TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8+ T cell exhaustion.

T cells expressing chimeric antigen receptors (CAR T cells) have shown impressive therapeutic efficacy against leukemias and lymphomas. However, they have not been as effective against solid tumors because they become hyporesponsive ("exhausted" or "dysfunctional") within the tumor microenvironment, with decreased cytokine production and increased expression of several inhibitory surface receptors. Here we define a transcriptional network that mediates CD8+ T cell exhaustion. We show that the high-mobility group (HMG)-box transcription factors TOX and TOX2, as well as members of the NR4A family of nuclear receptors, are targets of the calcium/calcineurin-regulated transcription factor NFAT, even in the absence of its partner AP-1 (FOS-JUN). Using a previously established CAR T cell model, we show that TOX and TOX2 are highly induced in CD8+ CAR+ PD-1high TIM3high ("exhausted") tumor-infiltrating lymphocytes (CAR TILs), and CAR TILs deficient in both TOX and TOX2 (Tox DKO) are more effective than wild-type (WT), TOX-deficient, or TOX2-deficient CAR TILs in suppressing tumor growth and prolonging survival of tumor-bearing mice. Like NR4A-deficient CAR TILs, Tox DKO CAR TILs show increased cytokine expression, decreased expression of inhibitory receptors, and increased accessibility of regions enriched for motifs that bind activation-associated nuclear factor κB (NFκB) and basic region-leucine zipper (bZIP) transcription factors. These data indicate that Tox and Nr4a transcription factors are critical for the transcriptional program of CD8+ T cell exhaustion downstream of NFAT. We provide evidence for positive regulation of NR4A by TOX and of TOX by NR4A, and suggest that disruption of TOX and NR4A expression or activity could be promising strategies for cancer immunotherapy.

Novel exons in the tbx5 gene locus generate protein isoforms with distinct expression domains and function.

TBX5 is the gene mutated in Holt-Oram syndrome, an autosomal dominant disorder with complex heart and limb deformities. Its protein product is a member of the T-box family of transcription factors and an evolutionarily conserved dosage-sensitive regulator of heart and limb development. Understanding TBX5 regulation is therefore of paramount importance. Here we uncover the existence of novel exons and provide evidence that TBX5 activity may be extensively regulated through alternative splicing to produce protein isoforms with differing N- and C-terminal domains. These isoforms are also present in human heart, indicative of an evolutionarily conserved regulatory mechanism. The newly identified isoforms have different transcriptional properties and can antagonize TBX5a target gene activation. Droplet Digital PCR as well as immunohistochemistry with isoform-specific antibodies reveal differential as well as overlapping expression domains. In particular, we find that the predominant isoform in skeletal myoblasts is Tbx5c, and we show that it is dramatically up-regulated in differentiating myotubes and is essential for myotube formation. Mechanistically, TBX5c antagonizes TBX5a activation of pro-proliferative signals such as IGF-1, FGF-10, and BMP4. The results provide new insight into Tbx5 regulation and function that will further our understanding of its role in health and disease. The finding of new exons in the Tbx5 locus may also be relevant to mutational screening especially in the 30% of Holt-Oram syndrome patients with no mutations in the known TBX5a exons.

Reptilian heart development and the molecular basis of cardiac chamber evolution.

The emergence of terrestrial life witnessed the need for more sophisticated circulatory systems. This has evolved in birds, mammals and crocodilians into complete septation of the heart into left and right sides, allowing separate pulmonary and systemic circulatory systems, a key requirement for the evolution of endothermy. However, the evolution of the amniote heart is poorly understood. Reptilian hearts have been the subject of debate in the context of the evolution of cardiac septation: do they possess a single ventricular chamber or two incompletely septated ventricles? Here we examine heart development in the red-eared slider turtle, Trachemys scripta elegans (a chelonian), and the green anole, Anolis carolinensis (a squamate), focusing on gene expression in the developing ventricles. Both reptiles initially form a ventricular chamber that homogenously expresses the T-box transcription factor gene Tbx5. In contrast, in birds and mammals, Tbx5 is restricted to left ventricle precursors. In later stages, Tbx5 expression in the turtle (but not anole) heart is gradually restricted to a distinct left ventricle, forming a left-right gradient. This suggests that Tbx5 expression was refined during evolution to pattern the ventricles. In support of this hypothesis, we show that loss of Tbx5 in the mouse ventricle results in a single chamber lacking distinct identity, indicating a requirement for Tbx5 in septation. Importantly, misexpression of Tbx5 throughout the developing myocardium to mimic the reptilian expression pattern also results in a single mispatterned ventricular chamber lacking septation. Thus ventricular septation is established by a steep and correctly positioned Tbx5 gradient. Our findings provide a molecular mechanism for the evolution of the amniote ventricle, and support the concept that altered expression of developmental regulators is a key mechanism of vertebrate evolution.

An endocardial pathway involving Tbx5, Gata4, and Nos3 required for atrial septum formation.

In humans, septal defects are among the most prevalent congenital heart diseases, but their cellular and molecular origins are not fully understood. We report that transcription factor Tbx5 is present in a subpopulation of endocardial cells and that its deletion therein results in fully penetrant, dose-dependent atrial septal defects in mice. Increased apoptosis of endocardial cells lacking Tbx5, as well as neighboring TBX5-positive myocardial cells of the atrial septum through activation of endocardial NOS (Nos3), is the underlying mechanism of disease. Compound Tbx5 and Nos3 haploinsufficiency in mice worsens the cardiac phenotype. The data identify a pathway for endocardial cell survival and unravel a cell-autonomous role for Tbx5 therein. The finding that Nos3, a gene regulated by many congenital heart disease risk factors including stress and diabetes, interacts genetically with Tbx5 provides a molecular framework to understand gene-environment interaction in the setting of human birth defects.

Pediatric ambulatory pertussis epidemiology in France, recent updates.

OBJECTIVES: Following various changes in the vaccine strategy in 2013 and the mandatory vaccination in 2018, we aimed to analyze the vaccination status, age, and source of contamination of pertussis and parapertussis cases in outpatient surveillance. PATIENTS AND METHODS: Confirmed pertussis and parapertussis cases were enrolled by 35 pediatricians. RESULTS: From 2014 to 2022, 73 confirmed cases of pertussis (n = 65) and parapertussis (n = 8) were reported. For children below 6 years of age, the number of cases with a 2 + 1 schedule (n = 22) was higher than that of those with a 3 + 1 schedule (n = 7). The age of cases with a 3 + 1 or a 2 + 1 schedule was not significantly different (3.8y ± 1.4 vs 4.2y ± 1.5). The main source of contamination was either adults or adolescents. CONCLUSION: Vaccination status and source of contamination are crucial to study the impact of vaccination recommendations.

Acute deletion of TET enzymes results in aneuploidy in mouse embryonic stem cells through decreased expression of Khdc3.

TET (Ten-Eleven Translocation) dioxygenases effect DNA demethylation through successive oxidation of the methyl group of 5-methylcytosine (5mC) in DNA. In humans and in mouse models, TET loss-of-function has been linked to DNA damage, genome instability and oncogenesis. Here we show that acute deletion of all three Tet genes, after brief exposure of triple-floxed, Cre-ERT2-expressing mouse embryonic stem cells (mESC) to 4-hydroxytamoxifen, results in chromosome mis-segregation and aneuploidy; moreover, embryos lacking all three TET proteins showed striking variation in blastomere numbers and nuclear morphology at the 8-cell stage. Transcriptional profiling revealed that mRNA encoding a KH-domain protein, Khdc3 (Filia), was downregulated in triple TET-deficient mESC, concomitantly with increased methylation of CpG dinucleotides in the vicinity of the Khdc3 gene. Restoring KHDC3 levels in triple Tet-deficient mESC prevented aneuploidy. Thus, TET proteins regulate Khdc3 gene expression, and TET deficiency results in mitotic infidelity and genome instability in mESC at least partly through decreased expression of KHDC3.

Roles of TET and TDG in DNA demethylation in proliferating and non-proliferating immune cells.

BACKGROUND: TET enzymes mediate DNA demethylation by oxidizing 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since these oxidized methylcytosines (oxi-mCs) are not recognized by the maintenance methyltransferase DNMT1, DNA demethylation can occur through "passive," replication-dependent dilution when cells divide. A distinct, replication-independent ("active") mechanism of DNA demethylation involves excision of 5fC and 5caC by the DNA repair enzyme thymine DNA glycosylase (TDG), followed by base excision repair. RESULTS: Here by analyzing inducible gene-disrupted mice, we show that DNA demethylation during primary T cell differentiation occurs mainly through passive replication-dependent dilution of all three oxi-mCs, with only a negligible contribution from TDG. In addition, by pyridine borane sequencing (PB-seq), a simple recently developed method that directly maps 5fC/5caC at single-base resolution, we detect the accumulation of 5fC/5caC in TDG-deleted T cells. We also quantify the occurrence of concordant demethylation within and near enhancer regions in the Il4 locus. In an independent system that does not involve cell division, macrophages treated with liposaccharide accumulate 5hmC at enhancers and show altered gene expression without DNA demethylation; loss of TET enzymes disrupts gene expression, but loss of TDG has no effect. We also observe that mice with long-term (1 year) deletion of Tdg are healthy and show normal survival and hematopoiesis. CONCLUSIONS: We have quantified the relative contributions of TET and TDG to cell differentiation and DNA demethylation at representative loci in proliferating T cells. We find that TET enzymes regulate T cell differentiation and DNA demethylation primarily through passive dilution of oxi-mCs. In contrast, while we observe a low level of active, replication-independent DNA demethylation mediated by TDG, this process does not appear to be essential for immune cell activation or differentiation.

[Awareness and adherence to Pertussis vaccination guidelines by occupational medicine physicians in Paris healthcare institution]. / Connaissance et application des recommandations vaccinales concernant la coqueluche par la médecine du travail des établissements de santé de Paris.

OBJECTIVE: A questionnaire was used on 44 public and private hospital physicians in Paris to evaluate their knowledge of and adherence to Vaccination Guidelines, three years after their introduction. RESULTS: Eighty per cent of the physicians answered and 92.5% were aware of the vaccination guidelines but only 2 out of 4 respected the targeted vaccination in young adults even when the vaccine was available. A policy of pertussis vaccination was applied only in 12 institutions, but even in these, the rate of vaccinated healthcare workers remained low or was not documented. CONCLUSION: Pertussis is a potential risk to newborns not or partially vaccinated in France. Even if the vaccine is available, adherence to pertussis vaccination guidelines must be improved. Efforts should be made to better publicize and apply pertussis vaccination guidelines.

Evaluation of an 'all-in-one' seven-day whole-genome sequencing solution in the investigation of a Staphylococcus aureus outbreak in a neonatal intensive care unit.

BACKGROUND: Meticillin-susceptible and -resistant Staphylococcus aureus (MSSA and MRSA) are responsible for outbreaks in intensive care units. MSSA infections have the same morbidity and mortality rate as MRSA infections but are studied less often. Whole-genome sequencing (WGS) is used increasingly for outbreak monitoring, but still requires specific installation and trained personnel to obtain and analyse the data. AIM: To evaluate the workflow and benefits of EpiSeq solution (bioMérieux, Marcy l'Etoile, France) in exploring the increased incidence of S. aureus bloodstream infections in a neonatal intensive care unit (NICU). METHODS: Four S. aureus bacteraemia isolates and 27 colonization isolates obtained between January and July 2016 were submitted to the 'all in one solution' EpiSeq [WGS, quality data assessment, multi-locus sequence typing (MLST), spa typing, virulome and resistome characterization, and phylogenetic tree construction]. More in-depth analyses were performed (whole-genome MLST and whole-genome single nucleotide polymorphism (wgSNP)] with BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium). FINDINGS: Nine different sequence types and 13 different spa types were found among the 31 isolates studied. Among those isolates, 11 (seven patients) were ST146 spa type t002, five (four patients) were ST30 and four (four patients) were ST398. The 11 ST146 isolates had a maximum of seven pairwise SNP differences. CONCLUSION: Use of EpiSeq solution allowed fast demonstration of the polyclonal profile of the MSSA population in neonates, and enabled the suspicion of a global outbreak to be ruled out. However, wgSNP analysis showed the transmission and persistence of one sequence type for over six months in the NICU, and enabled the infection control team to adapt its response.

Antibiothérapie des infections néonatales bactériennes précoces chez les nouveau-nés nés à partir de 34 semaines d'aménorrhée.

All newborns superior to 34 weeks of gestationnal age (GA) are concerned by these guidelines of the French Society of Neonatology and the French Society of Pediatrics. Only newborns at risk of Early-Onset Neonatal Bacterial Infection who are clinically symptomatic have to be treated with probabilistic antibiotherapy treatment. The antibiotic combination of choice is amoxicillin + gentamicin. The two exceptions that justify dual therapy with Cefotaxime and Gentamicin are the bacteriological documentation of Escherichia coli and/or the presence of clinical signs of severity. The continuation or not of the antibiotic treatment relies on the reevaluation of the clinical examination, the bacteriological culture results (blood culture and eventual Cerebro-Spinal-Fluid (CSF) culture) after 36-48 hours of treatment. Antibiotic treatment is continued for 7 days in case of positive blood culture, with amoxicillin alone if then bacterial species implicated are group B Streptococcus (GBS) or Listeria monocytogenes, with Cefotaxime alone in case of E.coli even if the strains are susceptible to aminopenicillins. In case of meningitis due to GBS or L. monocytogenes antibiotherapy is continued for 14 days with amoxicillin alone and 21 days of cefotaxime alone in case of E. coli meningitis. Specialist advice should be sought for other bacteria or extended-spectrum beat-lactamase-producing E. coli. and the absence of bacteriological documentation, according to the clinical evolution and the inflammatory parameters, it can be decided to continue or not the antibiotherapy.

Antimicrobial treatment of ENT infections.

ENT infections are the most common childhood infections and the leading causes of antibiotic prescriptions. These infections are mainly due to viruses and most of them (even if bacterial species are implicated) resolve spontaneously. Therefore, the first message is to not prescribe antibiotics in the following situations: common cold, non-streptococcal pharyngitis, laryngitis, non-purulent otitis media, etc. For sore throat/pharyngitis, the antibiotic treatment decision is based mainly on the use of group A streptococcus rapid diagnostic tests. For otitis media, only purulent forms occurring in children less than 2 years of age and most severe situations in older children should be treated with antibiotics. Amoxicillin is the first-line treatment for the vast majority of ENT infections requiring antibiotic treatment. Severe ENT infections (mastoiditis, epiglottitis, retro- and parapharyngeal abscesses, and ethmoiditis) are therapeutic emergencies requiring in most cases hospitalization and intravenous antibiotics.

[Impact of CRP rapid test in management of febrile children in paediatric emergency units of Ile-de-France]. / Impact de la protéine C-réactive (CRP) en microméthode sur la prise en charge des enfants fébriles aux urgences pédiatriques en Ile-de-France.

OBJECTIVE: Fever is a common cause of children visits to emergency units. Clinical evaluation does not always eliminate a bacterial infection. Among blood markers, several publications showed the interest of CRP. This study was undertaken to evaluate correlation between two techniques of CRP, one by usual technique at the laboratory and the other by a rapid test, and to evaluate the impact of this rapid test for febrile children at the emergency room, when a hospitalization was not immediately decided. MATERIAL AND METHODS: The study was undertaken in 2004-2005 in eight emergency paediatric units in Ile-de-France concerning febrile children during two periods. In period A, children had at the same time a CRP dosage through two methods, whereas in period B, only a rapid CRP test was first managed. The test used was NycoCard CRP Single test (Progen Biotechnique). RESULTS: Between September 2004 and June 2005, 572 children were included, 268 in period A and 304 in period B. Comparison of CRP results by the two methods showed for 247 children (93%) a fairly good linear correlation (r: 0.929). Blood cell count was the most often prescribed test (99.4 vs 10.5%). Conversely to chest radiography, blood culture, fibrinogen and urinary test were significantly most frequent in period A. The average cost of the additional examinations was 2.6 times more important during the first period. Duration of children management in the units was approximately two times shorter when rapid CRP test was used (199.7+/-92.8 vs 103.5+/-98.6 min). CONCLUSION: This study shows the interest of rapid CRP test for febrile children in the emergency units, and has to be confirmed in ambulatory paediatric practice.

[Management of intussusception in France in 2004: investigation of the Paediatric Infectious Diseases Group, the French Group of Paediatric Emergency and Reanimation, and the French Society of Paediatric Surgery]. / Modalités de prise en charge des invaginations intestinales aiguës en France en 2004: enquête commune du Groupe de pathologie infectieuse pédiatrique (GPIP), du Groupe francophone d'urgences et de réanimation pédiatrique (GFRUP) et de la Société française de chirurgie pédiatrique (SFCP).

OBJECTIVES: To describe the different pathways of management of intussusception (IS) in infants and children in metropolitan France and to identify paediatric emergency centres that might constitute a surveillance network for IS. MATERIAL AND METHODS: A questionnaire was sent to 273 paediatric emergency centres distributed across France in 2005. Modalities of diagnosis and treatment of IS had to be precised. RESULTS: One hundred and sixty-seven centres (61.2%) responded. The response was given by 131 paediatricians (78.4%) and 36 surgeons (21.6%) working in 38 universitary hospitals (22.7%) and 129 general hospitals (77.2%). The mean number of IS treated in each centre in 2004 was 11+/-13.5 (extr. 0 to 70; median 6). Diagnosis of IS required a collaboration between medical and surgical teams in 51.5% of the centres, but in 40.1% the sole medical team was in charge of the diagnosis. Ultrasonography is used for diagnosis by 98.8% of the centres. Reduction with hydrostatic enema and eventually surgery was performed in the same hospital in 44.3%. Other centres systematically or frequently transferred the patients for reduction, mostly towards universitary hospitals (90%). CONCLUSION: The procedures of IS diagnosis are the same everywhere in France but the pathways of therapeutic management do vary, depending on the availability of surgeons and anaesthetists trained in paediatrics on each site. These disparities will probably change with the implementation of the new plan for sanitary organization in children and adolescents in France. Labellized paediatric emergency centres will gather more surgical patients and could eventually constitute an effective surveillance network for IS.

[Pneumatic otoscopy in pediatrics]. / L'otoscopie pneumatique au quotidien.

Pneumatic otoscopy is based upon the reaction of the tympanic membrane after sending a small air volume. A normal mobility means that air is present in the middle ear. Instead absent or reduced mobility means that effusion is present. This technique improves the quality of ears clinical examination and is particularly useful for the diagnosis of suppurative and serous otitis media.

[Sciatic paralysis after a buttock intramuscular injection in children: an ongoing risk factor]. / Paralysie sciatique après injection intrafessière chez l'enfant: un risque toujours d'actualité.

Intramuscular injections are regularly recommended for the administration of certain drugs in children. This article underlines the fact that buttock intramuscular injection risks injury to the sciatic nerve, which may lead to lower limb palsy, most often presenting as paralytic drop foot. This condition rarely results from direct traumatic lesion of the sciatic nerve, but usually from the caustic effect of the injected drug. It may occur in older children and adolescents, as well as in infants and younger children. Therefore, the buttocks should not be used as an intramuscular injection site in children whatever their age. In the case of sciatic nerve injury following intramuscular injection, extrafascicular neurolysis may prevent the occurrence of paralysis.

[Group A streptococcal perineal infection in children]. / Infections périnéales à streptocoque du groupe A chez l'enfant.

Perineal diseases in children are usually caused by group A streptococcus (GAS). If the natural course of untreated cases is not known, it is well known that symptoms do not resolve spontaneously and can persist often for many months, until appropriate diagnosis and effective treatment are instituted. Furthermore, failures and recurrences after penicillin treatment are frequent. From 2009 to 2014, 165 perineal infections (median age: 48 months, extremes: 0.4-139) were enrolled by 15 pediatricians: 4 balanitis, 29 vulvo-vaginal diseases and 132 perianal infections. Painful defecation, anal fissures and macroscopic blood in stools were significantly more frequent in GAS perianal infections than negative GAS infections (p<0.01). The performance of GAS-rapid antigen test compared to the GAS culture was : sensitivity 97 % [CI 95 %: 89-100 %], specificity 76 % [CI 95 %: 66-84 %], negative predictive value 97 % [CI 95 %: 91-100 %], positive predictive value 71 % [CI 95 %: 60-80 %].

Computer-assisted segmentation of videocapsule images using alpha-divergence-based active contour in the framework of intestinal pathologies detection.

Visualization of the entire length of the gastrointestinal tract through natural orifices is a challenge for endoscopists. Videoendoscopy is currently the "gold standard" technique for diagnosis of different pathologies of the intestinal tract. Wireless capsule endoscopy (WCE) has been developed in the 1990s as an alternative to videoendoscopy to allow direct examination of the gastrointestinal tract without any need for sedation. Nevertheless, the systematic postexamination by the specialist of the 50,000 (for the small bowel) to 150,000 images (for the colon) of a complete acquisition using WCE remains time-consuming and challenging due to the poor quality of WCE images. In this paper, a semiautomatic segmentation for analysis of WCE images is proposed. Based on active contour segmentation, the proposed method introduces alpha-divergences, a flexible statistical similarity measure that gives a real flexibility to different types of gastrointestinal pathologies. Results of segmentation using the proposed approach are shown on different types of real-case examinations, from (multi)polyp(s) segmentation, to radiation enteritis delineation.

[Is the new vaccination schedule recommended in France adapted to premature babies?]. / Le nouveau calendrier vaccinal est-il adapté à l'ancien prématuré ?

The French 2013 immunization schedule having a goal of simplification with comparable efficacy, has decreased the number of injections and removed the injection performed at three months of age in the general population. Apart from the prevention of invasive pneumococcal infections for which it is recommended to maintain three dose primary immunization, vaccination of premature is not addressed in this new calendar. Can the extremely preterm infants (<33 weeks of gestational age) benefit from this new schedule or should we keep them in three injections schedule? The objective of this paper is to clarify this point through the data available in the literature. Children born prematurely and especially the "extremely premature" born before 33 weeks are at high risk of infections, some of them are preventable by immunization. Although there is no clinical evidence, for pertussis, pneumococcus, Haemophilus influenzae b, hepatitis B, whatever the immunogenicity criteria, immunogenicity is significantly lower in preterm than in term newborn after 3 doses primary schedule. This lower immunogenicity raises concerns about the transition to two doses, about the ability to give short term protection and booster responses. Given these data, GPIP takes the position for maintaining a primary 3-dose vaccination at 2.3 and 4 months for premature infants less than 33 weeks.