Intracellular trafficking of polyamidoamine-poly(ethylene glycol) block copolymers in DNA delivery

The delivery of nucleic acids has the potential to revolutionize medicine by allowing previously untreatable diseases to be clinically addressed. Viral delivery systems have shown immunogenicity and toxicity dangers, but synthetic vectors have lagged in transfection efficiency. Previously, we developed a modular, linear-dendritic block copolymer architecture with high gene transfection efficiency compared to commercial standards. This rationally designed system makes use of a cationic dendritic block to condense the anionic DNA and forms complexes with favorable endosomal escape properties. The linear block provides biocompatibility and protection from serum proteins, and can be functionalized with a targeting ligand. In this work, we quantitate performance of this system with respect to intracellular barriers to gene delivery using both high-throughput and traditional approaches. An image-based, high-throughput assay for endosomal escape is described and applied to the block copolymer system. Nuclear entry is demonstrated to be the most significant barrier to more efficient delivery and will be addressed in future versions of the system.     

Lysosomes co-localize with ryanodine receptor subtype 3 to form a trigger zone for calcium signalling by NAADP in rat pulmonary arterial smooth muscle

In arterial myocytes the Ca(2+) mobilizing messenger NAADP evokes spatially restricted Ca(2+) bursts from a lysosome-related store that are subsequently amplified into global Ca(2+) waves by Ca(2+)-induced Ca(2+)-release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs). Lysosomes facilitate this process by forming clusters that co-localize with a subpopulation of RyRs on the SR. We determine here whether RyR subtypes 1, 2 or 3 selectively co-localize with lysosomal clusters in pulmonary arterial myocytes using affinity purified specific antibodies. The density of: (1) alphalgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5mum of the nucleus) was approximately 4-fold greater than in the sub-plasmalemmal (within 1.5mum of the plasma membrane) and approximately 2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was approximately 4- and approximately 14-fold greater than that in the extra-perinuclear and sub-plasmalemmal regions, and approximately 2-fold greater than that for either RyR1 or RyR2; (3) despite there being no difference in the overall densities of fluorescent labelling of lysosomes and RyR subtypes between cells, co-localization with alphalgp120 labelling within the perinuclear region was approximately 2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between alphalgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30muM) abolished global Ca(2+) waves but not Ca(2+) bursts in response to intracellular dialysis of NAADP (10nM). We conclude that a subpopulation of lysosomes cluster in the perinuclear region of the cell and form junctions with SR containing a high density of RyR3 to comprise a trigger zone for Ca(2+) signalling by NAADP.     

Three-dimensional visualization of FKBP12.6 binding to an open conformation of cardiac ryanodine receptor

The cardiac isoform of the ryanodine receptor (RyR2) from dog binds predominantly a 12.6-kDa isoform of the FK506-binding protein (FKBP12.6), whereas RyR2 from other species binds both FKBP12.6 and the closely related isoform FKBP12. The role played by FKBP12.6 in modulating calcium release by RyR2 is unclear at present. We have used cryoelectron microscopy and three-dimensional (3D) reconstruction techniques to determine the binding position of FKBP12.6 on the surface of canine RyR2. Buffer conditions that should favor the "open" state of RyR2 were used. Quantitative comparison of 3D reconstructions of RyR2 in the presence and absence of FKBP12.6 reveals that FKBP12.6 binds along the sides of the square-shaped cytoplasmic region of the receptor, adjacent to domain 9, which forms part of the four clamp (corner-forming) structures. The location of the FKBP12.6 binding site on "open" RyR2 appears similar, but slightly displaced (by 1-2 nm) from that found previously for FKBP12 binding to the skeletal muscle ryanodine receptor that was in the buffer that favors the "closed" state. The conformation of RyR2 containing bound FKBP12.6 differs considerably from that depleted of FKBP12.6, particularly in the transmembrane region and in the clamp structures. The x-ray structure of FKBP12.6 was docked into the region of the 3D reconstruction that is attributable to bound FKBP12.6, to show the relative orientations of amino acid residues (Gln-31, Asn-32, Phe-59) that have been implicated as being critical in interactions with RyR2. A thorough understanding of the structural basis of RyR2-FKBP12.6 interaction should aid in understanding the roles that have been proposed for FKBP12.6 in heart failure and in certain forms of sudden cardiac death.     

In vitro and in vivo evaluation of topical formulations of Spantide II

The purpose of this study was to develop and evaluate topical formulations of Spantide II, a neurokinin-1 receptor (NK-1R) antagonist, for the treatment of inflammatory skin disorders. Spantide II lotion and gel was formulated with and without n-methyl-2-pyrrolidone (NMP) as a penetration enhancer. The release of Spantide II from gels was evaluated using microporous polyethylene and polypropylene membranes in a Franz Diffusion cell setup. In vitro percutaneous absorption of Spantide II from lotion and gel formulations was evaluated using the above setup by replacing the membranes with hairless rat skin. The in vivo anti-inflammatory activity of Spantide II formulations was evaluated in an allergic contact dermatitis (ACD) mouse model. Among different gels studied, PF127 gel showed highest (70-fold) release of Spantide II compared with hydroxypropyl methylcellulose (HPMC) and hydroxypropyl cellulose (HPC) gels. Lotion and gel formulations with or without NMP showed no detectable levels of Spantide II in the receiver compartment of the Franz diffusion cell until 24 hours. However, Spantide II showed significant retention in epidermis and dermis from lotion and gel formulations at 24 hours. The dermal levels increased ≈3.5- and 2-fold when the lotion and gel formulations contained NMP as compared with the formulation with no NMP (P<.05). The in vivo studies indicated that Spantide II formulations with NMP were effective in significantly reducing ACD response, similar to dexamethasone (0.5 mM). In conclusion, Spantide II was stable as a topical formulation and delivered to target skin tissue (epidermis and dermis) for the treatment of ACD. In addition this study supports the role of cutaneous neurosensory system in modulating inflammatory responses in the skin. Published: October 31, 2005     

Estimating Kidney Function in HIV-Infected Adults in Kenya: Comparison to a Direct Measure of Glomerular Filtration Rate by Iohexol Clearance

Background

More than two-thirds of the world''s HIV-positive individuals live in sub-Saharan Africa, where genetic susceptibility to kidney disease is high and resources for kidney disease screening and antiretroviral therapy (ART) toxicity monitoring are limited. Equations to estimate glomerular filtration rate (GFR) from serum creatinine were derived in Western populations and may be less accurate in this population.

Methods

We compared results from published GFR estimating equations with a direct measure of GFR by iohexol clearance in 99 HIV-infected, ART-naïve Kenyan adults. Iohexol concentration was measured from dried blood spots on filter paper. The bias ratio (mean of the ratio of estimated to measured GFR) and accuracy (percentage of estimates within 30% of the measured GFR) were calculated.

Results

The median age was 35 years, and 60% were women. The majority had asymptomatic HIV, with median CD4+ cell count of 355 cells/mm³. Median measured GFR was 115 mL/min/1.73 m². Overall accuracy was highest for the Chronic Kidney Disease Epidemiology Consortium (CKD-EPI) equation. Consistent with a prior report, bias and accuracy were improved by eliminating the coefficient for black race (85% of estimates within 30% of measured GFR). Accuracy of all equations was poor in participants with GFR 60–90 mL/min/1.73 m² (<65% of estimates within 30% of measured GFR), although this subgroup was too small to reach definitive conclusions.

Conclusions

Overall accuracy was highest for the CKD-EPI equation. Eliminating the coefficient for race further improved performance. Future studies are needed to determine the most accurate GFR estimate for use in individuals with GFR <90 mL/min/1.73 m², in whom accurate estimation of kidney function is important to guide drug dosing. Direct measurement of GFR by iohexol clearance using a filter paper based assay is feasible for research purposes in resource-limited settings, and could be used to develop more accurate GFR estimates in African populations.     

Impact of Helminth Diagnostic Test Performance on Estimation of Risk Factors and Outcomes in HIV-Positive Adults

Background

Traditional methods using microscopy for the detection of helminth infections have limited sensitivity. Polymerase chain reaction (PCR) assays enhance detection of helminths, particularly low burden infections. However, differences in test performance may modify the ability to detect associations between helminth infection, risk factors, and sequelae. We compared these associations using microscopy and PCR.

Methods

This cross-sectional study was nested within a randomized clinical trial conducted at 3 sites in Kenya. We performed microscopy and real-time multiplex PCR for the stool detection and quantification of Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale, Strongyloides stercoralis, and Schistosoma species. We utilized regression to evaluate associations between potential risk factors or outcomes and infection as detected by either method.

Results

Of 153 HIV-positive adults surveyed, 55(36.0%) and 20(13.1%) were positive for one or more helminth species by PCR and microscopy, respectively (p<0.001). PCR-detected infections were associated with farming (Prevalence Ratio 1.57, 95% CI: 1.02, 2.40), communal water source (PR 3.80, 95% CI: 1.01, 14.27), and no primary education (PR 1.54, 95% CI: 1.14, 2.33), whereas microscopy-detected infections were not associated with any risk factors under investigation. Microscopy-detected infections were associated with significantly lower hematocrit and hemoglobin (means of -3.56% and -0.77 g/dl) and a 48% higher risk of anemia (PR 1.48, 95% CI: 1.17, 1.88) compared to uninfected. Such associations were absent for PCR-detected infections unless infection intensity was considered, Infections diagnosed with either method were associated with increased risk of eosinophilia (PCR PR 2.42, 95% CI: 1.02, 5.76; microscopy PR 2.92, 95% CI: 1.29, 6.60).

Conclusion

Newer diagnostic methods, including PCR, improve the detection of helminth infections. This heightened sensitivity may improve the identification of risk factors for infection while reducing ability to discriminate infections associated with adverse clinical outcomes. Quantitative assays can be used to differentiate infection loads and discriminate infections associated with sequelae.     

Polymorphic ventricular tachycardia and abnormal Ca2+ handling in very-long-chain acyl-CoA dehydrogenase null mice

Patients with mutations in the mitochondrial very-long-chain acyl-CoA dehydrogenase (VLCAD) gene are at risk for cardiomyopathy, myocardial dysfunction, ventricular tachycardia (VT), and sudden cardiac death. The mechanism is not known. Here we report a novel mechanism of VT in mice lacking VLCAD (VLCAD(-/-)). These mice exhibited polymorphic VT and increased incidence of VT after isoproterenol infusion. Polymorphic VT was induced in 10 out of 12 VLCAD(-/-) mice (83%) when isoproterenol was used. One out of 10 VLCAD(-/-) mice with polymorphic VT had VT with the typical bidirectional morphology. At the molecular level, VLCAD(-/-) cardiomyocytes showed increased levels of cardiac ryanodine receptor 2, phospholamban, and calsequestrin with increased [(3)H]ryanodine binding in heart microsomes. At the single cardiomyocyte level, VLCAD(-/-) cardiomyocytes showed significant increase in diastolic indo 1 and fura 2 fluorescence, with increased Ca(2+) transient amplitude. These changes were associated with altered Ca(2+) dynamics, to include: faster sarcomere contraction, larger time derivative of the upstroke, and shorter time-to-minimum sarcomere length compared with VLCAD(+/+) control cells. The L-type Ca(2+) current characteristics were not different under voltage-clamp conditions in the two VLCAD genotypes. Sarcoplasmic reticulum Ca(2+) load measured as normalized integrated Na(+)/Ca(2+) exchange current after rapid caffeine application was increased by 48% in VLCAD(-/-) cells. We conclude that intracellular Ca(2+) handling represents a possible molecular mechanism of arrhythmias in mice and perhaps in VLCAD-deficient humans.     

Identification of MAGI-3 as a transforming growth factor-alpha tail binding protein

The cytoplasmic domain of the transforming growth factor-alpha precursor (proTGFalpha) contains a C-terminal PSD-95/SAP90, Discs Large, and Zona Occludens-1 (PDZ) recognition motif (TVV). By yeast two-hybrid screening of a mouse embryo library, we have found that a third member of a family of PDZ-containing proteins, membrane associated guanylate kinase inverted-3 (MAGI-3), binds to TGFalpha's TVV. MAGI-3 is widely expressed in multiple mouse tissues, including brain. Immunolocalization showed that MAGI-3 and TGFalpha were colocalized in neurons in the cortex and dentate gyrus, as well as in ependymal cells and some astrocytes. In vitro, proTGFalpha bound the PDZ-1 domain of MAGI-3 and MAGI-2, but not MAGI-1. MAGI-3 and the 17-kDa cell surface form of proTGFalpha interact transiently in MDCK cells stably transfected with both MAGI-3 and human proTGFalpha cDNAs. MAGI-3 and wild-type proTGFalpha colocalize at the cell surface. In contrast, MAGI-3 forms a stable complex with membrane-fixed TGFalpha early in the secretory pathway and interacts with immature and cell surface forms of membrane-fixed TGFalpha. Overexpression of MAGI-3 resulted in increased levels of TGFalpha in the basolateral medium of polarized MDCK cells, suggesting that MAGI-3 has a role in efficient trafficking of TGFalpha to the cell surface in polarized epithelial cells.