Dynamic shear analysis: a novel method to determine mechanical integrity of normal and torn human acetabular labra: Implications for prediction of outcome of repair.

OBJECTIVES: The aim of this study was to investigate the structural integrity of torn and non-torn human acetabular labral tissue. METHODS: A total of 47 human labral specimens were obtained from a biobank. These included 22 torn specimens and 25 control specimens from patients undergoing total hip arthroplasty with macroscopically normal labra. The specimens underwent dynamic shear analysis using a rheometer to measure storage modulus, as an indicator of structural integrity. RESULTS: There was a significant difference in the storage modulus between torn (mean modulus = 2144.08 Pa) and non-torn (3178.1 Pa) labra (p = 0.0001). CONCLUSION: The acetabular labrum of young patients with a tear has significantly reduced structural integrity compared with a non-torn labrum in older patients with end-stage osteoarthritis. This study contributes to the understanding of the biomechanics of labral tears, and the observation of reduced structural integrity in torn labra may explain why some repairs fail. Our data demonstrate that labral tears probably have a relatively narrow phenotype, presenting a basis for further investigations that will provide quantifiable data to support their classification and a means to develop a standardized surgical technique for their repair. This study also demonstrates the value of novel biomechanical testing methods in investigating pathological tissues of orthopaedic interest.Cite this article: A. K. Woods, J. Broomfield, P. Monk, F. Vollrath, S. Glyn-Jones. Dynamic shear analysis: a novel method to determine mechanical integrity of normal and torn human acetabular labra: Implications for prediction of outcome of repair. Bone Joint Res 2018;7:440-446. DOI: 10.1302/2046-3758.77.BJR-2017-0282.R2.

Contact investigations for outbreaks of Mycobacterium tuberculosis: advances through whole genome sequencing.

The control of tuberculosis depends on the identification and treatment of infectious patients and their contacts, who are currently identified through a combined approach of genotyping and epidemiological investigation. However, epidemiological data are often challenging to obtain, and genotyping data are difficult to interpret without them. Whole genome sequencing (WGS) technology is increasingly affordable, and offers the prospect of identifying plausible transmission events between patients without prior recourse to epidemiological data. We discuss the current approaches to tuberculosis control, and how WGS might advance public health efforts in the future.

Heterogeneity in the responses of human lung mast cells to stem cell factor.

BACKGROUND: Stem cell factor (SCF) is a growth factor that is involved in mast cell differentiation and proliferation. SCF primes human lung mast cells for enhanced responses to IgE-directed activation but is not generally recognized as a direct activator. SCF mediates its effects through c-kit. OBJECTIVE: The aim of this study was to reappraise the effects of SCF on human lung mast cells. METHODS: Mast cells were isolated from human lung. Mast cells were challenged with anti-IgE or SCF and the generation of histamine, cysteinyl-leukotrienes (cys-LTs) and prostaglandin D(2) (PGD(2) ) was assessed as was expression of the activation marker, CD63. The effects of c-kit inhibitors on mediator release were evaluated. RESULTS: Stem cell factor (10 ng/mL) alone was unable to induce mediator release but primed mast cells for enhanced IgE-dependent secretion. At higher concentrations (≥ 30 ng/mL), SCF had more varied effects and even when used alone was able to drive substantial levels of histamine release in about a third of all preparations studied. Similarly, SCF (100 ng/mL) alone was effective in stimulating the generation of cys-LTs in half of the preparations studied. SCF (100 ng/mL) was even more effective at stimulating PGD(2) generation as almost all preparations generated substantial quantities of the prostanoid. Mediator release induced by SCF was accompanied by the up-regulation of the activation marker, CD63. There was a positive correlation between the extent of mediator release induced by SCF and c-kit receptor expression. The effects of SCF on mediator release from mast cells were reversed by the c-kit inhibitor imatinib. CONCLUSIONS AND CLINICAL RELEVANCE: These data demonstrate that the responses of mast cells to SCF are heterogeneous. SCF can drive much greater levels of mediator release from mast cells, especially of PGD(2), than hitherto appreciated and this could be important in the context of respiratory diseases.

Protein engineering and preclinical development of a GM-CSF receptor antibody for the treatment of rheumatoid arthritis.

BACKGROUND AND PURPOSE: For antibody therapies against receptor targets, in vivo outcomes can be difficult to predict because of target-mediated clearance or antigen 'sink' effects. The purpose of this work was to engineer an antibody to the GM-CSF receptor α (GM-CSFRα) with pharmacological properties optimized for chronic, s.c. treatment of rheumatoid arthritis (RA) patients. EXPERIMENTAL APPROACH: We used an in silico model of receptor occupancy to guide the target affinity and a combinatorial phage display approach for affinity maturation. Mechanism of action and internalization assays were performed on the optimized antibody in vitro before refining the modelling predictions of the eventual dosing in man. Finally, in vivo pharmacology studies in cynomolgus monkeys were carried out to inform the predictions and support future clinical development. KEY RESULTS: Antibody potency was improved 8600-fold, and the target affinity was reached. The refined model predicted pharmacodynamic effects at doses as low as 1 mg kg(-1) and a study in cynomolgus monkeys confirmed in vivo efficacy at 1 mg kg(-1) dosing. CONCLUSIONS AND IMPLICATIONS: This rational approach to antibody drug discovery enabled the isolation of a potent molecule compatible with chronic, s.c. self-administration by RA patients. We believe this general approach enables the development of optimal biopharmaceuticals.

In vivo evaluation of edge-loading in metal-on-metal hip resurfacing patients with pseudotumours.

OBJECTIVES: Pseudotumours (abnormal peri-prosthetic soft-tissue reactions) following metal-on-metal hip resurfacing arthroplasty (MoMHRA) have been associated with elevated metal ion levels, suggesting that excessive wear may occur due to edge-loading of these MoM implants. This study aimed to quantify in vivo edge-loading in MoMHRA patients with and without pseudotumours during functional activities. METHODS: The duration and magnitude of edge-loading in vivo was quantified during functional activities by combining the dynamic hip joint segment contact force calculated from the three-dimensional (3D) motion analysis system with the 3D reconstruction of orientation of the acetabular component and each patient's specific hip joint centre, based on CT scans. RESULTS: Edge-loading in the hips with pseudotumours occurred with a four-fold increase in duration and magnitude of force compared with the hips without pseudotumours (p = 0.02). CONCLUSIONS: The study provides the first in vivo evidence to support that edge-loading is an important mechanism that leads to localised excessive wear (edge-wear), with subsequent elevation of metal ion levels in MoMHRA patients with pseudotumours.

Preclinical development of CAT-354, an IL-13 neutralizing antibody, for the treatment of severe uncontrolled asthma.

BACKGROUND AND PURPOSE: IL-13 is a pleiotropic Th2 cytokine considered likely to play a pivotal role in asthma. Here we describe the preclinical in vitro and in vivo characterization of CAT-354, an IL-13-neutralizing IgG4 monoclonal antibody (mAb), currently in clinical development. EXPERIMENTAL APPROACH: In vitro the potency, specificity and species selectivity of CAT-354 was assayed in TF-1 cells, human umbilical vein endothelial cells and HDLM-2 cells. The ability of CAT-354 to modulate disease-relevant mechanisms was tested in human cells measuring bronchial smooth muscle calcium flux induced by histamine, eotaxin generation by normal lung fibroblasts, CD23 upregulation in peripheral blood mononuclear cells and IgE production by B cells. In vivo CAT-354 was tested on human IL-13-induced air pouch inflammation in mice, ovalbumin-sensitization and challenge in IL-13 humanized mice and antigen challenge in cynomolgus monkeys. KEY RESULTS: CAT-354 has a 165 pM affinity for human IL-13 and functionally neutralized human, human variant associated with asthma and atopy (R130Q) and cynomolgus monkey, but not mouse, IL-13. CAT-354 did not neutralize human IL-4. In vitro CAT-354 functionally inhibited IL-13-induced eotaxin production, an analogue of smooth muscle airways hyperresponsiveness, CD23 upregulation and IgE production. In vivo in humanized mouse and cynomolgus monkey antigen challenge models CAT-354 inhibited airways hyperresponsiveness and bronchoalveolar lavage eosinophilia. CONCLUSIONS AND IMPLICATIONS: CAT-354 is a potent and selective IL-13-neutralizing IgG4 mAb. The preclinical data presented here support the trialling of this mAb in patients with moderate to severe uncontrolled asthma.

Embryonic stem cells and prospects for their use in regenerative medicine approaches to motor neurone disease.

Human embryonic stem cells are pluripotent cells with the potential to differentiate into any cell type in the presence of appropriate stimulatory factors and environmental cues. Their broad developmental potential has led to valuable insights into the principles of developmental and cell biology and to the proposed use of human embryonic stem cells or their differentiated progeny in regenerative medicine. This review focuses on the prospects for the use of embryonic stem cells in cell-based therapy for motor neurone disease or amyotrophic lateral sclerosis, a progressive neurodegenerative disease that specifically affects upper and lower motor neurones and leads ultimately to death from respiratory failure. Stem cell-derived motor neurones could conceivably be used to replace the degenerated cells, to provide authentic substrates for drug development and screening and for furthering our understanding of disease mechanisms. However, to reliably and accurately culture motor neurones, the complex pathways by which differentiation occurs in vivo must be understood and reiterated in vitro by embryonic stem cells. Here we discuss the need for new therapeutic strategies in the treatment of motor neurone disease, the developmental processes that result in motor neurone formation in vivo, a number of experimental approaches to motor neurone production in vitro and recent progress in the application of stem cells to the treatment and understanding of motor neurone disease.

Function, structure and therapeutic potential of complement C5a receptors.

Complement fragment (C)5a is a 74 residue pro-inflammatory polypeptide produced during activation of the complement cascade of serum proteins in response to foreign surfaces such as microorganisms and tissue damaged by physical or chemical injury. C5a binds to at least two seven-transmembrane domain receptors, C5aR (C5R1, CD88) and C5L2 (gpr77), expressed ubiquitously on a wide variety of cells but particularly on the surface of immune cells like macrophages, neutrophils and T cells. C5aR is a classical G protein-coupled receptor that signals through G alpha i and G alpha 16, whereas C5L2 does not appear to couple to G proteins and has no known signalling activity. Although C5a was first described as an anaphylatoxin and later as a leukocyte chemoattractant, the widespread expression of C5aR suggested more general functionality. Our understanding of the physiology of C5a has improved significantly in recent years through exploitation of receptor knockout and knocking mice, C5 and C5a antibodies, soluble recombinant C5a and C5a analogues and newly developed receptor antagonists. C5a is now also implicated in non-immunological functions associated with developmental biology, CNS development and neurodegeneration, tissue regeneration, and haematopoiesis. Combined receptor mutagenesis, molecular modelling, structure-activity relationship studies and species dependence for ligand potency on C5aR have been helpful for identifying ligand binding sites on the receptor and for defining mechanisms of receptor activation and inactivation. This review will highlight major developments in C5a receptor research that support C5aR as an important therapeutic target. The intriguing possibilities raised by the existence of a non-signalling C5a receptor are also discussed.

Mast cells express IL-13R alpha 1: IL-13 promotes human lung mast cell proliferation and Fc epsilon RI expression.

BACKGROUND: The Th2 cytokine interleukin (IL)-13 is implicated in the development of various allergic diseases including asthma. The IL-13 receptor, IL-13Ralpha1, is expressed on most leukocytes, except T-cells. Evidence to support IL-13Ralpha1 expression on mast cells is limited. METHODS: We investigated: (i) IL-13Ralpha1 expression by human lung mast cells (HLMC); (ii) the number of IL-13Ralpha1+ bronchial submucosal mast cells in subjects with asthma and normal controls and (iii) the effect of IL-13 priming on HLMC expression of high-affinity IgE receptor (FcepsilonRI), stem cell factor receptor (CD117), histamine release, proliferation, and survival. RESULTS: Human lung mast cell expressed IL-13Ralpha1 mRNA. IL-13Ralpha1 was highly expressed on the surface HLMC (82+/-9%). Bronchial submucosal mast cell IL-13Ralpha1 expression was higher in asthmatics (86+/-2%) than normal controls (78+/-2%; P=0.015). IL-13 priming for 30 min did not increase HLMC histamine release, in the presence or absence of SCF or in response to IgE/anti-IgE activation. IL-13 priming for 5 days upregulated HLMC FcepsilonRI expression (22% increase in fluorescent intensity; P=0.003), increased histamine release following IgE/anti-IgE activation by 56% (P=0.03) and increased proliferation by 50% (P=0.003) without affecting cell survival or CD117 expression. The IL-13 specific neutralizing antibody CAT-354 inhibited all IL-13 mediated effects. CONCLUSION: Human lung mast cell express IL-13Ralpha1 and activation by IL-13 for 5 days increased FcepsilonRI expression and proliferation. Histamine release was not affected by short-term priming with IL-13, but was upregulated by priming for 5 days suggesting that this effect was mediated by the increased FcepsilonRI expression. These data support the view that targeting IL-13 may be beneficial in the treatment of asthma.

Inhibition of human interleukin-13-induced respiratory and oesophageal inflammation by anti-human-interleukin-13 antibody (CAT-354).

BACKGROUND: Allergic asthma is a complex disorder characterized by local and systemic T helper type 2 -cell responses such as the production of IL-13, a cytokine associated with the induction of airway hyper-responsiveness (AHR), chronic pulmonary eosinophilia, airway mucus overproduction and eosinophilic oesophagitis. OBJECTIVE: Our study aimed to address the therapeutic potential of a human anti-human IL-13 IgG4 monoclonal antibody (CAT-354) in a murine model of respiratory and oesophageal inflammation induced by intratracheal human IL-13. METHODS: BALB/c mice were treated on days 1 and 3 with CAT-354 (intraperitoneal injection), and human IL-13 was injected intratracheally on days 2 and 4. AHR to methacholine, airway eosinophilia in bronchoalveolar lavage fluid, histologic analysis of goblet cell metaplasia and oesophageal eosinophilia were evaluated. RESULTS: Human IL-13 induced airway eosinophilia and goblet cell metaplasia in mice in a dose-dependent manner. Moreover, intratracheal dosing with 25 microg of human IL-13 was sufficient to induce AHR, goblet cell metaplasia and oesophageal eosinophilia. Pretreatment with CAT-354 significantly reduced AHR, airway eosinophilia and oesophageal eosinophilia. CONCLUSION: These results demonstrate that anti-human IL-13 (CAT-354) is a potential therapeutic treatment for allergic airway and oesophageal diseases.

Blunt trauma to the spleen: ultrasonographic findings.

The spleen is the most frequently injured organ in adults who sustain blunt abdominal trauma. Splenic trauma accounts for approximately 25% to 30% of all intra-abdominal injuries. The management of splenic injury has undergone rapid change over the last decade, with increasing emphasis on splenic salvage and non-operative management. Identifying the presence and degree of splenic injury is critical in triaging the management of patients. Imaging is integral in the identification of splenic injuries, both at the time of injury and during follow-up. Although CT remains the gold standard in blunt abdominal trauma, US continues to play an important role in assessing the traumatized spleen. This pictorial review illustrates the various ultrasonographic appearances of the traumatized spleen. Correlation with other imaging is presented and complications that occur during follow-up are described.

Clusters of meningococcal disease in school and preschool settings in England and Wales: what is the risk?

AIMS: To assess the risk of further cases in educational settings in order to inform policy on managing cases and clusters of meningococcal disease. METHODS: Between 1 April 1995 and 31 March 2001, surveillance in preschool and school settings in England and Wales identified 114 clusters of meningococcal disease. Twenty clusters were reported in preschool settings, 43 in primary, 46 in secondary, and five in independent schools. Seventy three clusters (64%) consisted of two or more confirmed cases, of which 30 had two or more serogroup C cases. Following the introduction of the national meningococcal serogroup C vaccination programme in 1999, no serogroup C clusters were observed between April 2000 and March 2001. RESULTS: The relative risk of further cases in the four weeks after a single case compared with the background rate was raised in all settings, ranging from RR 27.6 (95% CI 15.2 to 39.9) in preschool settings to RR 3.6 (95% CI 2.5 to 4.6) in secondary schools. Absolute risk estimates ranged from 70/100 000 in preschool settings to 3.0/100 000 in secondary schools. The relative risk of clustering was similar for serogroup B and C strains. Most (68%) second cases occurred within seven days of the first case. CONCLUSIONS: Although there was a higher risk of further cases of meningococcal disease in schools and especially in preschool settings, it is not known whether widespread antibiotic use after single cases reduces risk of further cases and if there is a real risk of harm. Evidence of risk reduction is needed to inform public health policy.

Risk factors for drug resistant tuberculosis in Leicestershire--poor adherence to treatment remains an important cause of resistance.

In the light of rising numbers of tuberculosis (TB) cases in the United Kingdom, the problem of anti-tubercular drug resistance remains a significant concern. Drug resistant TB cases are more difficult and costly to treat, and require appropriate treatment and control mechanisms. This matched case control study aimed to investigate risk factors for resistance in Leicestershire, using data for laboratory isolates of Mycobacterium tuberculosis identified from 1993 to 1998. Each case, defined as culture positive laboratory isolates resistant to at least one first-line drug, was matched to four fully sensitive controls on age, sex and ethnic group. Twenty-three cases and 81 controls were included in the analysis. Drug resistance in Leicestershire was found to be associated with poor adherence to treatment (OR 4.8, 95% CI 1.6-14.4, P=0.005) and with previous TB (OR 3.7, 95% CI 1.2-11.8, P=0.022). These findings emphasize the need to provide support to patients taking treatment in order to maximize adherence.

Tc-99m-labeled C5a and C5a des Arg74 for infection imaging.

UNLABELLED: The complement anaphylatoxin C5a and its natural metabolite C5a des Arg(74) (C5adR) are involved in several stages of the inflammatory process. Both act on a common receptor expressed on different cell types, including neutrophils and monocytes. The receptor binding affinity of C5a is in the nanomolar range and exceeds that of C5adR by 1-2 orders of magnitude. The biologic potency of C5a is considerably higher than that of C5adR. Here we tested both proteins labeled with (99m)Tc for imaging of infection. METHODS: The proteins were labeled with (99m)Tc via the hydrazinonicotinamide (HYNIC) chelator. The preparations were tested for imaging of infection in a rabbit model of intramuscular infection. Biodistribution of the radiolabel was determined by gamma-camera imaging and by counting dissected tissues at 5 h p.i. RESULTS: C5a and C5adR showed in vivo abscess uptakes of 0.12 and 0.025%ID/g, abscess/muscle ratios of 76 and 14, abscess/blood ratios of 9.1 and 2.6, and ROI derived target-to-background ratios of 5.9 and 2.1, respectively at 5 h p.i. CONCLUSION: For infection imaging (99m)Tc-labeled C5a showed excellent in vivo characteristics. However, C5a is a very bioactive protein, impeding its clinical use as an infection imaging agent. The naturally occurring partial agonist C5adR has less biological effect but showed suboptimal imaging characteristics. The present study showed that for adequate localization of a receptor binding ligand affinities for the receptor in the nanomolar range are required.

Mapping the ligand-binding site on the C5a receptor: arginine74 of C5a contacts aspartate282 of the C5a receptor.

The interaction between the anaphylatoxin C5a and its receptor involves two distinct sites. One site is formed by acidic residues at the receptor N-terminus and contributes to only ligand binding. The second site, responsible for activation, is less well defined. In this study, we demonstrate that the receptor residue D(282), near the extracellular face of transmembrane domain VII, is a component of the second ligand-binding site. Mutation of D(282) to A decreases the sensitivity of the receptor to activation by intact C5a but not by its less potent metabolite, C5adR(74), which lacks the C-terminal arginine(74). The mutation of the R(74) residue of C5a to A causes a 60-fold decrease in wild-type receptor sensitivity, but only a 2-fold decrease for the receptor mutated at D(282). In contrast, the mutation of R(74) to D makes C5a completely inactive on both wild-type and A(282) C5a receptors. The mutation of D(282) to R partly restores the response to C5a[D(74)], which is a more effective ligand than C5a at the mutant receptor. A peptide mimic of the C5a activation domain with a C-terminal R potently activates the wild type but is only a weak agonist at the mutant D(282)R-C5a receptor. Conversely, a peptide with D at the C-terminus is a more effective activator of D(282)R than wild-type C5a receptors. These data indicate that the R(74) side chain of C5a makes an interaction with receptor D(282) that is responsible for the higher potency of intact C5a versus that of C5adR(74).

Preventing secondary meningococcal disease in health care workers: recommendations of a working group of the PHLS meningococcus forum.

Based on new data on the risk of secondary meningococcal disease in health care workers, a review of published cases and an assessment of the available evidence, a change to the recommendations for giving chemoprophylaxis to health care workers in England and Wales is proposed. Previous guidance recommended prophylaxis only for those who had given mouth to mouth resuscitation. Chemoprophylaxis is now recommended for health care workers whose mouth or nose has been directly and heavily exposed to respiratory droplets/secretions from a case of meningococcal disease around the time of hospital admission. Wearing surgical face masks is encouraged to reduce risk of exposure.

Species dependence for binding of small molecule agonist and antagonists to the C5a receptor on polymorphonuclear leukocytes.

This study investigated the receptor binding affinities of a C5a agonist and cyclic antagonists for polymorphonuclear leukocytes (PMNs) isolated from human, sheep, pig, dog, rabbit, guinea pig, rat and mouse. The affinities of the two small molecule antagonists, F-[OPdChaWR] and AcF-[OPdChaWR], and the agonist, YSFKPMPLaR, revealed large differences in C5a receptor (C5aR) affinities between species. The antagonists bound to human, rat and dog PMNs with similar high affinities, but with lower affinities to PMNs from all other species. The C5a agonist also bound with varying affinities between species, but showed a different affinity profile to the antagonists. In contrast, recombinant human C5a had similar affinity for PMNs of all species investigated. The low correlation between the affinities of the antagonists and the agonist between species either suggests that different receptor residues are important for distinguishing between agonist/antagonist binding, or that the agonist and antagonist peptides bind to two distinct sites within the C5aR.