Identification and Monitoring of Host Cell Proteins by Mass Spectrometry Combined with High Performance Immunochemistry Testing

Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.     

MRI Plaque Imaging Detects Carotid Plaques with a High Risk for Future Cerebrovascular Events in Asymptomatic Patients

Purpose

The aim of this study was to investigate prospectively whether MRI plaque imaging can identify patients with asymptomatic carotid artery stenosis who have an increased risk for future cerebral events. MRI plaque imaging allows categorization of carotid stenosis into different lesion types (I–VIII). Within these lesion types, lesion types IV–V and VI are regarded as rupture-prone plaques, whereas the other lesion types represent stable ones.

Methods

Eighty-three consecutive patients (45 male (54.2%); age 54–88 years (mean 73.2 years)) presenting with an asymptomatic carotid stenosis of 50–99% according to ECST-criteria were recruited. Patients were imaged with a 1.5-T scanner. T1-, T2-, time-of-flight-, and proton-density weighted studies were performed. The carotid plaques were classified as lesion type I–VIII. Clinical endpoints were ischemic stroke, TIA or amaurosis fugax. Survival analysis and log rank test were used to ascertain statistical significance.

Results

Six out of 83 patients (7.2%) were excluded: 4 patients had insufficient MR image quality; 1 patient was lost-to-follow-up; 1 patient died shortly after the baseline MRI plaque imaging. The following results were obtained by analyzing the remaining 77 patients. The mean time of follow-up was 41.1 months.During follow-up, n = 9 (11.7%) ipsilateral ischemic cerebrovascular events occurred. Only patients presenting with the high-risk lesion types IV–V and VI developed an ipsilateral cerebrovascular event versus none of the patients presenting with the stable lesion types III, VII, and VIII (n = 9 (11.7%) vs. n = 0 (0%) during follow-up). Event-free survival was higher among patients with the MRI-defined stable lesion types (III, VII, and VIII) than in patients with the high-risk lesion types (IV–V and VI) (log rank test P<0.0001).

Conclusions

MRI plaque imaging has the potential to identify patients with asymptomatic carotid stenosis who are particularly at risk of developing future cerebral ischemia. MRI could improve selection criteria for invasive therapy in the future.     

Heart Rate Variability and Blood Pressure during Dynamic and Static Exercise at Similar Heart Rate Levels

Aim was to elucidate autonomic responses to dynamic and static (isometric) exercise of the lower limbs eliciting the same moderate heart rate (HR) response. Method: 23 males performed two kinds of voluntary exercise in a supine position at similar heart rates: static exercise (SE) of the lower limbs (static leg press) and dynamic exercise (DE) of the lower limbs (cycling). Subjective effort, systolic (SBP) and diastolic blood pressure (DBP), mean arterial pressure (MAP), rate pressure product (RPP) and the time between consecutive heart beats (RR-intervals) were measured. Time-domain (SDNN, RMSSD), frequency-domain (power in the low and high frequency band (LFP, HFP)) and geometric measures (SD1, SD2) as well as non-linear measures of regularity (approximate entropy (ApEn), sample entropy (SampEn) and correlation dimension D2) were calculated. Results: Although HR was similar during both exercise conditions (88±10 bpm), subjective effort, SBP, DBP, MAP and RPP were significantly enhanced during SE. HRV indicators representing overall variability (SDNN, SD 2) and vagal modulated variability (RMSSD, HFP, SD 1) were increased. LFP, thought to be modulated by both autonomic branches, tended to be higher during SE. ApEn and SampEn were decreased whereas D₂ was enhanced during SE. It can be concluded that autonomic control processes during SE and DE were qualitatively different despite similar heart rate levels. The differences were reflected by blood pressure and HRV indices. HRV-measures indicated a stronger vagal cardiac activity during SE, while blood pressure response indicated a stronger sympathetic efferent activity to the vessels. The elevated vagal cardiac activity during SE might be a response mechanism, compensating a possible co-activation of sympathetic cardiac efferents, as HR and LF/HF was similar and LFP tended to be higher. However, this conclusion must be drawn cautiously as there is no HRV-marker reflecting “pure” sympathetic cardiac activity.     

Praziquantel Treatment Decreases Schistosoma mansoni Genetic Diversity in Experimental Infections

Background

Schistosomiasis has a considerable impact on public health in many tropical and subtropical areas. In the new world, schistosomiasis is caused by the digenetic trematode Schistosoma mansoni. Chemotherapy is the main measure for controlling schistosomiasis, and the current drug of choice for treatment is praziquantel (PZQ). Although PZQ is efficient and safe, its repetitive large-scale use in endemic areas may lead to the selection of resistant strains. Isolates less susceptible to PZQ have been found in the field and selected for in the laboratory. The impact of selecting strains with a decreased susceptibility phenotype on disease dynamics and parasite population genetics is not fully understood. This study addresses the impact of PZQ pressure on the genetics of a laboratory population by analyzing frequency variations of polymorphic genetic markers.

Methodology

Infected mice were treated with increasing PZQ doses until the highest dose of 3×300 mg/Kg was reached. The effect of PZQ treatment on the parasite population was assessed using five polymorphic microsatellite markers. Parasitological and genetic data were compared with those of the untreated control. After six parasite generations submitted to treatment, it was possible to obtain a S. mansoni population with decreased susceptibility to PZQ. In our experiments we also observed that female worms were more susceptible to PZQ than male worms.

Conclusions

The selective pressure exerted by PZQ led to decreased genetic variability in S. mansoni and increased endogamy. The understanding of how S. mansoni populations respond to successive drug pressure has important implications on the appearance and maintenance of a PZQ resistance phenotype in endemic regions.     

Mate limitation in populations of the endangered Convolvulus lineatus L.: A case for genetic rescue?

In self-incompatible clonal plants, the spread of individual plants can exacerbate mate limitation to the point that it becomes a serious constraint on long-term population persistence, especially in small, isolated populations. In such species, it may be necessary to introduce new genetic material from other populations to restore seed production, a strategy termed “genetic rescue”. In this study we assess the potential pertinence of such genetic rescue in the clonal perennial plant Convolvulus lineatus L., whose populations are often highly reduced in spatial extent and are currently being fragmented by land development projects in Mediterranean France. To do so, we quantify fruit production in a range of populations of different size over four years and perform a series of hand-pollination experiments in natural populations to assess whether fruit set is limited by mate availability. We found that C. lineatus is a self-incompatible species that shows extremely low values of fruit set in natural populations and that a principal cause of this low fruit set is a lack of compatible pollen. This may be primarily due to clonal spread that causes individual populations to be comprised of patches containing one or very few incompatibility types. In small populations fragmented by human activities and which show an absence of fruit production, we thus argue that genetic rescue represents a promising conservation management strategy to avoid inevitable long-term future population decline. We discuss how best to introduce new genetic material into the study populations.     

Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-Reactive Neutralizing Human Monoclonal Antibody

The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.     

Emotion recognition in children and adolescents with attention-deficit/hyperactivity disorder (ADHD)

Children with attention-deficit/hyperactivity disorder (ADHD) are impaired in social adaptation and display deficits in social competence. Deficient emotion recognition has been discussed to underlie these social problems. However, comorbid conduct problems have not been considered in the majority of studies conducted so far, and the influence of medication on emotion recognition has rarely been studied. Here, emotion recognition performance was assessed in children with ADHD without medication compared with children with ADHD under stimulant medication and a matched control group. In order to rule out confounding by externalizing symptoms, children with comorbid conduct problems were excluded. Video clips with neutral faces developing a basic emotion (happiness, sadness, disgust, fear and anger) were presented in order to assess emotion recognition. Results indicated between-group differences neither concerning the number of correctly identified emotions nor concerning reaction times and their standard deviations. Thus, we suggest that ADHD per se is not associated with deficits in emotion recognition.     

Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types.     

Changing Mortality Profile among HIV-Infected Patients in Rio de Janeiro,Brazil: Shifting from AIDS to Non-AIDS Related Conditions in the HAART Era

Introduction

We describe temporal trends in the mortality rates and factors associated with AIDS and non-AIDS related mortality at the Evandro Chagas Clinical Research Institute (IPEC), Oswaldo Cruz Foundation (FIOCRUZ).

Methods

Adult patients enrolling from 1986 through 2009 with a minimum follow up of 60 days were included. Vital status was exhaustively checked using patients’ medical charts, through active contact with individuals and family members and by linkage with the Rio de Janeiro Mortality database using a previously validated algorithm. The CoDe protocol was used to establish the cause of death. Extended Cox proportional hazards models were used for multivariate modeling.

Results

A total of 3530 individuals met the inclusion criteria, out of which 868 (24.6%) deceased; median follow up per patient was 3.9 years (interquartile range 1.7–9.2 years). The dramatic decrease in the overall mortality rates was driven by AIDS-related causes that decreased from 9.19 deaths/100PYs n 1986–1991 to 1.35/100PYs in 2007–2009. Non-AIDS related mortality rates remained stable overtime, at around 1 death/100PYs. Immunodeficiency significantly increased the hazard of both AIDS-related and non-AIDS-related causes of death, while HAART use was strongly associated with a lower hazard of death from either cause.

Conclusions

Our results confirm the remarkable decrease in AIDS-related mortality as the HIV epidemic evolved and alerts to the conditions not traditionally related to HIV/AIDS which are now becoming more frequent, needing careful monitoring.