New Evidence for P-gp-Mediated Export of Amyloid-β PEPTIDES in Molecular, Blood-Brain Barrier and Neuronal Models

Defective clearance mechanisms lead to the accumulation of amyloid-beta (Aβ) peptides in the Alzheimer’s brain. Though predominantly generated in neurons, little is known about how these hydrophobic, aggregation-prone, and tightly membrane-associated peptides exit into the extracellular space where they deposit and propagate neurotoxicity. The ability for P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporter, to export Aβ across the blood-brain barrier (BBB) has previously been reported. However, controversies surrounding the P-gp–Aβ interaction persist. Here, molecular data affirm that both Aβ40 and Aβ42 peptide isoforms directly interact with and are substrates of P-gp. This was reinforced ex vivo by the inhibition of Aβ42 transport in brain capillaries from P-gp-knockout mice. Moreover, we explored whether P-gp could exert the same role in neurons. Comparison between non-neuronal CHO-APP and human neuroblastoma SK-N-SH cells revealed that P-gp is expressed and active in both cell types. Inhibiting P-gp activity using verapamil and nicardipine impaired Aβ40 and Aβ42 secretion from both cell types, as determined by ELISA. Collectively, these findings implicate P-gp in Aβ export from neurons, as well as across the BBB endothelium, and suggest that restoring or enhancing P-gp function could be a viable therapeutic approach for removing excess Aβ out of the brain in Alzheimer’s disease

New evidence for p-gp-mediated export of amyloid-β peptides in molecular, blood-brain barrier and neuronal models

Defective clearance mechanisms lead to the accumulation of amyloid-beta (Aβ) peptides in the Alzheimer's brain. Though predominantly generated in neurons, little is known about how these hydrophobic, aggregation-prone, and tightly membrane-associated peptides exit into the extracellular space where they deposit and propagate neurotoxicity. The ability for P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporter, to export Aβ across the blood-brain barrier (BBB) has previously been reported. However, controversies surrounding the P-gp–Aβ interaction persist. Here, molecular data affirm that both Aβ40 and Aβ42 peptide isoforms directly interact with and are substrates of P-gp. This was reinforced ex vivo by the inhibition of Aβ42 transport in brain capillaries from P-gp-knockout mice. Moreover, we explored whether P-gp could exert the same role in neurons. Comparison between non-neuronal CHO-APP and human neuroblastoma SK-N-SH cells revealed that P-gp is expressed and active in both cell types. Inhibiting P-gp activity using verapamil and nicardipine impaired Aβ40 and Aβ42 secretion from both cell types, as determined by ELISA. Collectively, these findings implicate P-gp in Aβ export from neurons, as well as across the BBB endothelium, and suggest that restoring or enhancing P-gp function could be a viable therapeutic approach for removing excess Aβ out of the brain in Alzheimer's disease.This research was funded by a seed grant from the University of Sydney, Australia. A.B.C. and A.Y. were recipients of Australian Government scholarships. A.M.S.H. was supported by grant number 2R01AG039621 from the National Institute on Aging. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Aging or the National Institutes of Health

Advanced methods in esophageal testing

Bibliography: p. 110-120Includes copy of ethics approval and copyright permissions. Original copies with original Partial Copyright Licence.Technical innovations for esophageal testing are continuously evolving to help people better understand and treat various esophageal disorders. In this work, efforts have been focused on the developments of two innovative systems for improving the diagnosis of gastroesophageal reflux disease (GERD) and esophageal motility disorders (EMDs). Determining the volume of gastroesophageal reflux (GER) is important for the diagnosis and treatment of GERD. However, a reliable diagnostic method for accomplishing this task is still missing. This research provided an integrated tool for automated monitoring of GER volume. A simple, low-cost ultrasonic probe was developed as a volumetric sensing component and it was combined with a commercially available multichannel intraluminal impedance catheter. The integrated tool was successfully tested in in-vitro laboratory and in-vivo human studies. In-vitro testing results demonstrated the accuracy of the volumetric measurement. In-vivo human testing was undertaken on two healthy human subjects. Results confirmed the feasibility of the proposed system for measuring volume of liquid GER in the vicinity of the ultrasonic probe. The proposed method paved the way towards an improved diagnosis and treatment of GERD. Ambulatory esophageal manometry has been considered the optimal technique for precise classification of various types of EMDs. However, current ambulatory manometry systems are not widely accepted due to their high costs, incomplete circumferential pressure sensing capabilities, and other practical issues. In this study, a novel manometry catheter containing a cost-effective pressure sensor capable of circumferential sensing was presented to overcome these limitations. A portable-size capacitance measurement device was used as an interface between the sensor and a graphical user interface on a personal computer. Experimental results confirmed the feasibility of the new manometry system for measuring circumferential pressure. This study provided a significant contribution towards a disposable esophageal manometry catheter to facilitate a low-cost and reliable ambulatory monitoring of esophageal motility

Exploiting the metabolic energy demands of drug efflux pumps provides a strategy to overcome multidrug resistance in cancer

Background: P-glycoprotein (P-gp) is a prevalent resistance mediator and it requires considerable cellular energy to ensure ATP dependent efflux of anticancer drugs. The glycolytic pathway generates the majority of catabolic energy in cancer cells; however, the high rates of P-gp activity places added strain on its inherently limited capacity to generate ATP. This is particularly relevant for compounds such as verapamil that are believed to trap P-gp in a futile transport process that requires continuing ATP consumption. Ultimately, this leads to cell death and the hypersensitivity of resistant cells to verapamil is termed collateral sensitivity. Results: We show that the addition of verapamil to resistant cells produces a prominent reduction in ATP levels that supports the idea of disrupted energy homeostasis. Even in the absence of verapamil, P-gp expressing cells display near maximal rates of glycolysis and oxidative phosphorylation, which prevents an adequate response to the demand for ATP to sustain transport activity. Moreover, the near perpetually maximal rate of oxidative phosphorylation in the presence of verapamil resulted in elevated levels of reactive oxygen species that affect cell survival and underscore collateral sensitivity. Conclusions: Our results demonstrate that the strained metabolic profiles of P-gp expressing resistant cancer cells can be overwhelmed by additional ATP demands. General significance: Consequently, collateral sensitising drugs may overcome the resistant phenotype by exploiting, rather than inhibiting, the energy demanding activity of pumps such as P-gp

A Novel Class of N-Sulfonyl and N-Sulfamoyl Noscapine Derivatives that Promote Mitotic Arrest in Cancer Cells

Noscapine displays weak anticancer efficacy and numerous research efforts have attempted to generate more potent noscapine analogues. These modifications included the replacement of the N‐methyl group in the 6′‐position with a range of substituents, where N‐ethylcarbamoyl substitution was observed to possess enhanced anticancer activity. Herein, we describe advances in this area, namely the synthesis and pharmacological evaluation of a series of N‐sulfonyl and N‐sulfamoyl noscapine derivatives. A number of these sulfonyl‐containing noscapinoids demonstrated improved activities compared to noscapine. ((R)‐5‐((S)‐4,5‐Dimethoxy‐1,3‐dihydroisobenzofuran‐1‐yl)‐4‐methoxy‐6‐((1‐methyl‐1H‐imidazol‐4‐yl)sulfonyl)‐5,6,7,8‐tetrahydro[1,3]dioxolo[4,5‐g]isoquinoline) (14 q) displayed sub‐micromolar activities of 560, 980, 271 and 443 nM against MCF‐7, PANC‐1, MDA‐MB‐435 and SK‐MEL‐5 cells, respectively. This antiproliferative effect was also maintained against drug‐resistant NCI/AdrRES cells despite high expression of the multidrug efflux pump, P‐glycoprotein

New Evidence for P-gp-Mediated Export of Amyloid-β Peptides in Molecular, Blood-Brain Barrier and Neuronal Models

Defective clearance mechanisms lead to the accumulation of amyloid-beta (Aβ) peptides in the Alzheimer’s brain. Though predominantly generated in neurons, little is known about how these hydrophobic, aggregation-prone, and tightly membrane-associated peptides exit into the extracellular space where they deposit and propagate neurotoxicity. The ability for P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporter, to export Aβ across the blood-brain barrier (BBB) has previously been reported. However, controversies surrounding the P-gp–Aβ interaction persist. Here, molecular data affirm that both Aβ40 and Aβ42 peptide isoforms directly interact with and are substrates of P-gp. This was reinforced ex vivo by the inhibition of Aβ42 transport in brain capillaries from P-gp-knockout mice. Moreover, we explored whether P-gp could exert the same role in neurons. Comparison between non-neuronal CHO-APP and human neuroblastoma SK-N-SH cells revealed that P-gp is expressed and active in both cell types. Inhibiting P-gp activity using verapamil and nicardipine impaired Aβ40 and Aβ42 secretion from both cell types, as determined by ELISA. Collectively, these findings implicate P-gp in Aβ export from neurons, as well as across the BBB endothelium, and suggest that restoring or enhancing P-gp function could be a viable therapeutic approach for removing excess Aβ out of the brain in Alzheimer’s disease

Enhanced mucosal immune responses induced by a combined candidate mucosal vaccine based on Hepatitis A virus and Hepatitis E virus structural proteins linked to tuftsin.

Hepatitis A virus (HAV) and Hepatitis E virus (HEV) are the most common causes of infectious hepatitis. These viruses are spread largely by the fecal-oral route and lead to clinically important disease in developing countries. To evaluate the potential of targeting hepatitis A and E infection simultaneously, a combined mucosal candidate vaccine was developed with the partial open reading frame 2 (ORF2) sequence (aa 368-607) of HEV (HE-ORF2) and partial virus protein 1 (VP1) sequence (aa 1-198) of HAV (HA-VP1), which included the viral neutralization epitopes. Tuftsin is an immunostimulatory peptide which can enhance the immunogenicity of a protein by targeting it to macrophages and dendritic cells. Here, we developed a novel combined protein vaccine by conjugating tuftsin to HE-ORF2 and HA-VP1 and used synthetic CpG oligodeoxynucleotides (ODNs) as the adjuvant. Subsequent experiments in BALB/c mice demonstrated that tuftsin enhanced the serum-specific IgG and IgA antibodies against HEV and HAV at the intestinal, vaginal and pulmonary interface when delivered intranasally. Moreover, mice from the intranasally immunized tuftsin group (HE-ORF2-tuftsin + HA-VP1-tuftsin + CpG) showed higher levels of IFN-γ-secreting splenocytes (Th1 response) and ratio of CD4+/CD8+ T cells than those of the no-tuftsin group (HE-ORF2 + HA-VP1 + CpG). Thus, the tuftsin group generated stronger humoral and cellular immune responses compared with the no-tuftsin group. Moreover, enhanced responses to the combined protein vaccine were obtained by intranasal immunization compared with intramuscular injection. By integrating HE-ORF2, HA-VP1 and tuftsin in a vaccine, this study validated an important concept for further development of a combined mucosal vaccine against hepatitis A and E infection

Immune responses to HBsAg conjugated to protein D of non-typeable Haemophilus influenzae in mice.

BACKGROUND:Hepatitis B vaccine that contains an aluminum hydroxide adjuvant induces apoptotic death of Hepa 1-6 cells. Difficult-to-degrade chemical additives in vaccines effectively enhance vaccine immunogenicity, but also affect the host tissue. Identification of bio-molecules that are readily degraded and compatible in vivo as an adjuvant is important for vaccine research. The hapten-carrier effect suggests that stimulation of helper T (Th) cells by carrier adjuvants is feasible. Protein D (PD) of non-typeable Haemophilus influenzae covalently conjugated to some polysaccharide vaccines has been confirmed to convert T-cell independent (TI) antigens into T-cell dependent (TD) antigens, and elicit strong T-cell responses ultimately. Herein, we would substitube PD for aluminum hydroxide adjuvant in Hepatitis B vaccine. METHODS AND RESULTS:Truncated PD (amino acids 20-364) was expressed in Escherichia coli and purified by (NH4)2SO4 precipitation and DEAE chromatography. After evaluation of antigenicity by western blotting, PD was covalently conjugated to yeast-derived recombinant HBsAg by cross-linking with glutaraldehyde. Intramuscular immunization with the conjugate induced higher level of HBsAg-specific antibody than did HBsAg alone (p < 0.05), and was comparable to commercial Hepatitis B vaccine. During the surveillance period (days 35-105), anti-HBs titers were hold high. Moreover, the conjugated vaccine enhanced Th1 immune responses, while Th2 responses were also activated and induced an antibody response, as determined by IFN-γ ELISPOT and IgG1/IgG2a ratio assays. CONCLUSIONS:Recombinant truncated PD covalently conjugated to HBsAg antigen enhanced the immunogenicity of the antigen in mice simultaneously by humoral and cellular immune response, which would facilitate therapeutic hepatitis B vaccines