The interleukin-1 cytokine family members: Role in cancer pathogenesis and potential therapeutic applications in cancer immunotherapy.

The interleukin-1 (IL-1) family is one of the first described cytokine families and consists of eight cytokines (IL-1ß, IL-1α, IL-18, IL-33, IL-36α, IL-36ß, IL-36γ and IL-37) and three receptor antagonists (IL-1Ra, IL-36Ra and IL-38). The family members are known to play an essential role in inflammation. The importance of inflammation in cancer has been well established in the past decades. This review sets out to give an overview of the role of each IL-1 family member in cancer pathogenesis and show their potential as potential anticancer drug candidates. First, the molecular structure is described. Next, both the pro- and anti-tumoral properties are highlighted. Additionally, a critical interpretation of current literature is given. To conclude, the IL-1 family is a toolbox with a collection of powerful tools that can be considered as potential drugs or drug targets.

Performance evaluation of the Hologic Aptima HCV Quant Dx assay for detection of HCV RNA from dried blood spots.

BACKGROUND: The availability of effective direct-acting antiviral therapy for hepatitis C virus (HCV) has led to a need for simplified diagnostic pathways. Barriers to treatment uptake, specifically in people who inject drugs and in remote and resource limited settings, may be overcome by utilizing novel collection methods, such as dried blood spots (DBS). However, there are currently no registered assays for HCV RNA testing from DBS samples. OBJECTIVES: To evaluate the sensitivity and specificity of the Aptima HCV Dx Quant assay for HCV RNA detection in DBS samples STUDY DESIGN: 107 paired venepuncture and DBS samples from HCV antibody positive individuals were analyzed for HCV RNA on the Aptima HCV Dx Quant and Roche CAP/CTM (gold standard) HCV assays. RESULTS: 78% (n=83) had detectable HCV RNA in plasma. Sensitivity of the Aptima assay for HCV RNA detection in DBS was 96.4% (95% CI 89.8-99.3%) and specificity was 95.8% (95% CI 78.8-99.9%). Sensitivity for HCV RNA detection in DBS using a quantitative threshold of ≥15 IU/mL in plasma was 95.1% (95% CI 88%-98.7%) and specificity was 96.0% (95% CI 79.7%-99.9%). The sensitivity of HCV RNA detection in DBS using a quantitative threshold of ≥1000 IU/mL (based on a clinically relevant threshold) was 100% (95% CI 95.3-100%) and specificity was 100% (95% CI 88.4-100%). CONCLUSIONS: Our data indicates that the Aptima HCV Dx Quant can detect active HCV infection from a DBS sample with good sensitivity and specificity, particularly when using a threshold of ≥1000 IU/mL.

[Immunotoxins and immunocytokines]. / Immunotoxines et immunocytokines.

Cytokines and biological toxins represent two potent classes of biomolecules that have long been explored for their potential as therapeutics. Considerable side effects and poor pharmacokinetics frequently observed with both have limited their broad application. Recombinant protein engineering has allowed the construction of immunocytokines and immunotoxins that seek to exploit the advantageous properties of immunoglobulins to address these issues. Whole antibodies, antibody fragments, constant domains and derivatives have been fused genetically to a range of cytokines and toxins. This review considers the strategies that have been employed and the problems sought to be resolved in the clinical evaluation of this class of biotherapeutic.

TITLE: Immunotoxines et immunocytokines. ABSTRACT: Les cytokines et les toxines biologiques représentent deux classes de biomolécules qui ont longtemps été explorées pour leur potentiel thérapeutique. Des effets secondaires considérables et des mauvaises propriétés pharmacocinétiques sont fréquemment observés chez chacune d'elles, ce qui limite leur application. L'ingénierie des protéines recombinantes a permis la création d'immunocytokines et d'immunotoxines qui visent à utiliser les propriétés avantageuses des immunoglobulines, pour résoudre ces problèmes. Des anticorps entiers, des fragments d'anticorps, des domaines constants et des dérivés ont été génétiquement fusionnés à une gamme de cytokines et de toxines. Cette revue présente les stratégies déployées et les problèmes à résoudre au cours de l'évaluation clinique pour cette classe de biothérapeutiques.

The Role of Surface Topography on Deformation-Induced Magnetization under Inhomogeneous Elastic-Plastic Deformation.

It is widely accepted that the magnetic state of a ferromagnetic material may be irreversibly altered by mechanical loading due to magnetoelastic effects. A novel standardized nondestructive testing (NDT) technique uses weak magnetic stray fields, which are assumed to arise from inhomogeneous deformation, for structural health monitoring (i.e., for detection and assessment of damage). However, the mechanical and microstructural complexity of damage has hitherto only been insufficiently considered. The aim of this study is to discuss the phenomenon of inhomogeneous "self-magnetization" of a polycrystalline ferromagnetic material under inhomogeneous deformation experimentally and with stronger material-mechanical focus. To this end, notched specimens were elastically and plastically deformed. Surface magnetic states were measured by a three-axis giant magnetoresistant (GMR) sensor and were compared with strain field (digital image correlation) and optical topography measurements. It is demonstrated that the stray fields do not solely form due to magnetoelastic effects. Instead, inhomogeneous plastic deformation causes topography, which is one of the main origins for the magnetic stray field formation. Additionally, if not considered, topography may falsify the magnetic signals due to variable lift-off values. The correlation of magnetic vector components with mechanical tensors, particularly for multiaxial stress/strain states and inhomogeneous elastic-plastic deformations remains an issue.

Diagnosis of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium: an observational study of testing patterns, prevalence and co-infection rates in northern New Zealand.

Background This study sought to determine community prevalence, epidemiology and testing patterns for sexually transmissible infections (STI) in northern New Zealand. METHODS: A total of 2643 samples submitted for STI testing between 26 November 2015 and 7 December 2015 underwent analysis by Aptima Combo 2 (Hologic, San Diego, CA, USA), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG) assays. Results were analysed by patient demographics. RESULTS: Four hundred and eleven pathogens were detected from 359 patients, with Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), TV, and MG detected in 178 (6.7%), 19 (0.7%), 80 (3%) and 134 (5.1%) samples respectively. With the exception of TV, STI prevalence was highest in people <25 years of age. Infection was more common in men for NG (odds ratio (OR) 5.05, P<0.001) and CT (OR 2.72, P<0.001). Maori and Pacific ethnicity were associated with increased risk of MG (OR 1.82, P=0.006,) TV (OR 6.1, P<0.001) and CT (OR 3.31, P<0.001) infection, and TV and NG infections were more prevalent as social deprivation increased. A mismatch between testing rates and prevalence of infection was seen, with fewer tests performed for males (OR 0.2, P<0.001) than females and no difference in testing of Maori and Pacific men (3064/100000) compared with men of European background (3181/100000, OR 0.96, P=0.76), despite an increased risk of disease. CONCLUSIONS: There are disparately low testing rates for STIs in certain high-risk groups in northern New Zealand.

NanoLuc Luciferase - A Multifunctional Tool for High Throughput Antibody Screening.

Based on the recent development of NanoLuc luciferase (Nluc), a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct Western blotting. The new Nluc protein fusion represents a "swiss army knife solution" for today and future high throughput antibody drug screenings.

Differential binding of RhoA, RhoB, and RhoC to protein kinase C-related kinase (PRK) isoforms PRK1, PRK2, and PRK3: PRKs have the highest affinity for RhoB.

Protein kinase C-related kinases (PRKs) are members of the protein kinase C superfamily of serine-threonine kinases and can be activated by binding to members of the Rho family of GTPases via a Rho-binding motif known as an HR1 domain. Three tandem HR1 domains reside at the N-terminus of the PRKs. We have assessed the ability of the HR1a and HR1b domains from the three PRK isoforms (PRK1, PRK2, and PRK3) to interact with the three Rho isoforms (RhoA, RhoB, and RhoC). The affinities of RhoA and RhoC for a construct encompassing both PRK1 HR1 domains were similar to those for the HR1a domain alone, suggesting that these interactions are mediated solely by the HR1a domain. The affinities of RhoB for both the PRK1 HR1a domain and the HR1ab didomain were higher than those of RhoA or RhoC. RhoB also bound more tightly to the didomain than to the HR1a domain alone, implicating the HR1b domain in the interaction. As compared with PRK1 HR1 domains, PRK2 and PRK3 domains bind less well to all Rho isoforms. Uniquely, however, the PRK3 domains display a specificity for RhoB that requires both the C-terminus of RhoB and the PRK3 HR1b domain. The thermal stability of the HR1a and HR1b domains was also investigated. The PRK2 HR1a domain was found to be the most thermally stable, while PRK2 HR1b, PRK3 HR1a, and PRK3 HR1b domains all exhibited lower melting temperatures, similar to that of the PRK1 HR1a domain. The lower thermal stability of the PRK2 and PRK3 HR1b domains may impart greater flexibility, driving their ability to interact with Rho isoforms.

Measurement of protein-ligand complex formation.

Experimental approaches to detect, measure, and quantify protein-ligand binding, along with their theoretical bases, are described. A range of methods for detection of protein-ligand interactions is summarized. Specific protocols are provided for a nonequilibrium procedure pull-down assay, for an equilibrium direct binding method and its modification into a competition-based measurement and for steady-state measurements based on the effects of ligands on enzyme catalysis.

Mutational analysis reveals a single binding interface between RhoA and its effector, PRK1.

Protein kinase C-related kinases (PRKs) are serine/threonine kinases that are members of the protein kinase C superfamily and can be activated by binding to members of the Rho family of small G proteins via a Rho binding motif known as an HR1 domain. The PRKs contain three tandem HR1 domains at their N-termini. The structure of the HR1a domain from PRK1 in complex with RhoA [Maesaki, R., et al. (1999) Mol. Cell 4, 793-803] identified two potential contact interfaces between the G protein and the HR1a domain. In this work, we have used an alanine scanning mutagenesis approach to identify whether both contact sites are used when the two proteins interact in solution and also whether HR1b, the second HR1 domain from PRK1, plays a role in binding to RhoA. The mutagenesis identified just one contact site as being relevant for binding of RhoA and HR1a in solution, and the HR1b domain was found not to contribute to RhoA binding. The folded state and thermal stability of the HR1a and HR1b domains were also investigated. HR1b was found to be more thermally stable than HR1a, and it is hypothesized that the differences in the biophysical properties of these two domains govern their interaction with small G proteins.

Validation of an optical microplate label-free platform in the screening of chemical libraries for direct binding to a nuclear receptor.

Optical microplate-based biosensors combine the advantages of label-free detection with industry-standard assay laboratory infrastructure and scalability. A plate-based label-free platform allows the same basic platform to be used to quantify molecular interactions of macromolecules and to screen and characterize drug-like small-molecule interactions. The ligand-binding domain of orphan estrogen-related nuclear receptor-γ (ERRγ) is utilized, as a model system of a challenging type of target, to illustrate the rapid development and utility of a range of biochemical assay formats on these biosensors. Formats in which either the domain, or a peptide derived from its cognate corepressor, RIP140, were immobilized were utilized. The direct binding of small drug molecules to the domain was characterized using immobilized domain. Subsequent addition of peptide distinguished whether compounds acted as either antagonists of peptide binding, or as agonists promoting a ternary complex. The format with peptide immobilized gave a more sensitive procedure for establishing the effect of compounds on the domain-peptide interaction. Using a direct-binding format, a diverse chemical library of 1,408 compounds in DMSO was screened for ability to bind to biosensors coated with ERRγ ligand-binding domain. Hits were then characterized using the other biosensor assay formats. The standard requirements for a full primary screening campaign were fulfilled by the acceptable hit-rate, quality-performance parameters, and throughput of the direct-binding assay format. Such a format allows direct screening of targets, such as orphan receptors, without the requirement for prior knowledge of a validated ligand.

[Protein scaffolds as alternatives to whole antibodies: from discovery research to clinical development]. / Ingénierie de charpentes protéiques comme alternative aux anticorps : de la recherche au développement clinique.

Recent advances in combinatorial protein engineering have made it possible to develop non-Ig protein scaffolds that can potentially substitute for most whole antibody-associated properties. These protein scaffolds display most of the binding properties associated with the variable domain of antibodies. In theory, many different natural human protein backbones are suitable to be used as recombinant templates for engineering ; in practice however, only a few have yielded the necessary properties to be translated into << druggable biologicals >>. Amongst these properties, potential broad specificities towards any kind of target, ease of production, small size, good tolerability and low immunogenicity are essential. Intellectual property is another key issue. In this review, a particular emphasis will be given to the most validated non-Ig scaffolds that have reached the clinical development phase.

The IQGAP1-Rac1 and IQGAP1-Cdc42 interactions: interfaces differ between the complexes.

IQGAP1 contains a domain related to the catalytic portion of the GTPase-activating proteins (GAPs) for the Ras small G proteins, yet it has no RasGAP activity and binds to the Rho family small G proteins Cdc42 and Rac1. It is thought that IQGAP1 is an effector of Rac1 and Cdc42, regulating cell-cell adhesion through the E-cadherin-catenin complex, which controls formation and maintenance of adherens junctions. This study investigates the binding interfaces of the Rac1-IQGAP1 and Cdc42-IQGAP1 complexes. We mutated Rac1 and Cdc42 and measured the effects of mutations on their affinity for IQGAP1. We have identified similarities and differences in the relative importance of residues used by Rac1 and Cdc42 to bind IQGAP1. Furthermore, the residues involved in the complexes formed with IQGAP1 differ from those formed with other effector proteins and GAPs. Relatively few mutations in switch I of Cdc42 or Rac1 affect IQGAP1 binding; only mutations in residues 32 and 36 significantly decrease affinity for IQGAP1. Switch II mutations also affect binding to IQGAP1 although the effects differ between Rac1 and Cdc42; mutation of either Asp-63, Arg-68, or Leu-70 abrogate Rac1 binding, whereas no switch II mutations affect Cdc42 binding to IQGAP1. The Rho family "insert loop" does not contribute to the binding affinity of Rac1/Cdc42 for IQGAP1. We also present thermodynamic data pertaining to the Rac1/Cdc42-RhoGAP complexes. Switch II contributes a large portion of the total binding energy to these complexes, whereas switch I mutations also affect binding. In addition we identify "cold spots" in the Rac1/Cdc42-RhoGAP/IQGAP1 interfaces. Competition data reveal that the binding sites for IQGAP1 and RhoGAP on the small G proteins overlap only partially. Overall, the data presented here suggest that, despite their 71% identity, Cdc42 and Rac1 appear to have only partially overlapping binding sites on IQGAP1, and each uses different determinants to achieve high affinity binding.

Double mutant cycle thermodynamic analysis of the hydrophobic Cdc42-ACK protein-protein interaction.

Protein-protein interactions such as those between small G proteins and their effector proteins control most cell signaling pathways and thereby govern many cellular processes in both normal and disease states. Each small G protein interacts with several effectors, some shared between similar G proteins and others unique to a single GTPase. Although there is knowledge of the structural basis of these interactions, there is limited understanding of their thermodynamic basis. This is particularly significant because of the intrinsic conformational flexibility of the interacting partners. Here we have conducted a double mutant thermodynamic cycle for two key hydrophobic interactions in the Cdc42-ACK interface: Val42Cdc42-Ile463ACK and Leu174Cdc42-Leu449ACK. Val42 and Leu174 are known to be energetically important in this complex from previous thermodynamic studies, and their respective partners were predicted from the structure of the complex. Such a study has not been hitherto performed on any hydrophobic protein-protein interaction. The results confirm that a significant proportion of the overall interaction is dependent upon these residues, but in neither case is the direct interaction between the side chains the predominant energetic force. Indeed, the interaction of the side chains of Val42 and Ile463 appears to exert an energetic penalty. Rather, the stabilization of the complex, which requires the presence of these two pairs of residues, appears to be due to conformational changes, or interactions, that are not easily visualized in the structure of the complexes. In this respect, it is noteworthy that isolated Cdc42 shows regions of disorder and isolated ACK has no stable tertiary structure, whereas the Cdc42-ACK complex has a well-defined quaternary structure. Such changes may well be critical for the known selectivity of Cdc42 and related proteins such as Rho and Rac, for their wide range of effectors.

Detection of HBsAg mutants in a population with a low prevalence of hepatitis B virus infection.

Two independent studies were conducted to evaluate performance of two HBsAg immunoassay products performed on the Abbott ARCHITECT and Bayer ADVIA Centaur immunoassay analyzers. One was a retrospective study of 484 stored samples and the second was a prospective study of 349 samples from random population. In the process of the evaluation, a number of discordant samples from HBsAg-positive patients were found which led to the discovery of a number of HBsAg mutants in the general Australian population. Following viral DNA sequencing, these were identified as HBsAg escape mutants. Whilst the existence of HBsAg mutants has been well documented in various regions of the world, this is surprising in an area of low endemicity and demonstrates the necessity of an HBsAg assay to detect mutants reliably in a diagnostic situation where HBsAg is used as the only marker to detect an HBV infection. These studies demonstrate the ability of the Abbott ARCHITECT and AxSYM HBsAg immunoassays to detect these HBsAg mutations which were not detected by the Bayer ADVIA Centaur.

Comparison of the Gen-Probe APTIMA Combo 2 assay to the AMPLICOR CT/NG assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine samples from Australian men and women.

The performance of the APTIMA Combo 2 assay (AC2) for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae infections in urine samples was compared to that of the AMPLICOR CT/NG assay (AMP). The AC2 performance was superior to that of AMP for both organisms in this study.

Use of various common isolation media to evaluate the new VITEK 2 colorimetric GN Card for identification of Burkholderia pseudomallei.

The use of automated systems in the modern microbiology laboratory is becoming commonplace as the pressure of cost containment impacts on staff resources. With increased international travel and threats of bioterrorism, recognition and accurate identification of organisms such as Burkholderia pseudomallei is important. In areas where this organism is endemic, identification is not usually problematic. This study evaluates the performance of the new VITEK 2 colorimetric GN card for the identification of this organism. A total of 103 previously identified clinical isolates were tested with the new card with isolates taken from MacConkey agar, Columbia horse blood agar, Columbia sheep blood agar, and Trypticase soy agar in order to determine identification performance and to see if there was any variability in results due to the agar. Columbia horse blood agar produced the highest rates of identification (81%), followed by Trypticase soy agar (78%), Columbia sheep blood agar (75%), and MacConkey agar (63%). There was considerable variability in some of the reactions obtained. Seven isolates failed to identify from any of the agars used. This study highlights issues with the identification of this organism with the new VITEK 2 GN card. Enhancements of the algorithm parameters for the GN card are warranted and are in progress. Laboratory personnel need to be aware of the current limitations with this GN card and the software (version 4.02 or older for the VITEK 2 60/XL and version 1.02 or older for VITEK 2 Compact) and rely on clinical history, a high index of suspicion, and basic microbiology tests to confirm the identification of this organism.

Specificity determinants on Cdc42 for binding its effector protein ACK.

Cdc42 and Rac are highly homologous members of the Rho family of small G proteins that interact with several downstream effector proteins thereby causing cytoskeletal rearrangements, cell proliferation, and differentiation. While some effectors, such as the tyrosine kinase, ACK, and the scaffold protein, WASP, are unique to Cdc42, others, such as the serine-threonine kinase, PAK, are shared with Rac. Previous mutagenesis studies identified Val42 and Leu174 as residues that selectively affect binding of Cdc42 to ACK and WASP but not to PAK. However, it is unclear whether these discriminatory residues are sufficient determinants of specificity. In this study we sought to introduce "gain-of function" mutations into Rac to allow it to bind to ACK and WASP, thereby revealing all specificity determinants. Thirteen mutations were made changing Rac residues to those in Cdc42. Equilibrium binding constants of all mutant Rac proteins to ACK, WASP, and PAK were measured. A combination of seven mutations (S41A, A42V, N43T, D47G, N52T, W56F, and R174L) was determined to be necessary to change the binding affinity of Rac for ACK from negligible (K(d) < 1 microM) to a comparable affinity to Cdc42 (K(d) 25 nM). These mutations are not confined to interface residues. We interpret these data to indicate the importance of the structure of regions of the protein distinct from the contact residues. None of these mutant Rac proteins bound WASP with a similar affinity to Cdc42. Hence, residues as yet unidentified, outside the interface, must be necessary for binding WASP.

A microarray analysis of potential genes underlying the neurosensitivity of mice to propofol.

Establishing the mechanism of action of general anesthetics at the molecular level is difficult because of the multiple targets with which these drugs are associated. Inbred short sleep (ISS) and long sleep (ILS) mice are differentially sensitive in response to ethanol and other sedative hypnotics and contain a single quantitative trait locus (Lorp1) that accounts for the genetic variance of loss-of-righting reflex in response to propofol (LORP). In this study, we used high-density oligonucleotide microarrays to identify global gene expression and candidate genes differentially expressed within the Lorp1 region that may give insight into the molecular mechanism underlying LORP. Microarray analysis was performed using Affymetrix MG-U74Av2 Genechips and a selection of differentially expressed genes was confirmed by semiquantitative reverse transcription-polymerase chain reaction. Global expression in the brains of ILS and ISS mice revealed 3423 genes that were significantly expressed, of which 139 (4%) were differentially expressed. Analysis of genes located within the Lorp1 region showed that 26 genes were significantly expressed and that just 2 genes (7%) were differentially expressed. These genes encoded for the proteins AWP1 (associated with protein kinase 1) and "BTB (POZ) domain containing 1," whose functions are largely uncharacterized. Genes differentially expressed outside Lorp1 included seven genes with previously characterized neuronal functions and thus stand out as additional candidate genes that may be involved in mediating the neurosensitivity differences between ISS and ILS.

Evidence for the presence of three distinct binding sites for the thioflavin T class of Alzheimer's disease PET imaging agents on beta-amyloid peptide fibrils.

Imaging the progression of Alzheimer's disease would greatly facilitate the discovery of therapeutics, and a wide range of ligands are currently under development for the detection of beta-amyloid peptide (Abeta)-containing plaques by using positron emission tomography. Here we report an in-depth characterization of the binding of seven previously described ligands to in vitro generated Abeta-(1-40) polymers. All of the compounds were derived from the benzothiazole compound thioflavin T and include 2-[4'-(methylamino)phenyl]benzothiazole and 2-(4'-dimethylamino-)phenyl-imidazo[1,2-a]-pyridine derivatives, 2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole and 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and a benzofuran compound (5-bromo-2-(4-dimethylaminophenyl)benzofuran). By using a range of fluorescent and radioligand binding assays, we find that these compounds display a more complex binding pattern than described previously and are consistent with three classes of binding sites on the Abeta fibrils. All of the compounds bound with very high affinity (low nm K(d)) to a low capacity site (BS3) (1 ligand-binding site per approximately 300 Abeta-(1-40) monomers) consistent with the previously recognized binding site for these compounds on the fibrils. However, the compounds also bound with high affinity (K(d) approximately 100 nm) to either one of two additional binding sites on the Abeta-(1-40) polymer. The properties of these sites, BS1 and BS2, suggest they are adjacent or partially overlapping and have a higher capacity than BS3, occurring every approximately 35 or every approximately 4 monomers of Abeta-(1-40)-peptide, respectively. Compounds appear to display selectivity for BS2 based on the presence of a halogen substitution (2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole, 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and 5-bromo-2-(4-dimethylaminophenyl)benzofuran) on their aromatic ring system. The presence of additional ligand-binding sites presents potential new targets for ligand development and may allow a more complete modeling of the current positron emission tomography data.