Classification of and risk factors for hematologic complications in a French national cohort of 102 patients with Shwachman-Diamond syndrome.

International audienceBACKGROUND: Patients with the Shwachman-Diamond syndrome often develop hematologic complications. No risk factors for these complications have so far been identified. The aim of this study was to classify the hematologic complications occurring in patients with Shwachman-Diamond syndrome and to investigate the risk factors for these complications. DESIGN AND METHODS: One hundred and two patients with Shwachman-Diamond syndrome, with a median follow-up of 11.6 years, were studied. Major hematologic complications were considered in the case of definitive severe cytopenia (i.e. anemia <7 g/dL or thrombocytopenia <20 × 10(9)/L), classified as malignant (myelodysplasia/leukemia) according to the 2008 World Health Organization classification or as non-malignant. RESULTS: Severe cytopenia was observed in 21 patients and classified as malignant severe cytopenia (n=9), non-malignant severe cytopenia (n=9) and malignant severe cytopenia preceded by non-malignant severe cytopenia (n=3). The 20-year cumulative risk of severe cytopenia was 24.3% (95% confidence interval: 15.3%-38.5%). Young age at first symptoms (<3 months) and low hematologic parameters both at diagnosis of the disease and during the follow-up were associated with severe hematologic complications (P<0.001). Fifteen novel SBDS mutations were identified. Genotype analysis showed no discernible prognostic value. CONCLUSIONS Patients with Shwachman-Diamond syndrome with very early symptoms or cytopenia at diagnosis (even mild anemia or thrombocytopenia) should be considered at a high risk of severe hematologic complications, malignant or non-malignant. Transient severe cytopenia or an indolent cytogenetic clone had no deleterious value

Exhaustion of bacteria-specific CD4 T cells and microbial translocation in common variable immunodeficiency disorders.

In the present study, we have investigated the functional profile of CD4 T cells from patients with common variable immunodeficiency (CVID), including production of cytokines and proliferation in response to bacteria and virus-derived antigens. We show that the functional impairment of CD4 T cells, including the reduced capacity to proliferate and to produce IFN-γ and IL-2, was restricted to bacteria-specific and not virus-specific CD4 T cells. High levels of endotoxins were found in the plasma of patients with CVID, suggesting that CD4 T cell dysfunction might be caused by bacterial translocation. Of note, endotoxemia was associated with significantly higher expression of programmed death 1 (PD-1) on CD4 T cells. The blockade of the PD-1-PD-L1/2 axis in vitro restored CD4 T cell proliferation capacity, thus indicating that PD-1 signaling negatively regulates CD4 T cell functions. Finally, we showed that intravenous immunoglobulin G (IVIG) treatment significantly reduced endotoxemia and the percentage of PD-1(+) CD4 T cells, and restored bacteria-specific CD4 T cell cytokine production and proliferation. In conclusion, the present study demonstrates that the CD4 T cell exhaustion and functional impairment observed in CVID patients is associated with bacterial translocation and that IVIG treatment resolves bacterial translocation and restores CD4 T cell functions

CD160-Associated CD8 T-Cell Functional Impairment Is Independent of PD-1 Expression.

Expression of co-inhibitory molecules is generally associated with T-cell dysfunction in chronic viral infections such as HIV or HCV. However, their relative contribution in the T-cell impairment remains unclear. In the present study, we have evaluated the impact of the expression of co-inhibitory molecules such as 2B4, PD-1 and CD160 on the functions of CD8 T-cells specific to influenza, EBV and CMV. We show that CD8 T-cell populations expressing CD160, but not PD-1, had reduced proliferation capacity and perforin expression, thus indicating that the functional impairment in CD160+ CD8 T cells may be independent of PD-1 expression. The blockade of CD160/CD160-ligand interaction restored CD8 T-cell proliferation capacity, and the extent of restoration directly correlated with the ex vivo proportion of CD160+ CD8 T cells suggesting that CD160 negatively regulates TCR-mediated signaling. Furthermore, CD160 expression was not up-regulated upon T-cell activation or proliferation as compared to PD-1. Taken together, these results provide evidence that CD160-associated CD8 T-cell functional impairment is independent of PD-1 expression

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Détection du (1,3)-b-D-glucane sérique pour le diagnostic des infections fongiques invasives : évaluation rétrospective au CHU de Besançon

International audienc

Human odontoblast-like cells produce nitric oxide with antibacterial activity upon TLR2 activation

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Holder pasteurization impacts the proteolysis, lipolysis and disintegration of human milk under in vitro dynamic term newborn digestion

International audienceWhen the mother’s own human milk is unavailable or limited, pasteurized human milk from milk banks is preferentially administered instead of infant formula, especially for vulnerable hospitalized neonates. Holder pasteurization (62.5 °C, 30 min) may alter human milk composition and structure, which may modulate its digestive behavior. An in vitro dynamic system was set up to simulate the gastrointestinal digestion of term newborns in order to compare the kinetics of lipolysis, proteolysis and structural disintegration of raw versus pasteurized human milk. Human milk from 5 donors was pooled. Half of the pool was either administrated raw (RHM) or pasteurized (PHM). Digestions were conducted at least in duplicate for RHM and PHM. Heat-induced protein aggregation was observed in PHM. During gastric digestion, β-casein was proteolyzed significantly faster for PHM than for RHM (p &lt; 0.05), whereas lactoferrin tended to be proteolyzed slower (p = 0.07) for PHM. Pasteurization selectively affected the intestinal release of some amino acids. At any time of the gastrointestinal digestion, the lipolysis of PHM was significantly lower than that of RHM, but no impact was observed on the profile of released fatty acids. RHM presented a structural destabilization after 60 min of gastric digestion, while there was no large variation for PHM. In the intestinal phase, the evolution of the particle sizes was rather similar. Overall, Holder pasteurization impacted the proteolysis, lipolysis and disintegration of human milk. However, this impact was limited and the physiologic and metabolic consequences remain to be investigated

Impact of pasteurization of human milk on preterm newborn in vitro digestion: Gastrointestinal disintegration, lipolysis and proteolysis

Human milk feeding is an important recommendation for preterm newborns considering their vulnerabilityand digestive immaturity. Holder pasteurization (62.5 C, 30 min) applied in milk banks modifies itsbiological quality and its microstructure. We investigated the impact of pasteurization of preterm humanmilk on its gastrointestinal kinetics of lipolysis, proteolysis and structural disintegration. An in vitrodynamic system was set up to simulate the gastrointestinal digestion of preterm newborns. A pool of pretermhuman milk was digested as raw or after Holder pasteurization. Pasteurization impacted themicrostructure of undigested human milk, its gastrointestinal disintegration and tended to limit theintestinal lipolysis. Furthermore, the gastrointestinal bioaccessibility of some fatty acids was decreasedby pasteurization, while the intestinal bioaccessibility of some amino acids was selectively modulated.The impact of pasteurization on the digestion of human milk may have nutritional relevance in vivoand potentially modulates preterm development and growth

Potentiel immunomodulateur des bactéries du lait maternel humain sur différents modèles cellulaires

International audienceBreastfeeding is recommended by the WHO for the first 6 months of life. Compared to infant formula (IF), human milk (HM) provides healthbenefits, including protection against intestinal and respiratory infections in early childhood and a lower risk of metabolic and immunediseases later in life. Discrepancies in health benefits are probably due to different nutritional and non-nutritional composition between IF andHM, the latter being more complex and richer in bioactive components.Our hypothesis is that HM health benefits come in part from the bacteria of the HM microbiota, which may promote the maturation of the immune system through their role on gut immune homeostasis. This study aimed to better understand the immunomodulatory role of HM bacteria. Bacteria were isolated from healthy breast milk and screened for their immunomodulatory potential on two different cellular models: PBMC (blood mononuclear cell) from 2 healthy donors and a multicellular model of intestinal epithelium enterocytes (Caco2), caliciform cells (HT 29-MTX), M cells (differentiated Caco2) and macrophages (THP-1).L'allaitement maternel est recommandé par l'OMS pour le nourrisson jusqu’à l’âge de 6 mois. Il apporte des bénéfices santé non retrouvés avec les préparations pour nourrissons. Notre hypothèse est que le microbiote du lait maternel humain (LH) participe aux bénéfices santé du LH en favorisant notamment la maturation du système immunitaire.Cette étude visait à mieux comprendre le rôle immunomodulateur des bactéries du LH. Des bactéries ont été isolées à partir de 28 échantillons de LH sains et leur potentiel immunomodulateur a été analysé sur deux modèles cellulaires (cellules immunitaires et un modèle quadricellulaire d'épithélium intestinal).1245 isolats répartis en 26 genres et 56 espèces ont été identifiés. La stimulation de la production de cytokines de 88 isolats, sélectionnés sur la base de leur prévalence et de leur abondance dans le LH, a été évaluée sur un modèle de cellules mononucléées du sang périphérique humain (PBMC). Les isolats ont été répartis en 4 groupes selon la réponse cytokinique des PBMC. Les propriétés immunomodulatrices (réponses IL-10 et TNF-α) de 28 souches représentatives ont ensuite été évaluées sur un modèle quadricellulaire d'épithélium intestinal constitué d’entérocytes (Caco2), de cellules caliciformes (HT 29-MTX), de cellules M, et de macrophages (THP-1). Le potentiel immunomodulateur des bactéries du LH était spécifique de l'espèce et de la souche. Certains isolats avaient un profil anti-inflammatoire, tandis que d’autres combinaient à la fois un profil anti et pro-inflammatoire, ces 2 types de profil pouvant présenter un intérêt pour favoriser la maturation du système immunitaire du nourrisson. Aucune bactérie n'a montré d'effets délétères majeurs (impact sur la barrière, translocation bactérienne forte) sur les 2 modèles cellulaires. Les effets synergiques de ces bactéries assemblées dans des consortia vont être étudiés sur le modèle quadricellulaire avant de caractériser leur impact physiologique in vivo

Potentiel immunomodulateur des bactéries du lait maternel humain sur différents modèles cellulaires

Projet ProlificInternational audienceIntroduction et but de l’étudeL'allaitement maternel est recommandé par l'Organisation Mondiale de la Santé pendant les six premiers mois de vie du nourrisson. Il renforce notamment les défenses immunitaires de l’enfant et diminue les risques de maladies métaboliques et immunitaires à l'âge adulte. De nombreux composés bioactifs spécifiquement apportés par le lait humain (LH) participent à ces bénéfices santé en favorisant le développement du système immunitaire et de la barrière intestinale du nourrisson et l’implantation d’un microbiote bénéfique. Notre hypothèse est que le microbiotenaturel du LH contribue à la mise en place de l’homéostasie intestinale du nourrisson. L’objectif de cette étude est de caractériser in vitro le rôleimmunomodulateur des bactéries du LH en utilisant 2 modèles cellulaires : des cellules immunitaires et un modèle quadricellulaire d'épithélium intestinal.Matériel et MéthodesVingt-huit échantillons de LH ont été collectés chez des femmes en bonne santé, à environ 1 mois de lactation. 1264 isolats bactériens appartenant à 29 genres et 56 espèces différentes ont été identifiés. Un premier modèle de cellules mononucléées du sang périphérique humain (PBMC) a été stimulé par 88 isolats sélectionnés sur la base de leur prévalence et de la représentativité des genres bactériens du microbiote du LH (en absence de toute infection). La réponse en IL-10 et TNF-α a permis de sélectionner 28 candidats d'intérêts dont les propriétés immunomodulatrices, la capacité de translocation et l‘impact sur la barrière ont été évalués avec un modèle quadricellulaire d'épithélium intestinal sur insert comportant un compartiment apical (dans lequel les bactéries sont déposées) constitué d’entérocytes (Caco2), de cellules caliciformes (HT 29-MTX) et de cellules M, et un compartiment basolatéral contenant des macrophages (THP-1 différenciés).Résultats et Analyses statistiquesLes profils cytokiniques obtenus avec les PBMC ont permis de classer les 88 isolats en 4 groupes en fonction de leur capacité à stimuler la sécrétion d'IL-10 (de 0 à 5532 pg/μL, médiane = 352 pg/μL) et/ou de TNF-α (de 12 à 24000 pg/μL, médiane = 3090 pg/μL) : anti-inflammatoire (20 isolats), pro-inflammatoire (10 isolats), immunostimulateur (49 isolats), et faiblement stimulateur (9 isolats). Parmi ceux-ci, 28 isolats avec des profils anti-flammatoires ou immunostimulateurs, susceptibles de stimuler le système immunitaire du nourrisson, ont été sélectionnés. Ces 28isolats montrent une faible capacité de translocation et aucun effet délétère sur la résistance transépithéliale évaluée avec le modèle quadricellulaire. La sécrétion d’IL10 dans le compartiment apical (de 0 à 234 pg/μL, médiane = 3 pg/μL) et basal (de 0 à 49 pg/μL, médiane = 18 pg/μL) et de TNF-α, essentiellement dans le compartiment basal (de 5 à 983 pg/μL ; médiane = 84 pg/μL) a permis de classer les 28 isolats en 3 groupes : anti-inflammatoire (5 isolats correspondants notamment aux genres Bifidobacterium, Cutibacterium, Streptococcus), immunostimulateur (4 isolats appartenant notamment aux genres Lactobacillus, Cutibacterium, Micrococcus), et faiblement stimulateur (19 isolats). L'étude del'expression de différents gènes d'intérêts (fonction barrière, système immunitaire) par les cellules du modèle quadricellulaire est en cours, afin d'affiner les propriétés des bactéries. ConclusionCette étude souligne le potentiel immunomodulateur des bactéries du LH, celui-ci dépend de l'espèce et de la souche. Certains isolats ont un profil anti-inflammatoire, tandis que d’autres combinent à la fois un profil anti et pro-inflammatoire, ces 2 types de profil pouvant présenter un intérêt pour lamaturation du système immunitaire du nourrisson. Les bactéries sélectionnées au long de l'étude n'ont pas montré d'effets délétères sur les modèles. Les travaux se poursuivent sur les effets synergiques de ces bactéries en les associant de manière à obtenir des consortia bactériens présentant des profils distincts : anti-inflammatoire ou immunostimulateur