.     

Invasive pneumococcal disease caused by ceftriaxone-resistant Streptococcus pneumoniae in Taiwan

Background

Invasive pneumococcal disease (IPD) was associated with mortality, but the risk factors associated with mortality remains controversial.

Adenovirus type 5 pseudotyped with adenovirus type 37 fiber uses sialic acid as a cellular receptor

For purposes of gene therapy, the tropism of adenovirus (Ad) serotype 5 vectors can be altered with fibers derived from alternative serotypes. However, there is currently limited information available on the cellular receptors used by the approximately 51 known Ad serotypes. Recently, alpha(2-->3)-linked sialic acid (2,3-SA) has been implicated as the cellular receptor for wild-type Ad37. However, some studies have demonstrated that wild-type Ad37 uses a 50-kDa protein and not sialic acid as its primary receptor for binding of human conjunctival cells. The sialic acid receptor has also been shown not to play a major role in the infection of these cells by an Ad5 virion pseudotyped with Ad37 fiber (Ad5.GFP.DeltaF/37F). In this study, we demonstrate that a similar virus (Ad5F37) can indeed use alpha(2-->3)-linked sialic acid as a cellular receptor. We also find that the receptor used by Ad5F37 is sensitive to proteases and that Ad5F37 can use integrin more efficiently than sialic acid for cell entry. Unlike Ad5 vectors, Ad5F37 does not efficiently employ the coxsackie and adenovirus receptor (CAR) to infect cells. Similar to Ad5, Ad5F37 infection of cells that form tight junctions can be enhanced by ethylenediaminetetraacetic acid (EDTA). These results have implications in the design of pseudotyped adenovirus vectors for gene therapy and may have particular use in the treatment of diseases involving breakdown of the blood-retinal barrier.     

Molecular mechanisms and functions of pyroptosis,inflammatory caspases and inflammasomes in infectious diseases

Cell death is a fundamental biological phenomenon that is essential for the survival and development of an organism. Emerging evidence also indicates that cell death contributes to immune defense against infectious diseases. Pyroptosis is a form of inflammatory programmed cell death pathway activated by human and mouse caspase-1, human caspase-4 and caspase-5, or mouse caspase-11. These inflammatory caspases are used by the host to control bacterial, viral, fungal, or protozoan pathogens. Pyroptosis requires cleavage and activation of the pore-forming effector protein gasdermin D by inflammatory caspases. Physical rupture of the cell causes release of the pro-inflammatory cytokines IL-1β and IL-18, alarmins and endogenous danger-associated molecular patterns, signifying the inflammatory potential of pyroptosis. Here, we describe the central role of inflammatory caspases and pyroptosis in mediating immunity to infection and clearance of pathogens.     

Regulation of calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1): review

The response of living cells to change in cell environment depends on the action of second messenger molecules. The two second messenger molecules cAMP and Ca2+ regulate a large number of eukaryotic cellular events. Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1) is one of the key enzymes involved in the complex interaction between cAMP and Ca2+ second messenger systems. Some PDE1 isozymes have similar kinetic and immunological properties but are differentially regulated by Ca2+ and calmodulin. Accumulating evidence suggests that the activity of PDE1 is selectively regulated by cross-talk between Ca2+ and cAMP signalling pathways. These isozymes are also further distinguished by various pharmacological agents. We have demonstrated a potentially novel regulation of PDE1 by calpain. This study suggests that limited proteolysis by calpain could be an alternative mechanism for the activation of PDE1. We have also shown PDE1 activity, expression and effect of calpain in the rat model in vitro of cardiac ischemia-reperfusion.