The interleukin-1 (IL-1) family is one of the first described cytokine families and consists of eight cytokines (IL-1β, IL-1α, IL-18, IL-33, IL-36α, IL-36β, 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.

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.

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.

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.

Recent therapeutics searching to reactivate and target the immune system to destroy tumors have demonstrated remarkable success in the treatment of patients with previously intractable disease such as metastatic melanoma. Current research is enlarging the spectrum of targets and strategies for enhancing the immune response against tumors, in order to further improve treatment efficacy. In that respect, ADAM17, has frequently been described for it’s over expression or over activation in the tumor microenvironment. ADAM17 is responsible for the shedding of a large number of receptors and ligands involved in immune tumor targeting and the deregulation of this activity should be considered when developing therapeutic strategies. The aim of this review is to summarize the spectrum of ADAM17 substrates and its potential role in tumor immune modulation.

ABSTRACT The detection of Mycoplasma genitalium was evaluated on 1,080 urine samples by the use of a Panther instrument. Overall sensitivity, specificity, positive predictive values, and negative predictive values were 100%, 99.4%, 93.6%, and 100%, respectively. Detection of M. genitalium by the use of the Panther transcription-mediated amplification assay offers a simple, accurate, and sensitive platform for diagnostic laboratories.

Introduction M. genitalium is an emerging sexually transmitted pathogen, with a strong association with urethritis, cervicitis and pelvic inflammatory disease. Detection of this bacterium by routine culture is not practical and routine diagnosis and screening for M. genitalium by molecular techniques has been hampered by lack of readily available commercial assays. A preliminary version of a commercial amplification assay is currently available on the Panther platform and was evaluated against an in-house qPCR assay currently in use for routine diagnostics. Methods Overall 1000 consecutive urine samples from men and women were utilised for this evaluation. Over the course of 3 months, urine samples were obtained from consecutive symptomatic men and women being screened for M. genitalium at Melbourne Sexual Health Centre, as well as women being screened prior to termination of pregnancy at the Royal Women’s Hospital, Melbourne. The primary Hologic assay targeting 80bp region of 16s rRNA was compared to the in-house diagnostic assay which targets a 517bp region of 16S gene, as well as a second 16s rRNA target available on the Hologic platform. Results The comparison of the two targets available on the Hologic platform showed very high correlation (k = 0.97 95% CI 0.93–1.00). Comparison of primary Hologic assay to in-house 16S qPCR assay, also showed very good correlation (K = 0.84 95% CI 0.75–0.93). Overall, both primary and secondary Hologic assays on Panther were more sensitive than the 16S qPCR for detection of M. genitalium in urine specimens. Conclusion The M. genitalium assay on the Hologic platform integrated well with the laboratory procedures allowing rapid testing and possibility of rapid and accurate reporting using integration with laboratory information system. Overall the Hologic assay for detection of M. genitalium offers a simple, accurate and sensitive platform for diagnostic laboratories for detection of this important upcoming pathogen. Disclosure of interest statement Hologic supplied the diagnostic kits and the Panther platform to conduct this study.

The objective of this study was to review the diagnostic accuracy of the Aptima Combo 2 (AC2) assay on the Panther TM system for reporting Neisseria gonorrhoeae (NG) detection from routine clinical samples. Results for samples routinely submitted for the diagnosis of NG infection from two laboratories in Australia were retrospectively reviewed from September 2011 to May 2013. Each sample was initially tested on a Panther instrument with the AC2 assay. Samples positive or equivocal for NG in AC2 were retested with the Aptima Neisseria gonorrhoeae assay (AGC) on the Panther instrument. AC2-NG positive results were considered false positive if the AGC result was negative. AC2 results were available for 91,217 samples, 1,434 of which were initially AC2 non-negative for NG and included an AGC result. The overall prevalence of NG was 1.5% (2.8% in males and 0.7% in females). Overall, 16% of all samples were from extragenital specimens. Overall, 0.02% of all samples (14/91,217) were false positives (0.05% of extragenital samples and 0.01% of urogenital samples). Without supplementary testing with the AGC assay, 14/91,217 false positive results would have been reported as positive, representing 0.02% of all samples tested and 1.0% of initially non-negative samples. This study demonstrates that the initial AC2 results from all sampling sites (urogenital and extragenital) can be accepted with high confidence.

Background Involvement of lower extremity joints in both rheumatoid arthritis (RA) and osteoarthritis (OA) is an important cause of declined functional status. Deterioration of skeletal muscle performance further contributes to disability. Quadriceps muscle plays an important role in most daily activities. Measuring thickness and pennation angle of quadriceps compartments can provide knowledge regarding quadriceps condition. Objectives Aim is to investigate structural and inflammatory changes in knee joint and architecture of quadriceps muscle in RA, knee OA patients and a group of healthy subjects. Methods Twenty RA patients and 20 knee OA patients with radiographic disease meeting Kellgren-Lawrence grade 2 and above were consecutively recruited, in absence of exclusion criteria. 20 healthy volunteers with similar demographics were recruited as control group. Knee symptoms and disability were evaluated by Lequesne Knee Index (LKI). Disease activity was evaluated by DAS28-CRP in RA patients. Quadriceps muscle was evaluated by ultrasonographic measurements of muscle thickness, subcutaneous fat tissue thickness and pennation angle of vastus muscles. Suprapatellar, lateral and medial compartments of knee joint were examined for effusion, synovial hypertrophy, power Doppler (PD) activity and degenerative changes. Results Demographics and clinical features of subjects and results of ultrasonographic evaluation are presented in Table 1. LKI scores were significantly higher in OA patients when compared to RA patients (p=0.004). Rate of degenerative changes was increased in RA and OA groups in comparison to controls (p=0.013). None of the subjects had PD activity. No differences were observed in thickness of rectus femoris, vastus muscles and subcutaneous fat between groups. Vastus medialis and intermedius pennation angles were reduced in OA and RA groups when compared to controls, but reached statistical significance only in vastus intermedius (3.56 ± 6.37 vs 4.89 ± 7.14 vs 7.29 ± 4.93, p=0.038). Conclusion In our study ultrasound evaluation findings of knee joint and quadriceps muscle were similar between knee OA and RA patients.VM and VI pennation were reduced when compared to healthy subjects in both groups. These findings may indicate a similar degree of functional impairment in OA and RA due to knee joint involvement. References [1]Hsieh LF, et al. Isokinetic and isometric testing of knee musculature in patients with rheumatoid arthritis with mild knee involvement. Arch Phys Med Rehabil. 1987;68:294-7. [2]Ruhdorfer A, et al. Association of knee pain with a reduction in thigh muscle strength - a cross-sectional analysis including 4553 osteoarthritis initiative participants. Osteoarthritis Cartilage. 2017;25:658-666. Acknowledgements I have no acknowledgements to declare. Disclosure of Interests None declared

Stefan R. Schmidt consolidates the hugely diverse field of fusion proteins and their application in the creation of biopharmaceuticals. The text is replete with case studies and clinical data that inform and intrigue the reader as to the myriad possibilities available when considering the creation of a fusion protein. This valuable text will serve the novice as a broad introduction or the seasoned professional as a thorough review of the state of the art. The first marketed therapeutic recombinant protein was human insulin (Humulin® R). Its approval in 1982 was followed by other such products, including erythropoietin (EPO), interferon (IFN), and tissue plasminogen activator (tPa). Since the 1980s, the number and general availability of recombinant products that replace natural proteins harvested from animal or human sources has increased considerably. Following the initial success, researchers started de novo designs of therapeutic proteins that do not occur in nature. The first of these new drugs to be approved was etanercept (Enbrel®), a fusion portion containing a section of the tumor necrosis factor (TNF) receptor fused to the Fc portion of human IgG1.

Introduction Trichomonas vaginalis (TV) is the most common non-viral sexually transmitted infection worldwide. Among Australian women, a wide variation in prevalence (0.38%–8.4%) using nucleic acid amplification tests (NAAT) has been reported. Our aim was to assess whether routine screening for TV in females is indicated in an urban Australian setting. Methods Females attending a sexual health clinic from July 2013–February 2014 who were tested for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) were eligible to have a TV test on the same specimen. Testing was performed by transcription-mediated amplification on female genital specimens using the Aptima Trichomonas vaginalis assay (Hologic Inc., United States). Characteristics of the study population were examined. Results During the study period, 393 women were tested for CT/NG on 471 occasions. TV tests were performed 347 (73.7%) of CT/NG specimens. There were no significant differences between women who had (n = 294), and did not have (n = 99), a TV test during the study period, except that women who had recent overseas sexual contact were less likely to be tested. Of the 347 tests, two TV infections were diagnosed, a positivity rate of 0.6% (95% CI 0.07–2.1%). Both cases were Australian-born with a history of injecting drug use in the past 12 months, Neither were sex workers and one identified as Aboriginal. One presented with post-coital bleeding, and TV was identified on wet film. The other reported pelvic symptoms, but was tested on outreach and no wet film microscopy was performed. Neither had concurrent CT/NG infections detected. Conclusion We found a low positivity rate of TV among female attendees. Both TV infections were in women who had symptoms suggestive of a sexually transmitted infection. Our findings are in accord with those from previous urban Australian studies and do not support routine TV screening for asymptomatic women in metropolitan Sydney. Disclosure of interest statement Aptima Trichomonas vaginalis assay testing kits were provided free by Hologic (Australia) Pty Ltd.

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.

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.

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.

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.

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.

D’importants progrès réalisés durant les deux dernières décennies dans le domaine du génie moléculaire des protéines ont permis l’émergence de nouvelles charpentes protéiques démontrant des propriétés de liaison sur une cible donnée comparables à la liaison d’un anticorps à sa cible. Ces molécules appelées scaffolds présentent l’essentiel des fonctions associées à la partie variable des anticorps. En théorie, de nombreuses protéines humaines possèdent une charpente susceptible d’être modifiée par ingénierie moléculaire : protéine de transport présentant une surface de contact plane, squelette peptidique rigide permettant l’insertion de boucles hypervariables, association de plusieurs domaines de liaison induisant des effets coopératifs. En pratique, seule une vingtaine de protéines présentent les propriétés nécessaires pour constituer de potentiels candidats thérapeutiques et une dizaine de scaffolds ont atteint à ce jour le stade des essais cliniques chez l’homme. Parmi leurs principales propriétés, une large diversité de protéines ciblées, une facilité de production liée à une structure tridimensionnelle simple, une petite taille, une stabilité importante, une bonne tolérance et une faible immunogénicité sont essentielles. La propriété intellectuelle associée au développement de ces scaffolds est également un aspect important, qui permet de contourner les brevets liés aux anticorps et à leurs dérivés. Cette minirevue se focalise plus particulièrement sur les scaffolds ayant débuté leur développement clinique.