Characterization of the complex formed between a potent neutralizing ovine-derived polyclonal anti-TNFα Fab fragment and human TNFα

TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the K_i in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays.     

Fatigue and functional performance of human biceps muscle following concentric or eccentric contractions.

A long-lasting fatigue was measured in human biceps muscle, following 40 maximal isokinetic concentric or eccentric contractions of the forearm, as the response to single-shock stimuli every minute for 4 h. This protocol allowed new observations on the early time course of long-lasting fatigue. Concentric contractions induced a novel progressive decline to 30.2% (SE 7.8, n = 7) of control at 23 min with complete recovery by 120 min. Eccentric contractions lead initially to a smaller force reduction of similar time course followed by a slower decline to 40.0% (SE 5.1, n = 7) control at 120 min with recovery less than half complete at 4 h. A 50-Hz test stimuli overcame both fatigues, identifying low-frequency fatigue. EMG recordings from the biceps muscle showed moderate (<20%) changes during the fatigue. A visual-tracking task showed no decrement in performance at the time of maximal fatigue of the single-shock response. Because the eccentric contractions have a similar activation, a larger force, but much smaller metabolic usage than concentric contractions, it is concluded that the initial decline is related to the effects of metabolites, whereas the slower phase after eccentric contractions is associated with higher mechanical stress.     

The challenge of invasive mosquito vectors in the U.K. during 2016–2018: a summary of the surveillance and control of Aedes albopictus

Mosquito‐borne diseases resulting from the expansion of two key vectors, Aedes aegypti and Aedes albopictus (Diptera: Culicidae), continue to challenge whole regions and continents around the globe. In recent years there have been human cases of disease associated with Chikungunya, dengue and Zika viruses. In Europe, the expansion of Ae. albopictus has resulted in local transmission of Chikungunya and dengue viruses. This paper considers the risk that Ae. aegypti and Ae. albopictus represent for the U.K. and details the results of mosquito surveillance activities. Surveillance was conducted at 34 points of entry, 12 sites serving vehicular traffic and two sites of used tyre importers. The most common native mosquito recorded was Culex pipiens s.l. (Diptera: Culicidae). The invasive mosquito Ae. albopictus was detected on three occasions in southern England (September 2016, July 2017 and July 2018) and subsequent control strategies were conducted. These latest surveillance results demonstrate ongoing incursions of Ae. albopictus into the U.K. via ground vehicular traffic, which can be expected to continue and increase as populations in nearby countries expand, particularly in France, which is the main source of ex‐continental traffic.     

Novel inhibitors of the αvβ3 integrin—lead identification strategy

A novel approach to inhibition of the αvβ3 integrin is described, which uses compounds designed to generate nM potency without using the arginine binding site.     

Repeated high-force eccentric exercise: effects on muscle pain and damage

Five women and three men (aged 24-43 yr) performed maximal eccentric contractions of the elbow flexors (for 20 min) on three occasions, spaced 2 wk apart. Muscle pain, strength and contractile properties, and plasma creatine kinase (CK) were studied before and after each exercise bout. Muscle tenderness was greatest after the first bout and thereafter progressively decreased. Very high plasma CK levels (1,500-11,000 IU/l) occurred after the first bout, but the second and third bouts did not significantly affect the plasma CK. After each bout the strength was reduced by approximately 50% and after 2 wk had only recovered to 80% of preexercise values. Each exercise bout produced a marked shift of the force-frequency curve to the right which took approximately 2 wk to recover. The recovery rate of both strength and force-frequency characteristics was faster after the second and third bouts. Since the adaptation occurred after the performance of maximal contractions it cannot have been a result of changes in motor unit recruitment. The observed training effect of repeated exercise was not a consequence of the muscle becoming either stronger or more resistant to fatigue.     

Expression and purification of biologically active IGF-binding proteins using the LCR/Mel expression system

The anabolic effects and bioavailability of insulin-like growth factors I and II (IGF-I, IGF-II) are regulated in part by a family of IGF-binding proteins (IGFBPs). There are six known members of the IGFBP family, which share distinct structural characteristics and functional activities. To study the binding properties of these proteins, we have expressed recombinant IGFBP-3 and IGFBP-4 using the LCR/Mel expression system. Using this system, we found that recombinant IGFBP-3 was secreted by Mel cells and had a glycosylation pattern similar to that of native IGFBP-3. Recombinant IGFBP-4 secreted from Mel cells had a molecular size identical to that of non-glycosylated native IGFBP-4. The binding kinetics of recombinant IGFBPs was measured using a solid-phase ligand-binding assay, an in vitro solution-binding assay, and a cellular proliferation assay. IGF-I bound with high affinity to recombinant IGFBP-3 and IGFBP-4 with K(D)s of <0.25 nmol. As reported for native IGFBPs, IGF-II bound with affinity higher than IGF-I to recombinant IGFBP-3 and IGFBP-4 (K(D) of <0.05 nmol). Recombinant IGFBP-3 and IGFBP-4 were found to inhibit the IGF-induced proliferation of an NIH3T3 cell line engineered to overexpress the IGF-I receptor. We have compared the binding kinetics of Mel cell-expressed IGFBPs with that of recombinant protein expressed in Escherichia coli and found them to be equivalent. Here, we show that the LCR/Mel expression system represents an effective route for expression of biologically active IGFBPs.     

Production of recombinant soluble human integrin alpha4beta1

Integrin alpha4beta1 is a major leukocyte adhesion receptor that is a key target for the development of anti-inflammatory therapeutics. With the dual long-term goals of developing a reagent for use in high-throughput inhibitor screening assays and for crystallisation trials and subsequent structure determination, we have generated a recombinant soluble alpha4beta1 receptor. Both subunits were truncated prior to the transmembrane domains by site-directed mutagenesis and expressed using baculovirus infection of insect cells. The molecular weights of the recombinant subunits were as expected for post-translationally unmodified protein. In addition, as observed for the native subunit, a proportion of the alpha4 subunit was proteolytically processed into two fragments. ELISA and solid phase ligand-binding assays were performed to investigate the folding and functionality of the soluble integrin. The data suggest that the receptor was correctly folded and that it bound recombinant ligands with similar kinetics to the native molecule.     

Skeletal muscle damage: a study of isotope uptake, enzyme efflux and pain after stepping

We have studied the occurrence of skeletal muscle uptake of 99mtechnetium pyrophosphate (Tc-PYP), creatine kinase (CK) release and muscle pain in normal subjects after exercise. Five subjects stepped on and off a high bench in such a way that one leg stepped up and the other down. Pain only developed in the muscles used for descending: quadriceps, adductors and gluteal muscles of one leg and the calf muscle of the other. A large rise in plasma CK occurred in four subjects but no increased Tc-PYP muscle uptake was seen in the quadriceps. In the four subjects with high CK effluxes, increased isotope uptake was seen in the thigh adductors used when stepping down; in the two subjects with the largest CK effluxes there was extensive uptake into the gluteal muscles. Muscle pain preceded and was not well correlated with either the magnitude of the enzyme release or the amount and distribution of increased muscle isotope uptake. We conclude that delayed onset muscle pain, the cause of which remains unknown, is a poor indicator of muscle damage as indicated by circulating muscle enzymes and muscle isotope uptake. Tc-PYP uptake by skeletal muscle can provide useful information about the localisation and time course of muscle damage.     

Design and Evaluation of Meningococcal Vaccines through Structure-Based Modification of Host and Pathogen Molecules

Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps.