Diagnostic Approach to Acute Liver Failure in Children: A Position Paper by the SIGENP Liver Disease Working Group.

Acute liver failure (ALF) is a clinical condition characterized by the abrupt onset of coagulopathy and biochemical evidence of hepatocellular injury, leading to rapid deterioration of liver cell function. In children, ALF has been characterized by raised transaminases, coagulopathy, and no known evidence of pre-existing chronic liver disease; unlike in adults, the presence of hepatic encephalopathy is not required to establish the diagnosis. Although rare, ALF has a high mortality rate without liver transplantation (LT). Etiology of ALF varies with age and geographical location, although it may remain indeterminate in a significant proportion of cases. However, identifying its etiology is crucial to undertake disease-specific management and evaluate indication to LT. In this position statement, the Liver Disease Working Group of the Italian Society of Gastroenterology, Hepatology and Nutrition (SIGENP) reviewed the most relevant studies on pediatric ALF to provide recommendations on etiology, clinical features and diagnostic work-up of neonates, infants and children presenting with ALF. Recommendations on medical management and transplant candidacy will be discussed in a following consensus conference.

Immunological fingerprint of 4CMenB recombinant antigens via protein microarray reveals key immunosignatures correlating with bactericidal activity.

Serogroup B meningococcus (MenB) is a leading cause of meningitis and sepsis across the world and vaccination is the most effective way to protect against this disease. 4CMenB is a multi-component vaccine against MenB, which is now licensed for use in subjects >2 months of age in several countries. In this study, we describe the development and use of an ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed.

Ultra-High-Field Targeted Imaging of Focal Cortical Dysplasia: The Intracortical Black Line Sign in Type IIb.

BACKGROUND AND PURPOSE: Conventional MR imaging has limitations in detecting focal cortical dysplasia. We assessed the added value of 7T in patients with histologically proved focal cortical dysplasia to highlight correlations between neuropathology and ultra-high-field imaging. MATERIALS AND METHODS: Between 2013 and 2019, we performed a standardized 7T MR imaging protocol in patients with drug-resistant focal epilepsy. We focused on 12 patients in whom postsurgical histopathology revealed focal cortical dysplasia and explored the diagnostic yield of preoperative 7T versus 1.5/3T MR imaging and the correlations of imaging findings with histopathology. We also assessed the relationship between epilepsy surgery outcome and the completeness of surgical removal of the MR imaging-visible structural abnormality. RESULTS: We observed clear abnormalities in 10/12 patients using 7T versus 9/12 revealed by 1.5/3T MR imaging. In patients with focal cortical dysplasia I, 7T MR imaging did not disclose morphologic abnormalities (n = 0/2). In patients with focal cortical dysplasia II, 7T uncovered morphologic signs that were not visible on clinical imaging in 1 patient with focal cortical dysplasia IIa (n = 1/4) and in all those with focal cortical dysplasia IIb (n = 6/6). T2*WI provided the highest added value, disclosing a peculiar intracortical hypointense band (black line) in 5/6 patients with focal cortical dysplasia IIb. The complete removal of the black line was associated with good postsurgical outcome (n = 4/5), while its incomplete removal yielded unsatisfactory results (n = 1/5). CONCLUSIONS: The high sensitivity of 7T T2*-weighted images provides an additional tool in defining potential morphologic markers of high epileptogenicity within the dysplastic tissue of focal cortical dysplasia IIb and will likely help to more precisely plan epilepsy surgery and explain surgical failures.

Human protective response induced by meningococcus B vaccine is mediated by the synergy of multiple bactericidal epitopes.

4CMenB is the first broad coverage vaccine for the prevention of invasive meningococcal disease caused by serogroup B strains. To gain a comprehensive picture of the antibody response induced upon 4CMenB vaccination and to obtain relevant translational information directly from human studies, we have isolated a panel of human monoclonal antibodies from adult vaccinees. Based on the Ig-gene sequence of the variable region, 37 antigen-specific monoclonal antibodies were identified and produced as recombinant Fab fragments, and a subset also produced as full length recombinant IgG1 and functionally characterized. We found that the monoclonal antibodies were cross-reactive against different antigen variants and recognized multiple epitopes on each of the antigens. Interestingly, synergy between antibodies targeting different epitopes enhanced the potency of the bactericidal response. This work represents the first extensive characterization of monoclonal antibodies generated in humans upon 4CMenB immunization and contributes to further unraveling the immunological and functional properties of the vaccine antigens. Moreover, understanding the mechanistic nature of protection induced by vaccination paves the way to more rational vaccine design and implementation.

Effectiveness of 3D T2-Weighted FLAIR FSE Sequences with Fat Suppression for Detection of Brain MR Imaging Signal Changes in Children.

BACKGROUND AND PURPOSE: T2-weighted FLAIR can be combined with 3D-FSE sequences with isotropic voxels, yielding higher signal-to-noise ratio than 2D-FLAIR. Our aim was to explore whether a T2-weighted FLAIR-volume isotropic turbo spin-echo acquisition sequence (FLAIR-VISTA) with fat suppression shows areas of abnormal brain T2 hyperintensities with better conspicuity in children than a single 2D-FLAIR sequence. MATERIALS AND METHODS: One week after a joint training session with 20 3T MR imaging examinations (8 under sedation), 3 radiologists independently evaluated the presence and conspicuity of abnormal areas of T2 hyperintensities of the brain in FLAIR-VISTA with fat suppression (sagittal source and axial and coronal reformatted images) and in axial 2D-FLAIR without fat suppression in a test set of 100 3T MR imaging examinations (34 under sedation) of patients 2-18 years of age performed for several clinical indications. Their agreement was measured with weighted κ statistics. RESULTS: Agreement was "substantial" (mean, 0.61 for 3 observers; range, 0.49-0.69 for observer pairs) for the presence of abnormal T2 hyperintensities and "fair" (mean, 0.29; range, 0.23-0.38) for the comparative evaluation of lesion conspicuity. In 21 of 23 examinations in which the 3 radiologists agreed on the presence of abnormal T2 hyperintensities, FLAIR-VISTA with fat suppression images were judged to show hyperintensities with better conspicuity than 2D-FLAIR. In 2 cases, conspicuity was equal, and in no case was conspicuity better in 2D-FLAIR. CONCLUSIONS: FLAIR-VISTA with fat suppression can replace the 2D-FLAIR sequence in brain MR imaging protocols for children.

Altered natural killer cells subsets distribution in children with hepatitis C following vertical transmission.

BACKGROUND: Natural killer (NK) cells number, phenotypes and function have been evaluated in many studies in adults with hepatitis C as compared with healthy controls or dynamically during interferon-based and interferon-free treatments. Overall, in adults with chronic infection number of circulating NK cells has been reported to be lower when compared to spontaneous resolvers and healthy subjects. Different studies yielded inconsistent findings due to patient and virus heterogeneity. AIM: To evaluate NK cells in children according to the different outcomes of the infection. METHODS: In this cross-sectional study, we examined numbers and phenotypes of circulating NK cells from a homogenous cohort of Italian children with vertically acquired hepatitis C. RESULTS: We compared 31 children who developed chronic infection with nine who presented spontaneous clearance and 13 controls. CD56(+) CD3(-) NK cell numbers were consistently lower in the persistently infected group (P = 0.03 and 0.04). This decrease was due to depletions of CD56(dim) NK cells (P = 0.03 chronic infection vs. spontaneous clearance), while CD56(bright) NK cells were expanded (P = 0.03). No significant difference was found in the frequencies of CD56(+) CD16(+) and CD56(dim) CD16(-) cells. Perforin expression was higher in children with chronic infection (P = 0.03 vs. spontaneous clearance). CONCLUSIONS: Altered NK cells number and phenotypes could impact the outcome of HCV infection in children following vertical transmission. This study suggests for the first time that NK cells cytolytic function, featured by CD56(dim) cells, contributes to the elimination of HCV in children presenting spontaneous clearance.

Validation of a food-frequency questionnaire for the assessment of calcium intake in schoolchildren aged 9-10 years.

Bone mass increases steadily until age 20-30 years, when peak bone mass (PBM) is acquired. Nutrition plays a critical role in achievement of the optimal genetically programmed PBM, with reduction in the risk of osteoporosis later in life. Intake of nutrients can be estimated through the use of various tools; typically, food-frequency questionnaires (FFQs) are used in epidemiologic studies. The aim of this study was to validate a 21-item, semiquantitative FFQ to assess important nutrient intakes for bone health in Italian schoolchildren 9-10 years of age. Relative validation was accomplished through comparison of the 7-days weighed food record (7D records) with an FFQ developed ad hoc, completed by a group of 75 Italian schoolchildren (36 females, 39 males). Agreement between the two methods was evaluated by Spearman's correlation test and Bland-Altman analysis applied on the data on intake of energy, macronutrients, and micronutrients. Particular attention was devoted to nutrients relevant for bone health. Good correlations between the two methods (FFQ and 7D records) were observed for all nutrients. In particular, mean dietary calcium intakes were 725.6 mg/day (95 % CI 683.2-768.1) from 7D records and 892.4 mg/day (95 % CI 844.6-940.2) from the FFQ. These results indicate that our FFQ for schoolchildren aged 9-10 years is highly acceptable as it is an accurate method that can be used in large-scale or epidemiological studies for the evaluation of nutrient intakes important for the prevention of osteoporosis in a similar population.

Validation of the Italian version of the Neurological Disorders Depression Inventory for Epilepsy (NDDI-E).

The Neurological Disorders Depression Inventory for Epilepsy (NDDI-E) was developed for the rapid detection of a major depressive episode in people with epilepsy. It has been proven to be a user-friendly screening instrument. This study describes the development, validation, and psychometric properties of the Italian version of the NDDI-E. A consecutive sample of 120 outpatients with epilepsy has been assessed using the M.I.N.I. Plus version 5.0.0 and the NDDI-E. All patients had no major difficulties in understanding or answering the questions of the Italian version. Cronbach's alpha coefficient was 0.851. Receiver operating characteristic analysis showed an area under the curve of 0.943 (CI95%=0.902-0.985; SE 0.021; p<0.001), a cut off score of 13, a sensitivity of 86.2%, a specificity of 89%, a positive predictive value of 71.4%, and a negative predictive value of 95.3%.

Gene expression profile in Neisseria meningitidis and Neisseria lactamica upon host-cell contact: from basic research to vaccine development.

Differential gene regulation in the human pathogen Neisseria meningitidis group B (MenB) and in Neisseria lactamica, a human commensal species, was studied by whole genome microarray after bacterial interaction with epithelial cells. Host-cell contact induced changes in the expression of 347 and 285 genes in MenB and N. lactamica, respectively. Of these, only 167 were common to MenB and N. lactamica, suggesting that a different subset of genes is activated by pathogens and commensals. Change in gene expression was stable over time in N. lactamica, but short-lived in MenB. A large part (greater than 30%) of the regulated genes encoded proteins with unknown function. Among the known genes, those coding for pili, capsule, protein synthesis, nucleotide synthesis, cell wall metabolism, ATP synthesis, and protein folding were down-regulated in MenB. Transporters for iron, chloride and sulfate, some known virulence factors, GAPDH and the entire pathway of selenocysteine biosynthesis were upregulated. Gene expression profiling indicates that approximately 40% of the regulated genes encode putative surface-associated proteins, suggesting that upon cell contact Neisseria undergoes substantial surface remodeling. This was confirmed by FACS analysis of adhering bacteria using mouse sera against a subset of recombinant proteins. Finally, a few surface-located, adhesion-activated antigens were capable of inducing bactericidal antibodies, indicating that microarray technology can be exploited for the identification of new vaccine candidates.

Oxidative stress signalling in the apoptosis of Jurkat T-lymphocytes.

The pathways of transduction of oxidative stress signals have been studied using the Jurkat T cell model. The oxidative stress was induced by exposure of the cells to 100 microM H(2)O(2). DNA damage was detected within 15 min after commencement of treatment. DNA damage repair occurred within about 1 h in cells exposed to oxidative stress for 15 min. In continuous exposure to stress, DNA repair was slower and control levels of DNA integrity were not reached. DNA repair did not involve gene transcription. H(2)O(2) at 100 microM caused cell death by necrosis as well as by apoptosis. Both these processes were induced by 15 min exposure to the stress stimulus. However, some important differences were found between necrosis and apoptosis. Necrosis was more rapid, began within an hour of treatment and continued to increase during the full duration of the experiment. But apoptosis was seen after 4 h from treatment and was conspicuous between 6 and 20 h after the start of treatment. The necrotic phase preceded apoptosis, although these did show an overlap. In the necrotic phase, Bcl-2, Caspase 8 genes were down regulated. The 6-20 h phase characterised by a marked increase in apoptosis is accompanied by the up regulation of both Bcl-2 and Caspase genes. Expression of the Fas and p53 genes was not altered in either phase. We also analysed the levels of expression of the scavenging genes whose gene products are involved in detoxification. No modulation of the antioxidant enzymes, catalase, Cu/Zn superoxide dismutase and glutathione peroxidase was detectable.

Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

Regulation of the human glut4 gene expression in tumor RD18 cell line.

We tested if glut4, the gene for muscle-specific glucose transporter, underwent some variations of expression in neoplastic cells. Our model was a rhabdomyosarcoma cell line (RD18) which retains the ability to differentiate along the myogenic pathway. Any definable changes of expression of glut4 in normal and RD18 cells were revealed by Northern blot analysis. In order to identify the transcriptional regulatory regions of the glut4 gene we performed a deletion analysis of the 5' flanking region. The downregulation which we found in the expression of this gene in RD18 cells could be related with the activity of a negative regulatory element.

Multi-plasmid DNA vaccination avoids antigenic competition and enhances immunogenicity of a poorly immunogenic plasmid.

DNA immunization is a very promising approach to the formulation of multivalent vaccines. However, little information is currently available on the immunogenicity of multi-plasmid formulations. To address this issue, we immunized mice with a combination of four plasmids encoding malarial antigens and we compared antibody responses with those obtained with single-plasmid injections. We found that when four plasmids encoding Plasmodium falciparum circumsporozoite protein, thrombospondin-related anonymous protein, major merozoite surface protein (MSP)1 and Pfs25 are co-injected into mice, Ab responses against each antigen are elicited at levels at least as high as the level obtained with single-plasmid injection. The quality of antibody production, as determined by isotype analysis, was similar when single- and multi-plasmid administrations were compared, indicating the priming of the same cytokine profile for CD4+ T helper cells. The sera from mice immunized with the four-plasmid formulation specifically recognized sporozoites, blood stage schizonts and gametes, indicating that DNA immunization induced antibody responses relevant to the native conformation. Finally and of particular interest, in the case of MSP1, the antibody response appears to be strongly potentiated by the presence of additional plasmids, indicating an adjuvant effect of DNA.

Computer sequence analysis of human highly conserved zinc finger modules.

We defined a sub-family of zinc finger proteins by computer analyses and comparisons of five new finger domains against protein databases. This subclass of the cysteine-cysteine/histidine-histidine motif shows additional well conserved amino acid patterns and belongs to the human kox and gli-Kruppel gene family, sharing also the same stretches of regulatory zinc finger-containing proteins of mouse and Xenopus. We particularly describe ZF6 cDNA which contains the most interesting sequence, encoding a putative multi-domain regulatory protein.

P1GF-saporin fusion protein: a potential anti-angiogenic agent.

Vascularization is an important step in tumor growth and metastasis. Tumor neovascularization can be considered, therefore, as a good target for antineoplastic therapy. In order to target saporin, a powerful plant toxin, in proximity of the tumor we fused the saporin coding sequence to that for placental growth factor-2 (P1GF-2). P1GF is an angiogenic factor involved in tumor neovascularization. The fusion protein P1GF-2-saporin was obtained by transient transfection of mammalian cells and released in the culture medium as a 57.5 kDa polypeptide. Selectivity and cytotoxic activity are reported as a preliminary step towards the evaluation of its in vivo antitumor activity.

Treatment of cryptorchidism by intramuscular administration of LHRH.

Ten prepubertal boys, aged 6-12 years, presenting with unilateral or bilateral cryptorchidism were treated with a single i.m. injection of 100 micrograms of synthetic LHRH. In addition to the clinical effect, pituitary responsiveness was studied. Immunoreactive FSH and LH in serum were measured by radioimmunoassay. One week after the treatment a complete descent of testes occurred in the four oldest patients. In another four patients, a clear-cut improvement in their condition was seen, and, in two patients, no effect was observed. In nine patients, a significant (p less than 0.001) rise in serum FSH and LH levels was observed. In one patient, serum gonadotropin levels remained unchanged. There was no correlation between the clinical effect and pituitary responsiveness. It was concluded that i.m. administration of LHRH offers advantages over the paranasal route since the dose required for a good effect appears to be considerably lower.