Perforation colique néonatale focale spontanée en dehors de la grande prématurité : rare et potentiellement insidieuse [Focal spontaneous colic perforation in term or near-term neonates: rare and potentially insidious].

Two cases of neonatal focal spontaneous colic perforations are reported. The 1st infant, born at 36 3/7 weeks gestational age, presented on day 3 with crying, abdominal distension, and liquid stools. Clinical examination showed a slightly irritable hypothermic (35.7 °C) infant with a distended abdomen and few bowel sounds. Blood tests were normal apart from an elevated C-reactive protein level (59 mg/l). The abdomen x-ray was erroneously considered normal. The infant's condition remained stable for nearly 3 days. After reviewing the initial x-ray, pneumoperitoneum was suspected and confirmed by a cross-table lateral abdominal x-ray. The infant was started on antibiotics and operated. Macroscopically, the entire gut was normal apart from a focal sigmoid perforation, which was stitched. A transmural colic biopsy revealed focal vascular dilation but was negative for necrotising enterocolitis or Hirschsprung disease. The infant recovered quickly. She is now a healthy, normal 3-year-old. The 2nd infant, born at 38 5/7 weeks gestational age, presented between day 1 and 2 with clinical signs of infection associated with slowly progressive ileus. The chest and abdomen x-ray was mistakenly considered normal. Frank septicemia developed. After reviewing the initial x-ray, pneumoperitoneum was suspected and confirmed by a cross-table lateral abdominal x-ray. The infant was operated. Macroscopically, the small intestine was normal, the ascending and transverse colons were dilated, and the descending and sigmoid colons were narrow. Three cecal perforations were discovered and stitched. An ileostomy and multiple colic biopsies were also performed. The postoperative course was complicated by persistent septic ileus due to descending and sigmoid colon leaks, which led to colic resections with end-to-end anastomosis. Rectal aspiration biopsies were also performed. At 1 month of age, the infant was discharged from the hospital. The ileostomy was closed in two steps at 2 and 5 months of age. A normal sweat test excluded cystic fibrosis. All colic and rectal biopsies revealed nonspecific inflammatory signs and excluded necrotizing enterocolitis and Hirschsprung disease. Nonspecific irregular thinning of muscularis mucosae and muscularis propria were observed in the two resected colic segments. The boy is now a healthy 7-year-old. The incidence of neonatal focal spontaneous colic perforations at term or close to term is unknown but probably very rare. Our department is the neonatal referral center for approximately 14,000 annual births. In the last 10 years (2000-2009), out of 5115 neonatal admissions in our unit, only ten cases have presented a neonatal spontaneous intestinal perforation, seven of ten in very-low-birth-weight infants and three of ten in term or near-term neonates (one with Hirschsprung disease and the two cases reported herein). In the same period, 108 infants suffered from necrotizing enterocolitis, seven of 108 were term infants and 6 out of 7 had a congenital heart disease. The medical literature is poor on the subject of focal spontaneous colic perforations at term; no risk factor is described. The most specific clinical sign seems to be the abdominal distension. The presence of pneumoperitoneum on an abdominal x-ray is the most sensitive paraclinical sign. In case of an intestinal perforation, surgery must be performed quickly. The vital prognosis seems to be good. The objective of this study was to draw pediatricians' attention to focal spontaneous colic perforations in term or close to term newborns. In the cases reported, the diagnostic delays could have been prevented if the entity - with its radiological manifestation - had been well known.

Requirement for CARMA1 in antigen receptor-induced NF-kappa B activation and lymphocyte proliferation.

Ligation of antigen receptors (TCR, BCR) on T and B lymphocytes leads to the activation of new transcriptional programs and cell cycle progression. Antigen receptor-mediated activation of NF-kappa B, required for proliferation of B and T cells, is disrupted in T cells lacking PKC theta and in B and T cells lacking Bcl10, a caspase recruitment domain (CARD)-containing adaptor protein. CARMA1 (also called CARD11 and Bimp3), the only lymphocyte-specific member in a family of membrane-associated guanylate kinase (MAGUK) scaffolding proteins that interact with Bcl10 by way of CARD-CARD interactions, is required for TCR-induced NF-kappa B activation in Jurkat T lymphoma cells. Here we show that T cells from mice lacking CARMA1 expression were defective in recruitment of Bcl10 to clustered TCR complexes and lipid rafts, in activation of NF-kappa B, and in induction of IL-2 production. Development of CD5(+) peritoneal B cells was disrupted in these mice, as was B cell proliferation in response to both BCR and CD40 ligation. Serum immunoglobulin levels were also markedly reduced in the mutant mice. Together, these results show that CARMA1 has a central role in antigen receptor signaling that results in activation and proliferation of both B and T lymphocytes.

Surface assembly of poly(I:C) on PEGylated microspheres to shield from adverse interactions with fibroblasts.

By expressing an array of pattern recognition receptors (PRRs), fibroblasts play an important role in stimulating and modulating the response of the innate immune system. The TLR3 ligand polyriboinosinic acid-polyribocytidylic acid, poly(I:C), a mimic of viral dsRNA, is a vaccine adjuvant candidate to activate professional antigen presenting cells (APCs). However, owing to its ligation with extracellular TLR3 on fibroblasts, subcutaneously administered poly(I:C) bears danger towards autoimmunity. It is thus in the interest of its clinical safety to deliver poly(I:C) in such a way that its activation of professional APCs is as efficacious as possible, whereas its interference with non-immune cells such as fibroblasts is controlled or even avoided. Complementary to our previous work with monocyte-derived dendritic cells (MoDCs), here we sought to control the delivery of poly(I:C) surface-assembled on microspheres to human foreskin fibroblasts (HFFs). Negatively charged polystyrene (PS) microspheres were equipped with a poly(ethylene glycol) (PEG) corona through electrostatically driven coatings with a series of polycationic poly(L-lysine)-graft-poly(ethylene glycol) copolymers, PLL-g-PEG, of varying grafting ratios g from 2.2 up to 22.7. Stable surface assembly of poly(I:C) was achieved by incubation of polymer-coated microspheres with aqueous poly(I:C) solutions. Notably, recognition of both surface-assembled and free poly(I:C) by extracellular TLR3 on HFFs halted their phagocytic activity. Ligation of surface-assembled poly(I:C) with extracellular TLR3 on HFFs could be controlled by tuning the grafting ratio g and thus the chain density of the PEG corona. When assembled on PLL-5.7-PEG-coated microspheres, poly(I:C) was blocked from triggering class I MHC molecule expression on HFFs. Secretion of interleukin (IL)-6 by HFFs after exposure to surface-assembled poly(I:C) was distinctly lower as compared to free poly(I:C). Overall, surface assembly of poly(I:C) may have potential to contribute to the clinical safety of this vaccine adjuvant candidate.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination.

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

G Protein-Coupled Receptor heterodimerization in the brain

G protein-coupled receptors (GPCRs) play critical roles in cellular processes and signaling and have been shown to form heteromers with diverge biochemical and/or pharmacological activities that are different from those of the corresponding monomers or homomers. However, despite extensive experimental results supporting the formation of GPCR heteromers in heterologous systems, the existence of such receptor heterocomplexes in the brain remains largely unknown, mostly because of the lack of appropriate methodology. Herein, we describe the in situ proximity ligation assay procedure underlining its high selectivity and sensitivity to image GPCR heteromers with confocal microscopy in brain sections. We describe here how the assay is performed and discuss advantages and disadvantages of this method compared with other available techniques.

Endogenous angiotensin II induces atherosclerotic plaque vulnerability and elicits a Th1 response in ApoE-/- mice.

Rupture of vulnerable plaques is the main cause of acute cardiovascular events. However, mechanisms responsible for transforming a stable into a vulnerable plaque remain elusive. Angiotensin II, a key regulator of blood pressure homeostasis, has a potential role in atherosclerosis. To study the contribution of angiotensin II in plaque vulnerability, we generated hypertensive hypercholesterolemic ApoE-/- mice with either normal or endogenously increased angiotensin II production (renovascular hypertension models). Hypertensive high angiotensin II ApoE-/- mice developed unstable plaques, whereas in hypertensive normal angiotensin II ApoE-/- mice plaques showed a stable phenotype. Vulnerable plaques from high angiotensin II ApoE-/- mice had thinner fibrous cap (P<0.01), larger lipid core (P<0.01), and increased macrophage content (P<0.01) than even more hypertensive but normal angiotensin II ApoE-/- mice. Moreover, in mice with high angiotensin II, a skewed T helper type 1-like phenotype was observed. Splenocytes from high angiotensin II ApoE-/- mice produced significantly higher amounts of interferon (IFN)-gamma than those from ApoE-/- mice with normal angiotensin II; secretion of IL4 and IL10 was not different. In addition, we provide evidence for a direct stimulating effect of angiotensin II on lymphocyte IFN-gamma production. These findings suggest a new mechanism in plaque vulnerability demonstrating that angiotensin II, within the context of hypertension and hypercholesterolemia, independently from its hemodynamic effect behaves as a local modulator promoting the induction of vulnerable plaques probably via a T helper switch.

Transmyocardial laser revascularisation in acutely ischaemic myocardium.

OBJECTIVE: Although recent experience suggests that transmyocardial laser revascularisation (TMLR) relieves angina, its mechanism of action remains undefined. We examined its functional effects and analysed its morphological features in an animal model of acute ischaemia. METHODS: A total of 15 pigs were randomised to ligation of left marginal arteries (infarction group, n = 5), to TMLR of the left lateral wall using a holmium:yttrium-aluminium garnet (Ho:YAG) laser (laser group, n = 5), and to both (laser-infarction group, n = 5). All the animals were sacrificed 1 month after the procedure. Haemodynamics and echocardiography with segmental wall motion score were carried out at both time intervals (scale 0-3: 0, normal; 1, hypokinesia; 2, akinesia; 3, dyskinesia). Histology of the involved area was analysed. RESULTS: Laser group showed no change of the segmental wall motion score of the involved area 30 min after the laser channels were made (score: 0 +/- 0). Infarction and laser infarction groups both showed a persistent and definitive increase of the segmental wall motion score (at 30 min: 1.6 +/- 0.3 and 2 +/- 0, respectively; at 1 month: 1.8 +/- 0.2 and 1.8 +/- 0.4, respectively). These increases were all statistically significant in comparison with baseline values (P < 0.5), however comparison between infarction and laser-infarction groups showed no significant difference. On macroscopic examination of the endocardial surface, no channel was opened. On histology, there were signs of neovascularisation around the channels in the laser group, whereas in the laser-infarction group the channels were embedded in the infarction scar. CONCLUSIONS: In this acute pig model, TMLR did not provide improvement of contractility of the ischaemic myocardium. To the degree that the present study pertains to the clinical setting, the results suggest that mechanisms other than blood flow through the channels should be considered, such as a laser-induced triggering of neovascularisation or neural destruction.

Loss of penicillin tolerance by inactivating the carbon catabolite repression determinant CcpA in Streptococcus gordonii.

OBJECTIVES: Antibiotic tolerance is a phenomenon allowing bacteria to withstand drug-induced killing. Here, we studied a penicillin-tolerant mutant of Streptococcus gordonii (Tol1), which was shown to be deregulated in the expression of the arginine deiminase operon (arc). arc was not directly responsible for tolerance, but is controlled by the global regulator CcpA. Therefore, we sought whether CcpA might be implicated in tolerance. METHODS: The ccpA gene was characterized and subsequently inactivated by PCR ligation mutagenesis in both the susceptible wild-type (WT) and Tol1. The minimal inhibitory concentration and time-kill curves for the strains were determined and the outcome of penicillin treatment in experimental endocarditis assessed. RESULTS: ccpA sequence and expression were similar between the WT and Tol1 strains. In killing assays, the WT lost 3.5 +/- 0.6 and 5.3 +/- 0.6 log(10) cfu/mL and Tol1 lost 0.4 +/- 0.2 and 1.4 +/- 0.9 log(10) cfu/mL after 24 and 48 h of penicillin exposure, respectively. Deletion of ccpA almost totally restored Tol1 kill susceptibility (loss of 2.5 +/- 0.7 and 4.9 +/- 0.7 log(10) cfu/mL at the same endpoints). In experimental endocarditis, penicillin treatment induced a significant reduction in vegetation bacterial densities between Tol1 (4.1 log(10) cfu/g) and Tol1DeltaccpA (2.4 log(10) cfu/g). Restitution of ccpA re-established the tolerant phenotype both in vitro and in vivo. CONCLUSIONS: CcpA, a global regulator of the carbon catabolite repression system, is implicated in penicillin tolerance both in vitro and in vivo. This links antibiotic survival to bacterial sugar metabolism. However, since ccpA sequence and expression were similar between the WT and Tol1 strains, other factors are probably involved in tolerance.

Distinctive role of integrin-mediated adhesion in TNF-induced PKB/Akt and NF-kappaB activation and endothelial cell survival.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine exerting pleiotropic effects on endothelial cells. Depending on the vascular context it can induce endothelial cell activation and survival or death. The microenvironmental cues determining whether endothelial cells will survive or die, however, have remained elusive. Here we report that integrin ligation acts permissive for TNF-induced protein kinase B (PKB/Akt) but not nuclear factor (NF)-kappaB activation. Concomitant activation of PKB/Akt and NF-kappaB is essential for the survival of endothelial cells exposed to TNF. Active PKB/Akt strengthens integrin-dependent endothelial cell adhesion, whereas disruption of actin stress fibers abolishes the protective effect of PKB/Akt. Integrin-mediated adhesion also represses TNF-induced JNK activation, but JNK activity is not required for cell death. The alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 sensitizes endothelial cells to TNF-dependent cytotoxicity and active PKB/Akt attenuates this effect. Interferon gamma synergistically enhanced TNF-induced endothelial cell death in all conditions tested. Taken together, these observations reveal a novel permissive role for integrins in TNF-induced PKB/Akt activation and prevention of TNF-induced death distinct of NF-kappaB, and implicate the actin cytoskeleton in PKB/Akt-mediated cell survival. The sensitizing effect of EMD121974 on TNF cytotoxicity may open new perspectives to the therapeutic use of TNF as anticancer agent.

An overview of L-2-hydroxyglutarate dehydrogenase gene (L2HGDH) variants: a genotype-phenotype study.

L-2-Hydroxyglutaric aciduria (L2HGA) is a rare, neurometabolic disorder with an autosomal recessive mode of inheritance. Affected individuals only have neurological manifestations, including psychomotor retardation, cerebellar ataxia, and more variably macrocephaly, or epilepsy. The diagnosis of L2HGA can be made based on magnetic resonance imaging (MRI), biochemical analysis, and mutational analysis of L2HGDH. About 200 patients with elevated concentrations of 2-hydroxyglutarate (2HG) in the urine were referred for chiral determination of 2HG and L2HGDH mutational analysis. All patients with increased L2HG (n=106; 83 families) were included. Clinical information on 61 patients was obtained via questionnaires. In 82 families the mutations were detected by direct sequence analysis and/or multiplex ligation dependent probe amplification (MLPA), including one case where MLPA was essential to detect the second allele. In another case RT-PCR followed by deep intronic sequencing was needed to detect the mutation. Thirty-five novel mutations as well as 35 reported mutations and 14 nondisease-related variants are reviewed and included in a novel Leiden Open source Variation Database (LOVD) for L2HGDH variants (http://www.LOVD.nl/L2HGDH). Every user can access the database and submit variants/patients. Furthermore, we report on the phenotype, including neurological manifestations and urinary levels of L2HG, and we evaluate the phenotype-genotype relationship.

Psoriasis triggered by toll-like receptor 7 agonist imiquimod in the presence of dermal plasmacytoid dendritic cell precursors.

BACKGROUND: It has been proposed that the innate immune system plays a central role in driving the autoimmune T-cell cascade leading to psoriasis; however, there is no direct evidence for this. OBSERVATIONS: We observed aggravation and spreading of a psoriatic plaque when treated topically with the toll-like receptor (TLR) 7 agonist imiquimod. The exacerbation of psoriasis was accompanied by a massive induction of lesional type I interferon activity, detected by MxA expression after imiquimod therapy. Since imiquimod induces large amounts of type I interferon production from TLR7-expressing plasmacytoid dendritic cell precursors (PDCs), the natural interferon-producing cells of the peripheral blood, we asked whether PDCs are present in psoriatic skin. We identified high numbers of PDCs in psoriatic skin lesions (up to 16% of the total dermal infiltrate) based on their coexpression of BDCA2 and CD123. By contrast, PDCs were present at very low levels in atopic dermatitis and not detected in normal human skin. CONCLUSIONS: This study shows that psoriasis can be driven by the innate immune system through TLR ligation. Furthermore, our finding that large numbers of PDCs infiltrate psoriatic skin suggests a role of lesional PDCs as type I interferon-producing targets for the TLR7 agonist imiquimod.

Ectopic lymphoid-organ development occurs through interleukin 7-mediated enhanced survival of lymphoid-tissue-inducer cells.

Development of Peyer's patches and lymph nodes requires the interaction between CD4+ CD3- IL-7Ralpha+ lymphoid-tissue inducer (LTi) and VCAM-1+ organizer cells. Here we showed that by promoting their survival, enhanced expression of interleukin-7 (IL-7) in transgenic mice resulted in accumulation of LTi cells. With increased IL-7 availability, de novo formation of VCAM-1+ Peyer's patch anlagen occurred along the entire fetal gut resulting in a 5-fold increase in Peyer's patch numbers. IL-7 overexpression also led to formation of multiple organized ectopic lymph nodes and cecal patches. After immunization, ectopic lymph nodes developed normal T cell-dependent B cell responses and germinal centers. Mice overexpressing IL-7 but lacking either RORgamma, a factor required for LTi cell generation, or lymphotoxin alpha1beta2 had neither Peyer's patches nor ectopic lymph nodes. Therefore, by controlling LTi cell numbers, IL-7 can regulate the formation of both normal and ectopic lymphoid organs.

Don't trust a vein graft to treat carotid aneurysm in patients with Behçet disease.

Extracranial carotid aneurysm is a rare vascular manifestation of Behçet disease. To our knowledge, only 32 cases have been reported. This article presents a complex case of a 28-year-old man who was first treated by vein graft reconstruction. At 12 months of follow-up, a nonanastomotic false aneurysm of the vein graft occurred and was treated by interposition of prosthetic graft. Two months later, an anastomotic pseudoaneurysm between the two grafts was excluded by two stent grafts. Based on our experience and a review of the literature, we compared the outcomes of prosthetic and autologous vein reconstructions and discussed the role of carotid ligation and immunosuppressive treatment.

Limited role of the c-Jun N-terminal kinase pathway in a neonatal rat model of cerebral hypoxia-ischemia

D-JNKI1, a cell-permeable peptide inhibitor of the c-Jun N-terminal kinase (JNK) pathway, has been shown to be a powerful neuroprotective agent after focal cerebral ischemia in adult mice and young rats. We have investigated the potential neuroprotective effect of D-JNKI1 and the involvement of the JNK pathway in a neonatal rat model of cerebral hypoxia-ischemia. Seven-day-old rats underwent a permanent ligation of the right common carotid artery followed by 2h of hypoxia (8% oxygen). Treatment with D-JNKI1 (0.3mg/kg intraperitoneally) significantly reduced early calpain activation, late caspase-3 activation and, in the thalamus, autophagosome formation, indicating an involvement of JNK in different types of cell death: necrotic, apoptotic and autophagic. However the size of the lesion was unchanged. Further analysis showed that neonatal hypoxia-ischemia induced an immediate decrease in JNK phosphorylation (reflecting mainly P-JNK1) followed by a slow progressive increase (including P-JNK3 54kDa), whereas c-jun and c-fos expression were both strongly activated immediately after hypoxia-ischemia. In conclusion, unlike in adult ischemic models, JNK is only moderately activated after severe cerebral hypoxia-ischemia in neonatal rats and the observed positive effects of D-JNKI1 are insufficient to give neuroprotection. Thus, for perinatal asphyxia, D-JNKI1 can only be considered in association with other therapies.