Identification of a New Stromal Cell Type Involved in the Regulation of Inflamed B Cell Follicles

Lymph node (LN) stromal cells provide survival signals and adhesive substrata to lymphocytes. During an immune response, B cell follicles enlarge, questioning how LN stromal cells manage these cellular demands. Herein, we used a murine fate mapping system to describe a new stromal cell type that resides in the T cell zone of resting LNs. We demonstrated that upon inflammation, B cell follicles progressively trespassed into the adjacent T cell zone and surrounded and converted these stromal cells into CXCL13 secreting cells that in return delineated the new boundaries of the growing follicle. Acute B cell ablation in inflamed LNs abolished CXCL13 secretion in these cells, while LT-β deficiency in B cells drastically affected this conversion. Altogether, we reveal the existence of a dormant stromal cell subset that can be functionally awakened by B cells to delineate the transient boundaries of their expanding territories upon inflammation.     

Association between Age and Severity to Leptospirosis in Children

Background

In endemic areas, leptospirosis is more common and more severe in adults compared with children. Reasons to explain this discrepancy remain unclear and limited data focusing on adolescents are available. The objective of the study was to describe disease spectrum and outcome differences in children and adolescents admitted for leptospirosis in a large at-risk population.

Methods

Clinical and laboratory data were obtained on hospitalized cases in New Caledonia from 2006 to 2012.

Results

Data of 60 patients <18 years of age (25 children under 14 and 35 adolescents aged 14 to 17) with confirmed leptospirosis were analyzed. Compared with children, adolescents presented more often with classic features of Weil disease (p = 0.02), combining hepatic and renal involvement with or without pulmonary participation. Jarisch-Herxheimer reactions were observed more often among adolescents (p<0.01). The overall case fatality rate was low (1 adolescent versus 0 children).

Conclusion

Severe leptospirosis in adolescents may be more likely to show adults'' characteristics compared with children. Further studies are required to explore age-dependant host factors, including puberty-related physiological changes.     

Life expectancy estimates as a key factor in over-treatment: The case of prostate cancer

Objective: To estimate the magnitude of over-diagnosis and of potential and actual over-treatment regarding prostate cancer, taking comorbidities into account. Materials and methods: We used a sample collected by the French cancer registries of 1840 cases (T1: 583; T2: 1257) diagnosed in 2001. The proportion of over-diagnosed and over-treated patients was estimated by comparing life expectancy (LE), including or not comorbidities, with natural LE with cancer, using several assumptions from the literature. We distinguished potential and actual over-treatment according to the treatment that patients actually received. Results: Among patients with T1 tumors the proportion of potential over-treatment using LE adjusted for comorbidity varied from 29.5% to 53.5%, using LE adjusted on comorbidities, and varied from 9.3% to 22.2% regarding actual over-treatment. Between 7.7% and 24.4% of patient's receiving a radical prostatectomy, and between 30.8% and 62.5% of those receiving radiotherapy, were over-treated. Among patients with T2 tumors, the proportions of potential and actual over-treatment were 0.9% and 2.0%. Two per cent of patients receiving a radical prostatectomy and 4.9% of those receiving radiotherapy were over-treated. Comorbidities dramatically increased these proportions to nearly 100% of patients, with more than two comorbidities being potentially over-treated and around 33% actually over-treated. Conclusions: According to the French incidence, 3200–4800 French patients may be over-treated, among whom a large proportion of patients had comorbidities. The real issue is to offer the most appropriate treatment to people with low-grade tumors and comorbidities.     

LCMV Glycosylation Modulates Viral Fitness and Cell Tropism

The glycoprotein (GP) of arenaviruses is glycosylated at 11 conserved N-glycosylation sites. We constructed recombinant lymphocytic choriomeningitis virus (rLCMV) featuring either additions or deletions of these N-glycans to investigate their role in the viral life cycle. N-glycosylation at two sites, T87 and S97, were found to be necessary to rescue rLCMV. Three of nine successfully rescued mutants, S116A, T234A, and S373A, under selective pressures in either epithelial, neuronal, or macrophage cells reverted to WT sequence. Of the seven stable N-glycan deletion mutants, five of these led to altered viral fitness and cell tropism, assessed as growth in either mouse primary cortical neurons or bone marrow derived macrophages. These results demonstrate that the deletion of N-glycans in LCMV GP may confer an advantage to the virus for infection of neurons but a disadvantage in macrophages.     

TLR9 activation induces normal neutrophil responses in a child with IRAK-4 deficiency: involvement of the direct PI3K pathway

Polymorphonuclear neutrophils (PMN) play a key role in innate immunity. Their activation and survival are tightly regulated by microbial products via pattern recognition receptors such as TLRs, which mediate recruitment of the IL-1R-associated kinase (IRAK) complex. We describe a new inherited IRAK-4 deficiency in a child with recurrent pyogenic bacterial infections. Analysis of the IRAK4 gene showed compound heterozygosity with two mutations: a missense mutation in the death domain of the protein (pArg12Cys) associated in cis-with a predicted benign variant (pArg391His); and a splice site mutation in intron 7 that led to the skipping of exon 7. A nontruncated IRAK-4 protein was detected by Western blotting. The patient's functional deficiency of IRAK-4 protein was confirmed by the absence of IRAK-1 phosphorylation after stimulation with all TLR agonists tested. The patient's PMNs showed strongly impaired responses (L-selectin and CD11b expression, oxidative burst, cytokine production, cell survival) to TLR agonists which engage TLR1/2, TLR2/6, TLR4, and TLR7/8; in contrast, the patient's PMN responses to CpG-DNA (TLR9) were normal, except for cytokine production. The surprisingly normal effect of CpG-DNA on PMN functions and apoptosis disappeared after pretreatment with PI3K inhibitors. Together, these results suggest the existence of an IRAK-4-independent TLR9-induced transduction pathway leading to PI3K activation. This alternative pathway may play a key role in PMN control of infections by microorganisms other than pyogenic bacteria in inherited IRAK-4 deficiency.     

Lipidomic analysis of Toxoplasma gondii reveals unusual polar lipids

Analysis of the polar lipids of Toxoplasma gondii by electrospray ionization tandem mass spectrometry provides a detailed picture of the lipid molecular species of this parasitic protozoan. Most notably, T. gondii contains a relatively high level, estimated to about 2% of the total polar lipid, of ceramide phosphoethanolamine. The ceramide phosphoethanolamine has a fatty amide profile with only 16- and 18-carbon species. Compared with the host fibroblasts in which it was grown, T. gondii also has higher levels of phosphatidylcholine but lower levels of sphingomyelin and phosphatidylserine. Analysis at the molecular species level indicated that T. gondii has greater amounts of shorter-chain fatty acid in its polar lipid molecular species than the host fibroblasts. Shorter-chain fatty acids with a combined total of 30 or fewer acyl carbons make up 21% of Toxoplasma's, but only 3% of the host's, diacyl phosphatidylcholine. Furthermore, diacyl phosphatidylcholine with two saturated acyl chains with 12, 14, or 16 carbons make up over 11% of parasite phosphatidylcholine but less than 3% of the host phosphatidylcholine molecular species. The distinctive T. gondii tachyzoite lipid profile may be particularly suited to the function of parasitic membranes and the interaction of the parasite with the host cell and the host's immune system. Combined with T. gondii genomic data, these lipidomic data will assist in elucidation of metabolic pathways for lipid biosynthesis in this important human pathogen.     

High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein—DNA complexes

DNA in living cells is generally processed via the generation and the protection of single-stranded DNA involving the binding of ssDNA-binding proteins (SSBs). The studies of SSB-binding mode transition and cooperativity are therefore critical to many cellular processes like DNA repair and replication. However, only a few atomic force microscopy (AFM) investigations of ssDNA nucleoprotein filaments have been conducted so far. The point is that adsorption of ssDN A–SSB complexes on mica, necessary for AFM imaging, is not an easy task. Here, we addressed this issue by using spermidine as a binding agent. This trivalent cation induces a stronger adsorption on mica than divalent cations, which are commonly used by AFM users but are ineffective in the adsorption of ssDNA–SSB complexes. At low spermidine concentration (<0.3mM), we obtained AFM images of ssDNA–SSB complexes (E. coli SSB, gp32 and yRPA) on mica at both low and high ionic strengths. In addition, partially or fully saturated nucleoprotein filaments were studied at various monovalent salt concentrations thus allowing the observation of SSB-binding mode transition. In association with conventional biochemical techniques, this work should make it possible to study the dynamics of DNA processes involving DNA–SSB complexes as intermediates by AFM.     

Sequence polymorphism-based identification and quantification of Vibrio nigripulchritudo at the species and subspecies level targeting an emerging pathogen for cultured shrimp in New Caledonia

In a previous study, we demonstrated the existence of an emerging cluster of Vibrio nigripulchritudo that proved to be associated with shrimp mortality events in New Caledonia. Using sequence polymorphisms evidenced in this previous MultiLocus Sequence Typing study, we developed two new quantitative PCR assays permitting the detection and quantification of V. nigripulchritudo at the genospecies level using SYBR Green I chemistry and at the emerging cluster level using Fluorescence Resonance Energy Transfer technology with hybridization probes. The use of this molecular diagnostic tool evidenced the colonization of the shrimp pond ecosystem by the pathogenic cluster at least at the onset of the disease. This new tool will allow better investigation of the dynamics of this bacterial pathogen in the shrimp farm ecosystem.     

Less favourable climates constrain demographic strategies in plants

Correlative species distribution models are based on the observed relationship between species’ occurrence and macroclimate or other environmental variables. In climates predicted less favourable populations are expected to decline, and in favourable climates they are expected to persist. However, little comparative empirical support exists for a relationship between predicted climate suitability and population performance. We found that the performance of 93 populations of 34 plant species worldwide – as measured by in situ population growth rate, its temporal variation and extinction risk – was not correlated with climate suitability. However, correlations of demographic processes underpinning population performance with climate suitability indicated both resistance and vulnerability pathways of population responses to climate: in less suitable climates, plants experienced greater retrogression (resistance pathway) and greater variability in some demographic rates (vulnerability pathway). While a range of demographic strategies occur within species’ climatic niches, demographic strategies are more constrained in climates predicted to be less suitable.     

Characterization of the Plasmodium falciparum and P. berghei glycerol 3‐phosphate acyltransferase involved in FASII fatty acid utilization in the malaria parasite apicoplast

Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti‐malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3‐phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N‐terminal targeting sequence to GFP and 3′ tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site‐directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3‐phosphate acyltransferase in malaria parasites.