Evaluation of variable new antigen receptors (vNARs) as a novel cathepsin S (CTSS) targeting strategy.

Aberrant activity of the cysteine protease Cathepsin S (CTSS) has been implicated across a wide range of pathologies. Notably in cancer, CTSS has been shown to promote tumour progression, primarily through facilitating invasion and migration of tumour cells and augmenting angiogenesis. Whilst an attractive therapeutic target, more efficacious CTSS inhibitors are required. Here, we investigated the potential application of Variable New Antigen Receptors (vNARs) as a novel inhibitory strategy. A panel of potential vNAR binders were identified following a phage display panning process against human recombinant proCTSS. These were subsequently expressed, purified and binding affinity confirmed by ELISA and SPR based approaches. Selected lead clones were taken forward and were shown to inhibit CTSS activity in recombinant enzyme activity assays. Further assessment demonstrated that our lead clones functioned by a novel inhibitory mechanism, by preventing the activation of proCTSS to the mature enzyme. Moreover, using an intrabody approach, we exhibited the ability to express these clones intracellularly and inhibit CTSS activity whilst lead clones were also noted to impede cell invasion in a tumour cell invasion assay. Collectively, these findings illustrate a novel mechanistic approach for inhibiting CTSS activity, with anti-CTSS vNAR clones possessing therapeutic potential in combating deleterious CTSS activity. Furthermore, this study exemplifies the potential of vNARs in targeting intracellular proteins, opening a range of previously "undruggable" targets for biologic-based therapy.

The role of Cathepsin S as a marker of prognosis and predictor of chemotherapy benefit in adjuvant CRC: a pilot study.

BACKGROUND: The aim of this pilot retrospective study was to investigate the immunohistochemical expression of Cathepsin S (CatS) in three cohorts of colorectal cancer (CRC) patients (n=560). METHODS: Prevalence and association with histopathological variables were assessed across all cohorts. Association with clinical outcomes was investigated in the Northern Ireland Adjuvant Chemotherapy Trial cohort (n=211), where stage II/III CRC patients were randomised between surgery-alone or surgery with adjuvant fluorouracil/folinic acid (FU/FA) treatment. RESULTS: Greater than 95% of tumours had detectable CatS expression with significantly increased staining in tumours compared with matched normal colon (P>0.001). Increasing CatS was associated with reduced recurrence-free survival (RFS; P=0.03) among patients treated with surgery alone. Adjuvant FU/FA significantly improved RFS (hazard ratio (HR), 0.33; 95% CI, 0.12-0.89) and overall survival (OS; HR, 0.25; 95% CI, 0.08-0.81) among 36 patients with high CatS. Treatment did not benefit the 66 patients with low CatS, with a RFS HR of 1.34 (95% CI, 0.60-3.19) and OS HR of 1.33 (95% CI, 0.56-3.15). Interaction between CatS and treatment status was significant for RFS (P=0.02) and OS (P=0.04) in a multivariate model adjusted for known prognostic markers. CONCLUSION: These results signify that CatS may be an important prognostic biomarker and predictive of response to adjuvant FU/FA in CRC.

A phase 1 study of a meningococcal native outer membrane vesicle vaccine made from a group B strain with deleted lpxL1 and synX, over-expressed factor H binding protein, two PorAs and stabilized OpcA expression.

This phase I clinical trial assessed the safety and immunogenicity of a native outer membrane vesicle (NOMV) vaccine prepared from an lpxL1(-) synX(-) mutant of strain 8570(B:4:P1.19,15:L8-5) of Neisseria meningitidis. Additional mutations enhance the expression of factor H binding protein variant 1 (fHbp v.1), stabilize expression of OpcA and introduce a second PorA (P1.22,14). Thirty-six volunteers were assigned to one of four dose groups (10, 25, 50 and 75 mcg, based on protein content) to receive three intramuscular injections at six week intervals with aluminum hydroxide adjuvant. Specific local and systemic adverse events were solicited by diary and at visits on days 2, 7, and 14 after each vaccination. Blood chemistries, complete blood count, and coagulation studies were measured on each vaccination day and again 2 and 14 days later. Blood for ELISA and serum bactericidal assays was drawn two and six weeks after each vaccination. The proportion of volunteers who developed a fourfold or greater increase in bactericidal activity to the wild type parent of the vaccine strain at two weeks after the third dose was 27 out of 34 (0.79, 95% C.I. 0.65-0.93). Against four other group B strains the response rate ranged from 41% to 82% indicating a good cross reactive antibody response. Depletion assays show contributions to bactericidal activity from antibodies to lipooligosaccharide (LOS), fHbp v.1 and OpcA.

Biomechanical and tissue handling property comparison of decellularized and cryopreserved tibialis anterior tendons following extreme incubation and rehydration.

Little is known regarding the biomechanical profiles and tissue handling properties of decellularized and cryopreserved human tibialis anterior tendons prepared as allografts for ACL reconstruction. This study compared allografts prepared using two extremes of the same cryoprotectant incubation and rehydration technique with a standardly prepared control group. Porcine tibiae with similar apparent BMD were randomly divided into three groups of ten specimens. Paired tendons were randomly divided into two experimental groups: Group 1 = 8 h incubation/15 min rehydration; Group 2 = 2 h incubation/1 h rehydration. Group 3 (control) consisted of ten standardly prepared tendons with 20 min rehydration. Tissue handling properties were graded during allograft preparation using a modified visual analog scale. Similar diameter allografts were fixed in matched diameter extraction drilled tibial tunnels with 35 mm long, 1 mm > tunnel diameter bioabsorbable interference screws. Potted constructs were mounted in a servo hydraulic device, pretensioned between 10-50 N at 0.1 Hz (10 cycles), and isometric pretensioned at 50 N for 1 min, prior to 500 submaximal loading cycles (50-250 N) at 0.5 Hz, and load to failure testing (20 mm/min). Constructs prepared under extreme conditions generally displayed comparable biomechanical properties to the control condition. Group 1 (8 h incubation/15 min rehydration)(-34 +/- 35 ms) and Group 2 (2 h incubation/1 h rehydration) (-22 +/- 38 ms) displayed smaller mean displacement-load peak phase timing differences over the initial ten cycles compared to Group 3 (control)(-42 +/- 49 ms), P = 0.004, suggesting greater relative construct stiffness. Group 1 (8 h incubation/15 min rehydration) (234.9 +/- 34 N/mm) and Group 2 (2 h incubation/1 h rehydration)(231.3 +/- 43 N/mm) displayed lower construct stiffness during load to failure testing than Group 3 (control)(284.5 +/- 25.2 N/mm), P = 0.003. Group 1 (8 h incubation/15 min rehydration) differed from Group 2 (2 h incubation/1 h rehydration) and Group 3 (control) for perceived tensile stiffness (2.4 +/- 2.0 vs. 7.0 +/- 0.5 and 7.9 +/- 0.3, respectively), compressive resilience (1.7 +/- 0.8 vs. 5.9 +/- 1.0 and 7.8 +/- 0.4, respectively), handling ease (2.8 +/- 1.0 vs. 6.5 +/- 0.5 and 7.0 +/- 0.7, respectively), color (2.6 +/- 0.8 vs. 4.7 +/- 0.7 and 5.1 +/- 0.3, respectively) and texture (4.0 +/- 0.8 vs. 6.2 +/- 0.8 and 6.8 +/- 0.8, respectively) (P < 0.0001). Group 2 (2 h incubation/1 h rehydration)(6.0 +/- 0.7 and 5.9 +/- 1.0, respectively) also differed from Group 3 (control)(6.8 +/- 0.8 and 7.8 +/- 0.4, respectively) for general "feel" and compressive resilience (P < 0.0001). Tensile stiffness and compressive resilience displayed moderate and weak relationships, respectively with displacement during submaximal cyclic loading (r2 = 0.78 and 0.58, respectively), stiffness (r2 = 0.33 and 0.44, respectively) and load at failure (r2 = 0.59 and 0.37, respectively) for Group 3 (control), but not for experimental Group 1 (8 h incubation/15 min rehydration) or Group 2 (2 h incubation/1 h rehydration). Knee surgeons should be aware that soft tissue tendon decellularization and cryopreservation may change the biomechanical stiffness, tissue handling properties, and relationships between these variables compared to standardly prepared allograft tissue.

Patients with diabetic nephropathy on renal replacement therapy in England and Wales.

BACKGROUND: The incidence of patients with diabetic nephropathy (DN) who start renal replacement therapy (RRT) is increasing. AIM: To describe the characteristics and survival of patients with DN starting RRT in the UK. DESIGN: Retrospective cohort study. METHODS: We analysed data for incident patients on RRT in centres participating in the Renal Association UK Renal Registry (UKRR), 1997 -2004, comparing DN vs. non-DN patients with regard to survival, social deprivation, ethnicity, gender, and age, using Cox regression models. RESULTS: DN was the most common renal disease (19%) in the 20 532 patients starting RRT. The majority of patients with DN (77%) were Caucasian. Within the Caucasian population, DN patients were more likely to be from a socially deprived area (p < 0.0001). About 20% were referred <3 months before starting RRT. The difference in crude survival was greatest in younger patients (5-year survival was 56% (DN) vs. 85% (non-DN) in patients aged 18-54 years, and 17% (DN) vs. 28% (non-DN) in patients aged >or=65 years). Despite adjusting for gender, age, treatment modality, social deprivation, referral and co-morbidities, the long-term prognosis for DN patients aged 18-54 years was worse (adjusted hazard ratio 2.13, 95%CI 1.23-3.67) than for older age groups. DISCUSSION: Patients with DN starting RRT are more likely to come from socially deprived areas. Relative risk of death is greatest in working-age DN patients and is not fully explained by recorded co-morbidity. This emphasizes the need for focused diabetes care in poorer areas, and assessment of quality of care of diabetic patients on RRT.

Biomechanical comparison between CentraLoc and Intrafix fixation of quadrupled semitendinosus-gracilis allografts in cadaveric tibiae with low bone mineral density.

Supplementary or back-up tibial tunnel fixation of a quadruple semitendinosus-gracilis (STG) graft is often performed when the knee surgeon questions the integrity of intra-tunnel fixation. Back-up fixation devices such as staples however may contribute to increased knee pain and dysfunction. Both primary extra-tunnel and intra-tunnel fixation devices may provide sufficient quadruple STG graft fixation in a tibial tunnel to preclude the need for back-up fixation. This biomechanical study compared the fixation of quadruple STG allografts in standard drilled tunnels prepared in low apparent bone mineral density (BMD) cadaveric tibiae using either an Intrafix device with primary intra-tunnel fixation in a region of predominantly cancellous trabecular bone, or a CentraLoc device with primary extra-tunnel fixation in a region of predominantly cortical bone. The study hypothesis was that the CentraLoc device would display superior fixation in these low apparent BMD cadaveric tibiae. Matched pair tibiae and quadruple STG allografts were divided into two groups of seven specimens each. Extraction drilled tunnels matched allograft diameter. Constructs were pretensioned on a servo hydraulic device between 10 and 50 N for 10 cycles and isometric pretensioned at 50 N for 1 min prior to undergoing 500 loading cycles (50-250 N) and load to failure testing (20 mm/min). The CentraLoc group displayed superior load at failure (448.4+/-171 N vs. 338.4+/-119 N, P=0.04) and survived more loading cycles (410+/-154 cycles vs. 196+/-230 cycles, P=0.04) than the Intrafix group. Most CentraLoc group specimens (6/7, 85.7%) failed by device pullout with intact quadruple STG allograft strands while all Intrafix group specimens (7/7, 100%) failed by slippage of one or more strands (P=0.005).

Detection of cathepsin S cysteine protease in human brain tumour microdialysates in vivo.

Microdialysis enables the chemistry of extracellular fluid in body tissues to be measured. Extracellular proteases such as the cysteine protease, cathepsin S (CatS), are thought to facilitate astrocytoma invasion. Microdialysates obtained from human brain tumours in vivo were subjected to cathepsin S activity and ELISA assays. Cathepsin S ELISA expression was detected in five out of 10 tumour microdialysates, while activity was detected in five out of 11 tumour microdialysates. Cathepsin S expression was also detected in microdialysate from the normal brain control although no activity was found in the same sample. While some refinements to the technique are necessary, the authors demonstrate the feasibility and safety of microdialysis in human astrocytomas in vivo. Characterisation of the extracellular environment of brain tumours in vivo using microdialysis may be a useful tool to identify the protease profile of brain tumours.

"Benign" hypertensive nephrosclerosis.

BACKGROUND: Whether benign hypertensive nephrosclerosis (BHN) causes end-stage renal failure (ESRF) is controversial. One reason for this is the lack of biopsy evidence confirming the clinical diagnosis in most cases. AIM: To investigate whether biopsy-proven BHN leads to ESRF. DESIGN: Retrospective analysis. METHODS: We analysed all cases of biopsy-proven BHN from a single centre over a period of 20 years (n = 60), followed-up for a mean +/- SD 6.7 +/- 5.5 years. RESULTS: Patients were divided into those with stable renal function (n = 17) and those with declining function (n = 43). Mean eGFR at the time of biopsy was lower in the declining function group (29 +/- 3 vs. 44 +/- 4 ml/min/1.73 m(2), serum creatinine 280 +/- 165 vs. 161 +/- 89 mumol/l, p < 0.001), of whom 72% progressed to ESRF. Median renal survival for the whole group was 6.8 years, with 5- and 10-year survivals of 56% and 35%, respectively. Renal survival was significantly affected by initial serum creatinine, and mean systolic and diastolic blood pressures during follow-up period. Mean protein excretion was higher in the declining group, but not significantly so. On multivariate analysis, only diastolic blood pressure during follow-up predicted renal survival (p = 0.017). Median patient survival for the whole group was 9.95 years post renal biopsy, with 5- and 10-year survivals of 70% and 49% respectively. Survival was affected by initial serum creatinine, initial serum albumin and mean systolic blood pressure during follow-up. On multivariate analysis, only initial serum creatinine was significantly correlated with survival (p = 0.017). DISCUSSION: Biopsy-proven BHN led to ESRF in a high percentage of our patients, and was associated with significant mortality.

Current chronic kidney disease practice patterns in the UK: a national survey.

BACKGROUND: There is an increasing focus on improving the detection and management of patients with chronic kidney disease (CKD). Data on CKD prevalence based on population sampling are now available, but there are few data about CKD patients attending nephrology services or how such services are organized. AIM: To survey services for CKD patients nationally. METHODS: A pre-piloted questionnaire was sent to all 72 renal units in the UK, referring to the situation in June 2004. RESULTS: Seventy units (97%) responded. The median ratio of prevalent CKD patients/prevalent renal replacement therapy (RRT) patients in the 25 units with data was 3.7 (IQR 2.7-5.7) and the median ratio of CKD stage 4 and 5 patients/prevalent RRT patients was 0.6 (IQR 0.4-1.1). This gives an estimated 140 000 CKD patients under the care of UK nephrologists, with 23 000 at CKD stage 4 or 5 (excluding those on RRT). Very few units had a full complement of the recommended multi-skilled renal team. Counsellors and psychologist were the most common perceived shortages. Of 70 responding units, 50 (74%) were using low clearance clinics for management of advanced CKD patients. Elective dialysis access services often had long delays, with median waiting time for vascular access ranging between 1 and 36 weeks, and for Tenchkoff catheter, between 0 and 12 weeks. DISCUSSION: CKD patients are a significant workload for UK nephrologists. Current provision of service is variable, and services need to be re-designed to cope with the expected future increase of referral of CKD patients.

Inequalities in glycaemic control in patients with Type 2 diabetes in primary care.

AIMS: To quantify relationships between patient and practice factors and glycaemic control in patients with Type 2 diabetes. METHODS: A cross-sectional study involving 1534 patients with Type 2 diabetes from 42 general practices in Nottingham, UK was undertaken. Patient characteristics were assessed by a clinical interview and case note review and practice characteristics by a questionnaire. The outcome measure was serum HbA(1c) concentration measured at entry to the study. Random effects linear regression was used to model patient and practice factors associated with glycaemic control. RESULTS: In multivariable regression analysis, HbA(1c) increased with increasing body mass index (BMI) [change in HbA(1c) for one unit increase in BMI: 0.03%, 95% confidence interval (CI) 0.01, 0.04], and was higher in those using oral medication (mean difference 0.75%, 95% CI 0.59, 0.92) or insulin compared with diet (mean difference 1.36%, 95% CI 1.10, 1.62). There was a dose-response relationship between HbA(1c) and increasing time since diagnosis. HbA(1c) was negatively associated with age (change per year -0.01%, 95% CI -0.02, -0.004). Patients registered at the most deprived practices had higher HbA(1c) values than those in the least deprived practices (mean difference 0.42%, 95% CI 0.14, 0.71), as did those in practices where annual reviews were carried out by the nurse alone (mean difference 0.24%, 95% CI 0.04, 0.44). CONCLUSIONS: Several patient and practice factors are related to glycaemic control. Poorer glycaemic control was associated with practice level deprivation and nurses undertaking annual reviews alone. Further research is required to explore outcomes of annual reviews undertaken by nurses alone. Greater resources may be needed by primary care teams working in deprived areas to address inequalities in diabetic control.

Trends in adult renal replacement therapy in the UK: 1982-2002.

BACKGROUND: Following the introduction of dialysis and transplantation for the treatment of established renal failure (ERF) 40 years ago, the UK failed to match the achievements of many other countries. AIM: To review progress with treatment for ERF in the UK in the past 20 years. DESIGN: Review of four cross-sectional national studies, and 1997-2002 annual UK Renal Registry data. METHODS: Data on UK patients on renal replacement treatment (RRT) were collated from three sources: European Registry reports for 1982-1990, surveys carried out within the UK in 1993, 1996, 1998 and 2002, and the UK Renal Registry database (1997-2002). Trends in acceptance and prevalence rates, median age, cause of ERF, and treatment modality were analysed and compared with current data from other countries. RESULTS: The UK annual acceptance rate for RRT increased from 20 per million population (pmp) in 1982 to 101 pmp in 2002. This growth was largely in those aged over 65 years, and in those with co-morbidity. Annual acceptance rates for ERF due to diabetes rose from 1.6 to 18 pmp. The prevalence of RRT increased from 157 pmp in 1982 to 626 pmp in 2002. Hospital haemodialysis has become the main modality, and is increasingly being provided in satellite units. Although rising, UK acceptance and prevalence rates are still lower than in many developed countries. DISCUSSION: Despite significant expansion in RRT services for adults in the UK over the last 20 years, there is evidence of unmet need, and need is expected to rise, due to demographic changes and trends in type 2 diabetes. Continuing growth in the already substantial investment in RRT will be needed, unless efforts to prevent the occurrence of ERF are successful.

Tracheobronchial malacia and stenosis in children in intensive care: bronchograms help to predict oucome.

BACKGROUND: Severe tracheobronchial malacia and stenosis are important causes of morbidity and mortality in children in intensive care, but little is known about how best to diagnose these conditions or determine their prognosis. METHODS: The records of all 62 children in whom one or both of these conditions had been diagnosed by contrast cinetracheobronchography in our intensive care unit in the period 1986-95 were studied. RESULTS: Seventy four per cent of the 62 children had congenital heart disease; none was a preterm baby with airways disease associated with prolonged ventilation. Fifteen of the children had airway stenosis without malacia; three died because of the stenosis and two died from other causes. Twenty eight of the 47 children with malacia died; only eight children survived without developmental or respiratory handicap. All children needing ventilation for malacia for longer than 14 consecutive days died if their bronchogram showed moderate or severe malacia of either main bronchus (15 cases), or malacia of any severity of both bronchi (three additional cases); all children needing ventilation for malacia for longer than 21 consecutive days died if their bronchogram showed malacia of any severity of the trachea or a main bronchus (three additional cases). These findings were strongly associated with a fatal outcome (p<0.00005); they were present in 21 children (all of whom died) and absent in 26 (of whom seven died, six from non-respiratory causes). They had a positive predictive value for death of 100%, but the lower limit of the 95% confidence interval was 83.9% so up to 16% of patients meeting the criteria might survive. CONCLUSION: In this series the findings on contrast cinetracheobronchography combined with the duration of ventilation provided a useful guide to the prognosis of children with tracheobronchomalacia. The information provided by bronchoscopy was less useful.

The effect of long, slow haemodialysis on patient survival.

BACKGROUND: Long, slow haemodialysis (24 h/week) has been associated with excellent patient survival and reduced cardiovascular mortality. Comparisons of patient survival have only been possible with registry data and other published series which do not control for individual patient characteristics. This retrospective study compares actuarial patient survival between a unit using long, slow haemodialysis (Tassin) and one employing 'conventional' haemodialysis (Nottingham). METHODS: All patients undergoing haemodialysis at each centre since 1980 were included (Tassin, 452 patients; Nottingham, 282 patients). Actuarial curves of patient survival were calculated by the life-table method and log rank test was used to compare data. Patients were grouped as follows: standard (SRD) and non-standard (NSRD) renal diseases; diabetics and non-diabetics; patients with and without cardiovascular antecedents; risk stratification based on age and comorbidity. RESULTS: Overall survival was significantly better in Tassin. This difference was also noted for patients with SRD and non-diabetics (both P < 0.001) and for those with (P = 0.007) and without (P < 0.001) cardiovascular antecedents. Survival did not differ significantly for NSRD and diabetics. Survival was better in Tassin in low-risk (P < 0.001) and medium-risk (P < 0.001) groups, but not for high-risk (risk stratification). CONCLUSIONS: Overall survival is increased on long, slow haemodialysis. Although the benefits are seen in the most favourable prognostic categories, they are also present in patients with comorbid illness (medium-risk group) and pre-existing cardiovascular disease.

Management of hypertension in patients developing end-stage renal failure.

The benefits of treating hypertension include preventing or delaying the progression of chronic renal failure, and reducing the cardiovascular complications of patients with renal disease. We examined how well hypertension had been managed in all 145 patients from a single health district who started dialysis during a 3-year period. Data relating to management of hypertension, including all blood pressure readings, were obtained from their general practice and hospital case notes. The anonymized data were reviewed by two independent assessors against a set of standards based on the British Hypertension Society guidelines. There was close agreement between the assessors. Complete records were obtained in 98.5% of cases. Of the 145 patients, 107 (76.4%) were hypertensive before developing end-stage renal failure. There were departures from standards in all categories of care: 24.3% in detection/diagnosis, 29% in investigation, 22.4% in referring to a nephrologist, and 17% in follow-up. The British Hypertension Society recommended standard for diastolic blood pressure of 90 mmHg was achieved in only 45%. In 32%, the assessors independently concluded that poor blood pressure control might have affected adversely the progression of renal failure. New methods of dealing with these problems are required and possible approaches are discussed.