Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway

Background

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.

Methods

Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.

Results

A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.

Conclusions

The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.

Trial registration

NHS Health Research Authority (13/LO/0256).     

Tumor delivery of ultrasound contrast agents using Shiga toxin B subunit

The present study demonstrates the targeting of ultrasound contrast agents to human xenograft tumors by exploiting the overexpression of the glycolipid Gb3 in neovasculature. To this end, microbubbles were functionalized with a natural Gb3 ligand, the B subunit of the Shiga toxin (STxB). The targeting of Gb3-expressing tumor cells by STxB microbubbles was first shown by flow cytometry and fluorescence microscopy. A significantly higher proportion of STxB microbubbles were associated with Gb3-expressing tumor cells compared to cells in which Gb3 expression was inhibited. Moreover, ultrasonic imaging of culture plates showed a 12 dB contrast enhancement in average backscattered acoustic intensity on the surface of Gb3-expressing cells compared to Gb3-negative cells. Also, a 18 dB contrast enhancement was found in favor of STxB microbubbles compared to unspecific microbubbles. Microbubble signal intensity in subcutaneous tumors in mice was more than twice as high after the injection of STxB-functionalized microbubbles compared to the injection of unspecific microbubbles. These in vitro and in vivo experiments demonstrated that STxB-functionalized microbubbles bind specifically to cells expressing the Gb3 glycolipid. The cell-binding moieties of toxins thus appear as a new group of ligands for angiogenesis imaging with ultrasound.     

Comparison of crystal structures of human type 3 3alpha-hydroxysteroid dehydrogenase reveals an "induced-fit" mechanism and a conserved basic motif involved in the binding of androgen

The aldo-keto reductase (AKR) human type 3 3alpha-hydroxysteroid dehydrogenase (h3alpha-HSD3, AKR1C2) plays a crucial role in the regulation of the intracellular concentrations of testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), two steroids directly linked to the etiology and the progression of many prostate diseases and cancer. This enzyme also binds many structurally different molecules such as 4-hydroxynonenal, polycyclic aromatic hydrocarbons, and indanone. To understand the mechanism underlying the plasticity of its substrate-binding site, we solved the binary complex structure of h3alpha-HSD3-NADP(H) at 1.9 A resolution. During the refinement process, we found acetate and citrate molecules deeply engulfed in the steroid-binding cavity. Superimposition of this structure with the h3alpha-HSD3-NADP(H)-testosterone/acetate ternary complex structure reveals that one of the mobile loops forming the binding cavity operates a slight contraction movement against the citrate molecule while the side chains of many residues undergo numerous conformational changes, probably to create an optimal binding site for the citrate. These structural changes, which altogether cause a reduction of the substrate-binding cavity volume (from 776 A(3) in the presence of testosterone/acetate to 704 A(3) in the acetate/citrate complex), are reminiscent of the "induced-fit" mechanism previously proposed for the aldose reductase, another member of the AKR superfamily. We also found that the replacement of residues Arg(301) and Arg(304), localized near the steroid-binding cavity, significantly affects the 3alpha-HSD activity of this enzyme toward 5alpha-DHT and completely abolishes its 17beta-HSD activity on 4-dione. All these results have thus been used to reevaluate the binding mode of this enzyme for androgens.     

Loop relaxation, a mechanism that explains the reduced specificity of rabbit 20alpha-hydroxysteroid dehydrogenase, a member of the aldo-keto reductase superfamily

The aldo-keto reductase rabbit 20alpha-hydroxysteroid dehydrogenase (rb20alpha-HSD; AKR1C5) is less selective than other HSDs, since it exerts its activity both on androgens (C19 steroids) and progestins (C21 steroids). In order to identify the molecular determinants responsible for this reduced selectivity, binary (NADPH) and ternary (NADP(+)/testosterone) complex structures were solved to 1.32A and 2.08A resolution, respectively. Inspection of the cofactor-binding cavity led to the identification of a new interaction between side-chains of residues His222 and Lys270, which cover the central phosphate chain of the cofactor, reminiscent of the "safety-belt" found in other aldo-keto reductases. Testosterone is stabilized by a phenol/benzene tunnel composed of side-chains of numerous residues, among which Phe54, which forces the steroid to take up an orientation markedly contrasting with that found in HSD ternary complexes reported. Combining structural, site-directed mutagenesis, kinetic and fluorescence titration studies, we found that the selectivity of rb20alpha-HSD is mediated by (i) the relaxation of loop B (residues 223-230), partly controlled by the nature of residue 230, (ii) the nature of the residue found at position 54, and (iii) the residues found in the C-terminal tail of the protein especially the side-chain of the amino acid 306.     

Proteins associated with the exon junction complex also control the alternative splicing of apoptotic regulators

Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x(S) splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.     

Linking selenium biogeochemistry to the sulfur-dependent biological detoxification of arsenic

Geochemistry often reveals unexpected (anti)correlations. Arsenic (As) and selenium (Se) are cases in point. We explore the hypothesis that bacteria living in an As-replete environment recruited a biological process involving Se and sulfur to fulfil their need for As detoxification. In analogy with the formation of arsenolipids and arsenosugars, which are common non-toxic As metabolites derived from microbial and plant metabolism, we attempt to explain the prevalence of novel sulfur-containing As derivatives, in particular monothioarsenate, in the aqueous environment. Thiolated-As species have been overlooked so far mainly because of the difficulty of their identification. Based on comparative genomics, we propose a scenario where SelD and SelU proteins, commonly used to make selenophosphate and modify transfer RNA, have been recruited to make monothioarsenate, a relatively innocuous arsenical. This hypothesis is discussed in terms of the relative geochemical distribution of Se and As.     

Ligand-specific recycling profiles determine distinct potential for chronic analgesic tolerance of delta-opioid receptor (DOPr) agonists

δ-opioid receptor (DOPr) agonists have analgesic efficacy in chronic pain models but development of tolerance limits their use for long-term pain management. Although agonist potential for inducing acute analgesic tolerance has been associated with distinct patterns of DOPr internalization, the association between trafficking and chronic tolerance remains ill-defined. In a rat model of streptozotocin (STZ)-induced diabetic neuropathy, deltorphin II and TIPP produced sustained analgesia  following daily (intrathecal) i.t. injections over six days, whereas similar treatment with SNC-80 or SB235863 led to progressive tolerance and loss of the analgesic response. Trafficking assays in murine neuron cultures showed no association between the magnitude of ligand-induced sequestration and development of chronic tolerance. Instead, ligands that supported DOPr recycling were also the ones producing sustained analgesia over 6-day treatment. Moreover, endosomal endothelin-converting enzyme 2 (ECE2) blocker 663444 prevented DOPr recycling by deltorphin II and TIPP and precipitated tolerance by these ligands. In conclusion, agonists, which support DOPr recycling, avoid development of analgesic tolerance over repeated administration.     

Temporal Phases in Apoptosis Defined by the Actions of Src Homology 2 Domains, Ceramide, Bcl-2, Interleukin-1β Converting Enzyme Family Proteases, and a Dense Membrane Fraction

We have begun to explore the mechanisms of apoptosis using a cell-free system based on extracts from Xenopus eggs. Nuclei assembled or placed in these extracts undergo the morphological changes typical of apoptosis and eventually disintegrate. We used this system to investigate the potential involvement in apoptosis of proteins containing Src homology 2 (SH2) domains, which are known to interact with specific tyrosine-phosphorylated ligands. SH2 domains from a number of signaling proteins, including Lck, Src, and Abl, inhibited apoptosis when present at concentrations of 10–100 nM. The inhibition was dependent on specific interaction with endogenous tyrosine-phosphorylated ligands. A synthetic peptide ligand for Src family SH2 domains also inhibited apoptosis in a phosphotyrosine-dependent manner. Kinetic analysis defined three phases in the apoptotic process occurring in this cell-free system. SH2 domains and ceramide act throughout the first 60–90 min of the process (the “initiation” phase). Next, Bcl-2, interleukin-1β converting enzyme family(CPP32-like) proteases, and the heavy membrane fraction act in a period occurring ~90–120 min after the start of incubation (the “sentencing” phase). In the final phase (“execution”), the process of active nuclear destruction ensues.