The relationship between ADAMTS13 genotype and phenotype in congenital thrombotic thrombocytopenic purpura and characterisation of ADAMTS13 mutants

Congenital thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy, usually involving ADAMTS13 gene defects. ADAMTS13 processes the multimeric plasma glycoprotein Von Willebrand factor making it less reactive to platelets. Patients differ in terms of disease severity and evidence suggests a relationship between ADAMTS13 genotype and disease phenotype. Over 140 mutations have been identified in patients but only ~30% of these has been expressed in vitro. The aim of this thesis was to study certain ADAMTS13 mutations identified in a homozygous form in congenital TTP patients to assess in vitro their effect on the secretion and activity of ADAMTS13 and to assess their contribution to disease phenotype. ADAMTS13 mutants (p.R102H, p.I143T and p.Y570C) and wild type (WT) were expressed in HEK293T cells. The p.R102H mutation partially affected the secretion of ADAMTS13 and reduced the catalytic efficiency of the mutant but not to the extent predicted based upon levels measured in patient plasma. Expressing this mutant with three ADAMTS13 polymorphisms (p.Q448E, p.P618A and p.A900V) which were also identified in the patient with this mutation further reduced the secretion and activity of ADAMTS13. When these three polymorphisms were expressed separately in WT ADAMTS13, the p.P618A polymorphism reduced the secretion and subsequently the activity of ADAMTS13 suggesting that this polymorphism in particular was responsible for the reduction observed. These results highlight the importance of ADAMTS13 polymorphisms. The p.I143T and p.Y570C mutations severely affected ADAMTS13 secretion. Immunofluorescence studies showed localisation of these mutants within the ER but less extensive localisation within the cis Golgi compared to WT ADAMTS13. The p.I143T mutant was characterised further and was shown to be degraded by the cell proteasome. Addition of a chemical chaperone (betaine) appeared to rescue the secretion defect caused by the p.I143T mutation. This may have future therapeutic implications for the treatment of some congenital TTP patients

Autoantibodies enhance ADAMTS-13 clearance in patients with immune thrombotic thrombocytopenic purpura

Background Severe deficiency in ADAMTS-13 (<10%) and the loss of von Willebrand factor–cleaving function can precipitate microvascular thrombosis associated with thrombotic thrombocytopenic purpura (TTP). Patients with immune-mediated TTP (iTTP) have anti-ADAMTS-13 immunoglobulin G antibodies that inhibit ADAMTS-13 function and/or increase ADAMTS-13 clearance. Patients with iTTP are treated primarily by plasma exchange (PEX), often in combination with adjunct therapies that target either the von Willebrand factor-dependent microvascular thrombotic processes (caplacizumab) or the autoimmune components (steroids or rituximab) of the disease. Objectives To investigate the contributions of autoantibody-mediated ADAMTS-13 clearance and inhibition in patients with iTTP at presentation and through the course of the PEX therapy. Patients/Methods Anti-ADAMTS-13 immunoglobulin G antibodies, ADAMTS-13 antigen, and activity were measured before and after each PEX in 17 patients with iTTP and 20 acute TTP episodes. Results At presentation, 14 out of 15 patients with iTTP had ADAMTS-13 antigen levels of <10%, suggesting a major contribution of ADAMTS-13 clearance to the deficiency state. After the first PEX, both ADAMTS-13 antigen and activity levels increased similarly, and the anti-ADAMTS-13 autoantibody titer decreased in all patients, revealing ADAMTS-13 inhibition to be a modest modifier of the ADAMTS-13 function in iTTP. Analysis of ADAMTS-13 antigen levels between consecutive PEX treatments revealed that the rate of ADAMTS-13 clearance in 9 out of 14 patients analyzed was 4- to 10-fold faster than the estimated normal rate of clearance. Conclusion These data reveal, both at presentation and during PEX treatment, that antibody-mediated clearance of ADAMTS-13 is the major pathogenic mechanism that causes ADAMTS-13 deficiency in iTTP. Understanding the kinetics of ADAMTS-13 clearance in iTTP may now enable further optimization of treatment of patients with iTTP

Comparative Study of the Effects of Different Growth Hormone Doses on Growth and Spatial Performance of Hypophysectomized Rats

This study was designed to examine the effects of recombinant human growth hormone replacement on somatic growth and cognitive function in hypophysectomized (HYPOX) female Sprague-Dawley rats. Rats (5 per group) were randomized by weight to 3 experimental groups: group 1, administered 200 µg/kg of GH once daily for 9 days; group 2, administered 200 µg/kg of GH twice daily; and group 3, administered saline daily. Somatic growth was evaluated by measurement of body weight daily and of the width of the proximal tibial growth plate of the HYPOX rats. Cognitive function was evaluated using the Morris water maze (MWM) test. The results indicated that GH replacement therapy in HYPOX rats promoted an increase in the body weight and the width of the tibial growth plate in a dose-dependent manner. On the third day of the MWM test, the escape latency in the GH-treated groups 1 and 2 was significantly shorter than that in the control rats (P<0.001 and P=0.032, respectively), suggesting that rhGH improved spatial memory acquisition in the MWM test. Therefore it is concluded that rhGH replacement therapy in HYPOX rats stimulates an increase in somatic growth in a dose-dependent manner and also has beneficial effects on cognitive functions

Interactions between Multiple Recruitment Drivers: Post-Settlement Predation Mortality and Flow-Mediated Recruitment

Dispersal is a primary driver in shaping the future distribution of species in both terrestrial and marine systems. Physical transport by advection can regulate the distance travelled and rate of propagule supply to a habitat but post-settlement processes such as predation can decouple supply from recruitment. The effect of flow-mediated recruitment and predation on the recruitment success of an intertidal species, the eastern oyster Crassostrea virginica was evaluated in two-replicated field experiments. Two key crab species were manipulated to test predator identity effects on oyster mortality.Recruitment was ∼58% higher in high flow compared to low flow, but predation masked those differences. Predation mortality was primarily attributed to the blue crab Callinectes sapidus, whilst the mud crab Panopeus herbstii had no effect on recruit mortality. Recruit mortality from predation was high when recruit densities were high, but when recruit density was low, predation effects were not seen. Under high recruitment (supply), predation determined maximum population size and in low flow environments, recruitment success is likely determined by a combination of recruitment and resource limitation but not predation.Four processes are demonstrated: (1) Increases in flow rate positively affect recruitment success; (2) In high flow (recruitment) environments, resource availability is less important than predation; (3) predation is an important source of recruit mortality, but is dependent upon recruit density; and (4) recruitment and/or resource limitation is likely a major driver of population structure and functioning, modifying the interaction between predators and prey. Simultaneous testing of flow-mediated recruitment and predation was required to differentiate between the role of each process in determining population size. Our results reinforce the importance of propagule pressure, predation and post-settlement mortality as important determinants of population growth and persistence, but demonstrate that they should not be considered mutually exclusive

Evolutionary Pathways of the Pandemic Influenza A (H1N1) 2009 in the UK

The emergence of the influenza (H1N1) 2009 virus provided a unique opportunity to study the evolution of a pandemic virus following its introduction into the human population. Virological and clinical surveillance in the UK were comprehensive during the first and second waves of the pandemic in 2009, with extensive laboratory confirmation of infection allowing a detailed sampling of representative circulating viruses. We sequenced the complete coding region of the haemagglutinin (HA) segment of 685 H1N1 pandemic viruses selected without bias during two waves of pandemic in the UK (April-December 2009). Phylogenetic analysis showed that although temporal accumulation of amino acid changes was observed in the HA sequences, the overall diversity was less than that typically seen for seasonal influenza A H1N1 or H3N2. There was co-circulation of multiple variants as characterised by signature amino acid changes in the HA. A specific substitution (S203T) became predominant both in UK and global isolates. No antigenic drift occurred during 2009 as viruses with greater than four-fold reduction in their haemagglutination inhibition (HI) titre (“low reactors”) were detected in a low proportion (3%) and occurred sporadically. Although some limited antigenic divergence in viruses with four-fold reduction in HI titre might be related to the presence of 203T, additional studies are needed to test this hypothesis

Development of a novel monoclonal antibody with reactivity to a wide range of Venezuelan equine encephalitis virus strains

<p>Abstract</p> <p>Background</p> <p>There is currently a requirement for antiviral therapies capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV), as a licensed vaccine is not available for general human use. Monoclonal antibodies are increasingly being developed as therapeutics and are potential treatments for VEEV as they have been shown to be protective in the mouse model of disease. However, to be truly effective, the antibody should recognise multiple strains of VEEV and broadly reactive monoclonal antibodies are rarely and only coincidentally isolated using classical hybridoma technology.</p> <p>Results</p> <p>In this work, methods were developed to reliably derive broadly reactive murine antibodies. A phage library was created that expressed single chain variable fragments (scFv) isolated from mice immunised with multiple strains of VEEV. A broadly reactive scFv was identified and incorporated into a murine IgG2a framework. This novel antibody retained the broad reactivity exhibited by the scFv but did not possess virus neutralising activity. However, the antibody was still able to protect mice against VEEV disease induced by strain TrD when administered 24 h prior to challenge.</p> <p>Conclusion</p> <p>A monoclonal antibody possessing reactivity to a wide range of VEEV strains may be of benefit as a generic antiviral therapy. However, humanisation of the murine antibody will be required before it can be tested in humans.</p> <p>Crown Copyright © 2009</p

Increased Local Retention of Reef Coral Larvae as a Result of Ocean Warming

Climate change will alter many aspects of the ecology of organisms, including dispersal patterns and population connectivity. Understanding these changes is essential to predict future species distributions, estimate potential for adaptation, and design effective networks of protected areas. In marine environments, dispersal is often accomplished by larvae. At higher temperatures, larvae develop faster, but suffer higher mortality, making the effect of temperature on dispersal difficult to predict. Here, we experimentally calibrate the effect of temperature on larval survival and settlement in a dynamic model of coral dispersal. Our findings imply that most reefs globally will experience several-fold increases in local retention of larvae due to ocean warming. This increase will be particularly pronounced for reefs with mean water residence times comparable to the time required for species to become competent to settle. Higher local retention rates strengthen the link between abundance and recruitment at the reef scale, suggesting that populations will be more responsive to local conservation actions. Higher rates of local retention and mortality will weaken connectivity between populations, and thus potentially retard recovery following severe disturbances that substantially deplete local populations. Conversely, on isolated reefs that are dependent on replenishment from local broodstock, increases in local retention may hasten recovery

Illustrating risk difference and number needed to treat from a randomized controlled trial of spinal manipulation for cervicogenic headache

<p>Abstract</p> <p>Background</p> <p>The number needed to treat (NNT) for one participant to benefit is considered a useful, clinically meaningful way of reporting binary outcomes from randomized trials. Analysis of continuous data from our randomized controlled trial has previously demonstrated a significant and clinically important difference favoring spinal manipulation over a light massage control.</p> <p>Methods</p> <p>Eighty participants were randomized to receive spinal manipulation or a light massage control (n = 40/group). Improvements in cervicogenic headache pain (primary outcome), disability, and number in prior four weeks were dichotomized into binary outcomes at two thresholds: 30% representing minimal clinically important change and 50% representing clinical success. Groups were compared at 12 and 24-week follow-up using binomial regression (generalized linear models) to compute the adjusted risk difference (RD) between groups and number needed to treat (NNT) after adjusting for baseline differences between groups. Results were compared to logistic regression results.</p> <p>Results</p> <p>For headache pain, clinically important improvement (30% or 50%) was more likely for spinal manipulation: adjusted RD = 17% to 27% and NNT = 3.8 to 5.8 (p = .005 to .028). Some statistically significant results favoring manipulation were found for headache disability and number.</p> <p>Conclusion</p> <p>Spinal manipulation demonstrated a benefit in terms of a clinically important improvement of cervicogenic headache pain. The use of adjusted NNT is recommended; however, adjusted RD may be easier to interpret than NNT. The study demonstrated how results may depend on the threshold for dichotomizing variables into binary outcomes.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov NLM identifier NCT00246350.</p

The survey of serum retinol of the children aged 0~4 years in Zhejiang Province, China

<p>Abstract</p> <p>Background</p> <p>Vitamin A can have a positive impact on growth and development of children, but vitamin A deficiency (VAD) was found to be a public health problem in Zhejiang Province, China in 1998. There have been no studies on this topic in Zhejiang Province recently. This study was designed to evaluate the serum retinol levels of children aged 0~4 years in Zhejiang Province, southeast China. This epidemiological data will help design supplementation strategies for vitamin A in high-risk groups and improve their vitamin A status.</p> <p>Methods</p> <p>Children were randomly recruited for this study using a stratified sampling method. A blood sample was collected from each child. Assessment included C-reactive protein (CRP), serum retinol measured with HPLC and a questionnaire completed providing for family information and nutritional status. Logistic regression analysis was used to evaluate the risk factors for VAD in children.</p> <p>Results</p> <p>A group of 357 subjects aged 1 day to 4 years were recruited. The mean plasma retinol concentration was 1.653 (sd 0.47) μmol/L. There were 3.08% (11/357) of children affected with VAD, and 7.28% (26/357) of children had low vitamin A status, but none of the children showed any clinical symptoms of VAD. There was no significant difference in the levels of plasma retinol and the incidence rate of VAD between male and female children. Multivariate logistic regression analysis showed that living in urban region, having parents with good education and taking vitamin A capsule regularly prevented children from VAD, whereas being young (less than 2 years old) was a risk factor.</p> <p>Conclusion</p> <p>Low vitamin A status remains a nutritional problem in Zhejiang Province. The high-risk group in this study were young, dwelled in rural regions, had parents with poor education and did not take a regular vitamin A containing supplement.</p

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration