Lipid and Carbohydrate Modifications of α-Galactosylceramide Differently Influence Mouse and Human Type I Natural Killer T Cell Activation

The ability of different glycosphingolipids (GSLs) to activate type I natural killer T cells (NKT cells) has been known for 2 decades. The possible therapeutic use of these GSLs has been studied in many ways; however, studies are needed in which the efficacy of promising GSLs is compared under identical conditions. Here, we compare five unique GSLs structurally derived from α-galactosylceramide. We employed biophysical and biological assays, as well as x-ray crystallography to study the impact of the chemical modifications of the antigen on type I NKT cell activation. Although all glycolipids are bound by the T cell receptor of type I NKT cells in real time binding assays with high affinity, only a few activate type I NKT cells in in vivo or in vitro experiments. The differences in biological responses are likely a result of different pharmacokinetic properties of each lipid, which carry modifications at different parts of the molecule. Our results indicate a need to perform a variety of assays to ascertain the therapeutic potential of type I NKT cell GSL activators.     

The Paired Basic Amino Acid-cleaving Enzyme 4 (PACE4) Is Involved in the Maturation of Insulin Receptor Isoform B: AN OPPORTUNITY TO REDUCE THE SPECIFIC INSULIN RECEPTOR-DEPENDENT EFFECTS OF INSULIN-LIKE GROWTH FACTOR 2 (IGF2)*

Gaining the full activity of the insulin receptor (IR) requires the proteolytic cleavage of its proform by intra-Golgi furin-like activity. In mammalian cells, IR is expressed as two isoforms (IRB and IRA) that are responsible for insulin action. However, only IRA transmits the growth-promoting and mitogenic effects of insulin-like growth factor 2. Here we demonstrate that the two IR isoforms are similarly cleaved by furin, but when this furin-dependent maturation is inefficient, IR proforms move to the cell surface where the proprotein convertase PACE4 selectively supports IRB maturation. Therefore, in situations of impaired furin activity, the proteolytic maturation of IRB is greater than that of IRA, and accordingly, the amount of phosphorylated IRB is also greater than that of IRA. We highlight the ability of a particular proprotein convertase inhibitor to effectively reduce the maturation of IRA and its associated mitogenic signaling without altering the signals emanating from IRB. In conclusion, the selective PACE4-dependent maturation of IRB occurs when furin activity is reduced; accordingly, the pharmacological inhibition of furin reduces IRA maturation and its mitogenic potential without altering the insulin effects.     

Selective testing strategies for diagnosing group A streptococcal infection in children with pharyngitis: a systematic review and prospective multicentre external validation study

Background:Several clinical prediction rules for diagnosing group A streptococcal infection in children with pharyngitis are available. We aimed to compare the diagnostic accuracy of rules-based selective testing strategies in a prospective cohort of children with pharyngitis.Methods:We identified clinical prediction rules through a systematic search of MEDLINE and Embase (1975–2014), which we then validated in a prospective cohort involving French children who presented with pharyngitis during a 1-year period (2010–2011). We diagnosed infection with group A streptococcus using two throat swabs: one obtained for a rapid antigen detection test (StreptAtest, Dectrapharm) and one obtained for culture (reference standard). We validated rules-based selective testing strategies as follows: low risk of group A streptococcal infection, no further testing or antibiotic therapy needed; intermediate risk of infection, rapid antigen detection for all patients and antibiotic therapy for those with a positive test result; and high risk of infection, empiric antibiotic treatment.Results:We identified 8 clinical prediction rules, 6 of which could be prospectively validated. Sensitivity and specificity of rules-based selective testing strategies ranged from 66% (95% confidence interval [CI] 61–72) to 94% (95% CI 92–97) and from 40% (95% CI 35–45) to 88% (95% CI 85–91), respectively. Use of rapid antigen detection testing following the clinical prediction rule ranged from 24% (95% CI 21–27) to 86% (95% CI 84–89). None of the rules-based selective testing strategies achieved our diagnostic accuracy target (sensitivity and specificity > 85%).Interpretation:Rules-based selective testing strategies did not show sufficient diagnostic accuracy in this study population. The relevance of clinical prediction rules for determining which children with pharyngitis should undergo a rapid antigen detection test remains questionable.Pharyngitis accounts for about 6% of visits by children to primary care physicians each year in high-income nations.¹ Group A streptococcus is found in 30%–40% of cases of childhood pharyngitis; the remaining cases are considered viral.² Antibiotic treatment is indicated for group A streptococcal infection to prevent suppurative (e.g., retropharyngeal abscess and quinsy) and nonsuppurative complications (e.g., acute rheumatic fever and rheumatic heart disease) and to reduce the duration of symptoms and the spread of the condition.³ In settings where poststreptococcal diseases have become uncommon, such as Western Europe and North America,⁴ the public health goal is shifting from preventing complications to minimizing the inappropriate use of antibiotic agents to contain antimicrobial resistance.⁵ However, 60%–70% of the visits by children with pharyngitis to American primary care physicians result in antibiotic agents being prescribed.^6–8Because signs and symptoms of streptococcal and viral pharyngitis overlap, most experts recommend that the diagnosis of group A streptococcal infection be confirmed by a throat culture or rapid antigen detection test.^9–13 Whereas European guidelines suggest all children with pharyngitis undergo such testing,¹⁴ North American guidelines recommend that clinicians select patients on the basis of clinical and epidemiologic grounds.^11–13 Currently, there is no guidance from the Canadian Medical Association or Canadian Paediatric Society for the management of pharyngitis.Various clinical prediction rules that combine signs and symptoms have been proposed to help clinicians define groups of patients according to the clinical likelihood of group A streptococcal infection.^15–18 These rules aim to identify patients at low risk in whom the disease can be managed without further testing and without antibiotic treatment, and patients at high risk who could receive empiric antibiotic treatment without testing.¹⁶ Clinical prediction rules for pharyngitis have not been sufficiently validated for clinical practice and have never been compared head-to-head in a single pediatric population from a high-income country.¹⁸The purpose of our study was to externally validate and directly compare the diagnostic accuracy of relevant rules-based selective testing strategies with original data from a French prospective multicentre cohort of children with pharyngitis. To optimize this validation study, we first conducted a systematic review of existing clinical prediction rules.     

Genetic Evolution of Mycobacterium bovis Causing Tuberculosis in Livestock and Wildlife in France since 1978

To study the dynamics of bovine tuberculosis (bTB) in France, 4,654 M. bovis strains isolated mainly from livestock and wildlife since 1978 were characterized by spoligotyping and MLVA based on MIRU-VNTR. In our study spoligotyping allowed the discrimination of 176 types although 3 spoligotypes are predominant and account for more than half of the total strain population: SB0120 (26%), SB0134 (11%) and SB0121 (6%). In addition, 11% of the isolates, principally from Southern France, showing close spoligotypes and MIRU-VNTR types have been gathered in a family designated as the “F4-family”. MLVA typing allowed extensive discrimination, particularly for strains with predominant spoligotypes, with a total of 498 genotypes, several of which were highly regionalized. The similarity of the strains’ genetic relationships based on spoligotyping and MIRU-VNTR markers supports the co-existence of different clonal populations within the French M. bovis population. A genetic evolution of the strains was observed both geographically and in time. Indeed, as a result of the reduction of bTB due to the national control campaigns, a large reduction of the strains’ genetic variability took place in the last ten years. However, in the regions were bTB is highly prevalent at present, cases in both livestock and in wildlife are due to the spread of unique local genotype profiles. Our results show that the highly discriminating genotyping tools used in this study for molecular studies of bTB are useful for addressing pending questions, which would lead to a better insight into the epidemiology of the disease, and for finding proper solutions for its sustainable control in France.     

Sequential FOLFIRI.3 + Gemcitabine Improves Health-Related Quality of Life Deterioration-Free Survival of Patients with Metastatic Pancreatic Adenocarcinoma: A Randomized Phase II Trial

BackgroundA randomized multicenter phase II trial was conducted to assess the sequential treatment strategy using FOLFIRI.3 and gemcitabine alternately (Arm 2) compared to gemcitabine alone (Arm 1) in patients with metastatic non pre-treated pancreatic adenocarcinoma. The primary endpoint was the progression-free survival (PFS) rate at 6 months. It concludes that the sequential treatment strategy appears to be feasible and effective with a PFS rate of 43.5% in Arm 2 at 6 months (26.1% in Arm 1). This paper reports the results of the longitudinal analysis of the health-related quality of life (HRQoL) as a secondary endpoint of this study.MethodsHRQoL was evaluated using the EORTC QLQ-C30 at baseline and every two months until the end of the study or death. HRQoL deterioration-free survival (QFS) was defined as the time from randomization to a first significant deterioration as compared to the baseline score with no further significant improvement, or death. A propensity score was estimated comparing characteristics of partial and complete responders. Analyses were repeated with inverse probability weighting method using the propensity score. Multivariate Cox regression analyses were performed to identify independent factors influencing QFS.Results98 patients were included between 2007 and 2011. Adjusting on the propensity score, patients of Arm 2 presented a longer QFS of Global Health Status (Hazard Ratio: 0.52 [0.31-0.85]), emotional functioning (0.35 [0.21–0.59]) and pain (0.50 [0.31 – 0.81]) than those of Arm 1.ConclusionPatients of Arm 2 presented a better HRQoL with a longer QFS than those of Arm 1. Moreover, the propensity score method allows to take into account the missing data depending on patients’ characteristics.

Trial registration information

Eudract N° 2006-005703-34. (Name of the Trial: FIRGEM).     

To What Extent is Primate Second Molar Enamel Occlusal Morphology Shaped by the Enamel-Dentine Junction?

The form of two hard tissues of the mammalian tooth, dentine and enamel, is the result of a combination of the phylogenetic inheritance of dental traits and the adaptive selection of these traits during evolution. Recent decades have been significant in unveiling developmental processes controlling tooth morphogenesis, dental variation and the origination of dental novelties. The enamel-dentine junction constitutes a precursor for the morphology of the outer enamel surface through growth of the enamel cap which may go along with the addition of original features. The relative contribution of these two tooth components to morphological variation and their respective response to natural selection is a major issue in paleoanthropology. This study will determine how much enamel morphology relies on the form of the enamel-dentine junction. The outer occlusal enamel surface and the enamel-dentine junction surface of 76 primate second upper molars are represented by polygonal meshes and investigated using tridimensional topometrical analysis. Quantitative criteria (elevation, inclination, orientation, curvature and occlusal patch count) are introduced to show that the enamel-dentine junction significantly constrains the topographical properties of the outer enamel surface. Our results show a significant correlation for elevation, orientation, inclination, curvature and occlusal complexity between the outer enamel surface and the enamel dentine junction for all studied primate taxa with the exception of four modern humans for curvature (p<0.05). Moreover, we show that, for all selected topometrical parameters apart from occlusal patch count, the recorded correlations significantly decrease along with enamel thickening in our sample. While preserving tooth integrity by providing resistance to wear and fractures, the variation of enamel thickness may modify the curvature present at the occlusal enamel surface in relation to enamel-dentine junction, potentially modifying dental functionalities such as blunt versus sharp dental tools. In terms of natural selection, there is a balance between increasing tooth resistance and maintaining efficient dental tools. In this sense the enamel cap acts as a functional buffer for the molar occlusal pattern. In primates, results suggest a primary emergence of dental novelties on the enamel-dentine junction and a secondary transposition of these novelties with no or minor modifications of dental functionalities by the enamel cap. Whereas enamel crenations have been reported by previous studies, our analysis do not support the presence of enamel tubercles without dentine relief nuclei. As is, the enamel cap is, at most, a secondary source of morphological novelty.     

Cold Atmospheric Plasma Induces a Predominantly Necrotic Cell Death via the Microenvironment

Introduction

Cold plasma is a partially ionized gas generated by an electric field at atmospheric pressure that was initially used in medicine for decontamination and sterilization of inert surfaces. There is currently growing interest in using cold plasma for more direct medical applications, mainly due to the possibility of tuning it to obtain selective biological effects in absence of toxicity for surrounding normal tissues,. While the therapeutic potential of cold plasma in chronic wound, blood coagulation, and cancer treatment is beginning to be documented, information on plasma/cell interaction is so far limited and controversial.

Methods and Results

Using normal primary human fibroblast cultures isolated from oral tissue, we sought to decipher the effects on cell behavior of a proprietary cold plasma device generating guided ionization waves carried by helium. In this model, cold plasma treatment induces a predominantly necrotic cell death. Interestingly, death is not triggered by a direct interaction of the cold plasma with cells, but rather via a transient modification in the microenvironment. We show that modification of the microenvironment redox status suppresses treatment toxicity and protects cells from death. Moreover, necrosis is not accidental and seems to be an active response to an environmental cue, as its execution can be inhibited to rescue cells.

Conclusion

These observations will need to be taken into account when studying in vitro plasma/cell interaction and may have implications for the design and future evaluation of the efficacy and safety of this new treatment strategy.     

(R)-NODAGA-PSMA: A Versatile Precursor for Radiometal Labeling and Nuclear Imaging of PSMA-Positive Tumors

Purpose

The present study aims at developing and evaluating an urea-based prostate specific membrane antigen (PSMA) inhibitor suitable for labeling with ¹¹¹In for SPECT and intraoperative applications as well as ⁶⁸Ga and ⁶⁴Cu for PET imaging.

Methods

The PSMA-based inhibitor-lysine-urea-glutamate-coupled to the spacer Phe-Phe-D-Lys(suberoyl) and functionalized with the enantiomerically pure prochelator (R)-1-(1-carboxy-3-carbotertbutoxypropyl)-4,7-carbotartbutoxymethyl)-1,4,7-triazacyclononane ((R)-NODAGA(tBu)₃), to obtain (R)-NODAGA-Phe-Phe-D-Lys(suberoyl)-Lys-urea-Glu (CC34). CC34 was labeled with ¹¹¹In, ⁶⁸Ga and ⁶⁴Cu. The radioconjugates were further evaluated in vitro and in vivo in LNCaP xenografts by biodistribution and PET studies. Biodistribution studies were also performed with ⁶⁸Ga-HBED-CC-PSMA (HBED-CC: N,N′-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid) and ¹¹¹In-PSMA-617 for comparison.

Results

⁶⁸Ga-CC34, ⁶⁴Cu-CC34, and ¹¹¹In-CC34 were prepared in radiochemical purity >95%. ^68/natGa-CC34, ^64/natCu-CC34, ^111/natIn-CC34, ^68/natGa-HBED-CC-PSMA, and ^111/natIn-PSMA-617 exhibited high affinity for the LNCaP cells, with K_d values of 19.3±2.5 nM, 27.5±2.7 nM, 5.5±0.9 nM, 2.9±0.6 nM and 5.4±0.8 nM, respectively. They revealed comparable internalization profiles with approximately 75% of the total cell associated activity internalized after 3 h of incubation. ⁶⁸Ga-CC34 showed very high stability after its administration in mice. Tumor uptake of ⁶⁸Ga-CC34 (14.5±2.9% IA/g) in LNCaP xenografts at 1 h p.i. was comparable to ⁶⁸Ga-HBED-CC-PSMA (15.8±1.4% IA/g) (P = 0.67). The tumor-to-normal tissue ratios at 1 and 2 h p.i of ⁶⁸Ga-CC34 were also comparable to ⁶⁸Ga-HBED-CC-PSMA (P>0.05). Tumor uptake of ¹¹¹In-CC34 (28.5±2.6% IA/g) at 1 h p.i. was lower than ¹¹¹In-PSMA-617 (52.1±6.5% IA/g) (P = 0.02). The acquisition of PET-images with ⁶⁴Cu-CC34 at later time points showed wash-out from the kidneys, while tumor uptake still remained relatively high. This resulted in an increased tumor-to-kidney ratio over time.

Conclusions

⁶⁸Ga-CC34 is comparable to ⁶⁸Ga-HBED-CC-PSMA in terms of tumor uptake and tumor to normal tissue ratios. ⁶⁴Cu-CC34 could enable high contrast imaging of PSMA positive tissues characterized by elevated expression of PSMA or when delayed imaging is required. ⁶⁴Cu-CC34 is currently being prepared for clinical translation.     

Implementation of Motor Imagery during Specific Aerobic Training Session in Young Tennis Players

The aim of this study was to investigate the effects of implementing motor imagery (MI) during specific tennis high intensity intermittent training (HIIT) sessions on groundstroke performance in young elite tennis players. Stroke accuracy and ball velocity of forehand and backhand drives were evaluated in ten young tennis players, immediately before and after having randomly performed two HIIT sessions. One session included MI exercises during the recovery phases, while the other included verbal encouragements for physical efforts and served as control condition. Results revealed that similar cardiac demand was observed during both sessions, while implementing MI maintained groundstroke accuracy. Embedding MI during HIIT enabled the development of physical fitness and the preservation of stroke performance. These findings bring new insight to tennis and conditioning coaches in order to fulfil the benefits of specific playing HIIT sessions, and therefore to optimise the training time.     

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.