Simultaneous use of DGGE and DHPLC to screen TP53 mutations in cancers of the esophagus and cardia from a European high incidence area (Lower Normandy,France)

We investigated TP53 mutation patterns in cancers of the esophagus and cardia of patients coming from Lower Normandy, a region situated in the highest incidence area in Europe. To screen tumor samples, we first used denaturing gradient gel electrophoresis (DGGE), a well-characterized technique which constituted our reference method. Then the results were compared with those obtained by denaturing high performance liquid chromatography (DHPLC), a recent and automatic screening technology. Analysis of the TP53 mutations profile showed that the detected alterations were mainly point mutations. Ninety-seven percent (33/34) of esophageal squamous cell carcinoma samples presented at least one mutation or polymorphism. The proportion of somatic, non-silent and sequence-confirmed mutations was 76% (26/34). The most common substitutions were G-->A transitions, which could be related to nitrosamines, acetaldehyde or factors prone to producing mucosal irritation, like hot beverages. G-->T transversions, which were also frequently detected, could originate from benzo[a]pyrene in tobacco smoke. A-->T transversions were not revealed in our series, which constitutes a discordance with mutational spectra already performed in north-western France. Concerning adenocarcinoma of the esophagus and cardia, the alteration frequency was 69% (11/16), with a majority of G-->A transitions at CpG dinucleotides. They are probably related to endogenous process mediated by inflammatory diseases like gastro-esophageal reflux and Barrett's esophagus. The main advantage provided by DHPLC was its ease of application. However, the optimization steps turned out to be quite critical, especially for sequences with high melting temperatures embedded in lower melting temperature fragments. Considering only the common sequences analyzed by the two techniques, four of the 46 positive samples detected by DGGE were not revealed by DHPLC. This result stresses the limited sensitivity of DHPLC compared with DGGE under the conditions described in this study.     

Fatty acid profiles,antioxidant status,and growth of preterm infants fed diets without or with long-chain polyunsaturated fatty acids

Summary. Long-chain polyunsaturated fatty acids (LCP) are considered conditionally essential nutrients for the infant born prematurely, and attempts are being made to match fatty acid profiles of formula and breast fed infants. In this double-blind, randomized study we investigated the effects of a formula enriched with both n-6 and n-3 LCP on plasma fatty acid profiles, antioxidant status and growth of premature infants. 29 infants received either a formula devoid of LCP or a LCP supplemented formula (0.5 g/100 g fat linoleic acid metabolites, 0.8 g/100 g fat -linolenic acid metabolites). 17 breast fed infants served as a control group. At study entry as well as two and four weeks later, plasma and urine samples were collected, growth data obtained and food tolerance was documented. At the end of the four week study period, plasma docosahexaenoic acid (DHA) levels of supplemented infants were significantly higher than those of unsupplemented infants and similar to those of infants fed human milk. Plasma n-6 LCP concentrations including arachidonic acid (AA) were similar between groups. The plasma -tocopherol levels of breast fed and supplemented infants were similar and tended to be lower than in infants fed the formula devoid of LCP. Urinary malondialdehyde (MDA) excretion of formula fed infants was significantly higher compared to infants fed human milk, but did not differ between the two formula groups. Parameters of growth and milk tolerance did not differ between groups. Our results demonstrate that plasma LCP levels similar to those of breast fed infants can be achieved with the LCP supplemented formula used in this trial, without evidence of adverse effects of the LCP enrichment.AA arachidonic acid - ALA -linolenic acid - DGLA dihomo--linolenic acid - DHA docosahexaenoic acid - EPA eicosapentaenoic acid - F formula devoid of LCP - GLA -linolenic acid - HM human milk - LA linoleic acid - LCP long-chain polyunsaturated fatty acids - LCP-F formula enriched with LCP - MDA malondialdehyde - PC phosphatidylcholine - PCA postconceptional age - PE phosphatidylethanolamine - PUFA polyunsaturated fatty acid     

Persistent cortical activity: mechanisms of generation and effects on neuronal excitability

Local cortical networks in the prefrontal cortex and visual cortex are capable of spontaneously generating sustained activity for periods of seconds or longer. This sustained activity is generated through recurrent excitation between pyramidal cells that is controlled by feedback inhibition and can have both a rapid onset and a rapid offset. The period of activity is associated with a marked increase in neuronal responsiveness to the intracellular injection of current pulses, especially those of smaller amplitude. Independently mimicking the depolarization, increase in membrane conductance and increase in noise associated with sustained activity revealed that the depolarization is largely responsible for the increase in neuronal responsiveness, although an increase in membrane noise also facilitates responses to small inputs. These results indicate that the persistent activity associated with the performance of working memory tasks may be generated largely through recurrent networks. They also suggest that feedback pathways, such as those involved in selective attention, may exert a powerful influence on neuronal responsiveness through synaptic bombardment.     

Invasive explorations in children younger than 3years

PurposeIn children with drug-resistant focal epilepsy who are candidates for surgery, invasive exploration is sometimes required. However, this is being controversially discussed for children younger than 3 years. The question of its necessity, feasibility and its risks is often raised, since it concerns primarily lesional epilepsy and a lesionectomy might be proposed right away. However, this attitude does not take into account the specificities of epilepsy at this age, including poor specificity of electroclinical semiology and the ongoing myelination challenging the interpretation of magnetic resonance imaging (MRI).MethodsWe retrospectively studied the records of children with drug-resistant epilepsy who were younger than 3 years of age at the time of their invasive exploration at our institution from 2000 to 2009. We reviewed the clinical, imaging and electrophysiological data, and included post-operative outcome for those who underwent surgery.Key findings26 Children met the inclusion criteria. All had drug-resistant epilepsy that started at an average of 5.2 months (range 0–20 months) with multiple daily seizures in all and developmental delay in 16. The average age at the time of exploration was 21.8 months (range 5–35). In 20 children, subdural electrodes in combination with two or three depth electrodes were implanted, and in six children aged over 2 years a stereo-electro-encephalography (SEEG) was performed. SEEG was considered technically difficult to achieve before the age of 2 years. The tolerance of invasive exploration was good with a 3% morbidity consisting of one subdural hematoma during exploration by subdural electrodes, evacuated without any particular sequelae. In 25 patients, the exploration permitted to propose a focal resection. The surgical intervention was in the frontal lobe in 12 cases, the parietal lobe in six, the occipital lobe in two patients, and the temporal lobe in one child who underwent an additional resection. Four children had a resection of two or three lobes. Five underwent a second surgery, following a second invasive exploration. Histologically, the resected tissue revealed focal cortical dysplasia in 21 cases (including three patients with tuberous sclerosis), two post-ischemic lesions, one dysembryoplastic neuroepithelial tumor, and one gangliglioma associated with dysplasia. The mean postoperative follow-up period was 51 months (range 4–110). For the children operated on twice, follow-up was counted from the second surgery on. Seventeen children (68%) had an outcome of Engel class 1. In five (20%), seizure frequency was significantly improved (Engel class 3). In two of three patients without improvement in seizure frequency (Engel class 4), a new SEEG is planned and the third is presently a candidate for hemispherotomy.SignificanceInvasive exploration is feasible, well tolerated and carries a low morbidity in children under 3 years of age. At this age, it is indicated for drug-resistant lesional epilepsy associated with developmental delay. It permits delineating the lesion, which is not possible with MRI. The choice of the technique is in part age-dependent. The discussion of its indication arises in the same way as in the older child.     

Intraoperative blood loss during decompressive craniectomy for intractable intracranial hypertension after severe traumatic brain injury in children

Purpose

There are no data available on the risk of intraoperative bleeding during decompressive craniectomy (DC) after traumatic brain injury (TBI) in children. The objectives of this study were to assess the risk of intraoperative bleeding during DC for intractable intracranial hypertension after TBI, to identify potential factors associated with the risk of bleeding during DC, and to assess the impact of DC on systemic and cerebral hemodynamics and on coagulation.

Methods

Twelve children were identified as having undergone DC after TBI from April 2009 to June 2013 in our center. Subjects were allocated into two groups according to the percentage of blood loss (IBL) during the intraoperative period (<or ≥50 % of the estimated blood volume (EBV)).

Results

The median IBL during DC was 49 [17–349] % of the EBV. Children with an IBL?≥?50 % of EBV had higher preoperative intracranial pressure (ICP) (p?=?0.03) and international normalized ratio (INR) (p?=?0.02) than those with an IBL?<?50 % of EBV. DC induced significant decreases in ICP (p?=?0.0005), mean arterial pressure (p?=?0.01), and a significant increase in norepinephrine flow rate (p?=?0.04) between the immediate pre- and postoperative periods.

Conclusions

DC allows a significant decrease in ICP after severe pediatric TBI but is a surgical procedure at a high risk of bleeding. High ICP and INR during the immediate preoperative period are the main factors associated with increased IBL during DC. Further studies are needed to confirm our results and to assess the impact of the amount of IBL on the postoperative survival and functional outcome.     

Dietary intake and plasma concentrations of PUFA and LC-PUFA in breastfed and formula fed infants under real-life conditions

Background  

The breastfed infant is usually used as standard for formula feeding, also regarding long-chain polyunsaturated fatty acids (LC-PUFA). Here, plasma fatty acid concentrations in formula fed infants and the effects of LC-PUFA supplementation were investigated under real-life conditions.     

Frequencies and demographic determinants of breastfeeding and DHA supplementation in a nationwide sample of mothers in Germany

Purpose

German guidelines recommend breast milk as ideal for infant’s nutrition, supporting exclusive breastfeeding for at least 4 months. Moreover, in mothers with insufficient fish intake, DHA status may be improved by supplementation during pregnancy and lactation. However, little is known on current rates of breastfeeding and DHA supplementation in Germany. The objective of this study was to analyse frequencies and demographic determinants of breastfeeding and DHA supplementation in Germany.

Methods

Data derived from a nationwide consumer survey of 986 mothers with children between 5 and 36 months of age in Germany.

Results

78.3 % reported that they ever breastfed their children, and 55.6 % of the mothers exclusively breastfed for at least 4 months. Mothers who did not breastfeed were less likely to be informed by their paediatrician or midwife and were more often not informed at all; 27.8 % of mothers used DHA supplements during pregnancy, 16.8 % postnatal. DHA supplementation was more common in women with a high versus a low fish intake. The social status was the major determinant of breastfeeding initiation and exclusivity and also DHA supplementation.

Conclusion

Breastfeeding initiation and duration of exclusive breastfeeding in Germany need to be improved. Professional counselling and support, with a focus on mothers from lower social classes, appears necessary to increase current rates of breastfeeding initiation, duration, and exclusiveness, but also to ensure a sufficient supply with DHA in pregnant and lactating women, particularly in women with low fish consumption.     

Protein kinase C-theta is required for development of experimental cerebral malaria

Cerebral malaria is the most severe neurologic complication in children and young adults infected with Plasmodium falciparum. T-cell activation is required for development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (CM). To characterize the T-cell activation pathway involved, the role of protein kinase C-theta (PKC-θ) in experimental CM development was examined. PKC-θ-deficient mice are resistant to CM development. In the absence of PKC-θ, no neurologic sign of CM developed after blood stage PbA infection. Resistance of PKC-θ-deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and magnetic resonance angiography, whereas wild-type mice developed distinct microvascular pathology. Recruitment and activation of CD8(+) T cells, and ICAM-1 and CD69 expression were reduced in the brain of resistant mice; however, the pulmonary inflammation and edema associated with PbA infection were still present in the absence of functional PKC-θ. Resistant PKC-θ-deficient mice developed high parasitemia, and died at 3 weeks with severe anemia. Therefore, PKC-θ signaling is crucial for recruitment of CD8(+) T cells and development of brain microvascular pathology resulting in fatal experimental CM, and may represent a novel therapeutic target of CM.     

Resolution of thromboemboli in patients with acute pulmonary embolism: a systematic review

STUDY OBJECTIVES: Much attention has been paid in recent years to optimizing the diagnosis of acute pulmonary embolism (PE). However, little is known about the changes in clot burden that occur at the level of the pulmonary arteries after documented PE. It is often problematic to distinguish between a new or residual defect on lung scintigraphy or helical CT. This may lead to falsely labeling patients with residual PE as having recurrent PE and consequent unnecessary treatment changes. DESIGN: We performed a systematic analysis of studies of imaging tests (radionuclide and CT) evaluating resolution rate of PE with independent assessment of predefined methodologic criteria by two investigators. RESULTS: We identified 29 clinical studies. Of these, 25 studies were excluded and 4 studies were included in our review. Because studies differed largely in patient selection, duration of anticoagulation, and timing of follow-up, the studies were not pooled but briefly described. The percentage of patients with residual pulmonary thrombi was 87% at 8 days after diagnosis, 68% after 6 weeks, 65% after 3 months, 57% after 6 months, and 52% after 11 months. DISCUSSION: This review shows that complete resolution of PE is not routinely achieved between 8 days and 11 months after diagnosis. More than 50% of patients with PE still have defects 6 months after diagnosis, after which resolution of thrombi appears to reach a plateau phase. Physicians should be aware of the high percentage of incomplete resolution of pulmonary emboli. Routine re-imaging after cessation of anticoagulant therapy in patients with PE to obtain a new baseline could be considered.     

Prospective associations between serum biomarkers of lipid metabolism and overall,breast and prostate cancer risk

Experimental studies provided evidence about mechanisms by which cholesterol, especially high density lipoprotein cholesterol (HDL-C), could influence carcinogenesis, notably through antioxidant and anti-inflammatory properties. However, prospective studies that investigated the associations between specific lipid metabolism biomarkers and cancer risk provided inconsistent results. The objective was to investigate the prospective associations between total cholesterol (T-C), HDL-C, low density lipoprotein cholesterol, apolipoproteins A1 (apoA1) and B, and triglycerides and overall, breast and prostate cancer risk. Analyses were performed on 7,557 subjects of the Supplémentation en Vitamines et Minéraux Antioxydants Study, a nationwide French cohort study. Biomarkers of lipid metabolism were measured at baseline and analyzed regarding the risk of first primary incident cancer (N = 514 cases diagnosed during follow-up, 1994–2007), using Cox proportional hazards models. T-C was inversely associated with overall (HR_{1mmol/L increment} = 0.91, 95 % CI 0.82–1.00; P = 0.04) and breast (HR_{1mmol/L increment} = 0.83, 95 % CI 0.69–0.99; P = 0.04) cancer risk. HDL-C was also inversely associated with overall (HR_{1mmol/L increment} = 0.61, 95 % CI 0.46–0.82; P = 0.0008) and breast (HR_{1mmol/L increment} = 0.48, 95 % CI 0.28–0.83; P = 0.009) cancer risk. Consistently, apoA1 was inversely associated with overall (HR_{1g/L increment} = 0.56, 95 % CI 0.39–0.82; P = 0.003) and breast (HR_{1g/L increment} = 0.36, 95 % CI 0.18–0.73; P = 0.004) cancer risk. This prospective study suggests that pre-diagnostic serum levels of T-C, HDL-C and ApoA1 are associated with decreased overall and breast cancer risk. The confirmation of a role of cholesterol components in cancer development, by further large prospective and experimental studies, may have important implications in terms of public health, since cholesterol is already crucial in cardiovascular prevention.