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.

Humanization of care in pediatric wards: differences between perceptions of users and staff according to department type.

BACKGROUND: As the quality and quantity of patient-centered care may be perceived differently by recipients and independent observers, assessment of humanization of pediatric care remains an elusive issue. Herein we aim to analyze differences between the degrees of verified existing vs. perceived humanization issues of a pediatric ward. Furthermore, we examine whether there is concurrence between the degrees of humanization perceived by users (parents/visitors) vs. staff members. METHODS: The study was conducted in the pediatric wards of seven medical centers of the Campania region (Italy) categorized as general (n = 4), children's (n = 1), and university (n = 2) hospitals. The degree of existing humanization was assessed by a multidisciplinary focus group for each hospital through a pediatric care-oriented checklist specifically developed to individuate the most critical areas (i.e., those with scores < 2.5). The degree of perceived humanization was assessed through four indicators: well-being, social aspects, safety and security, and health promotion. RESULTS: The focus groups showed that critical areas common to all centers were mainly concerned with welfare, mediation, translation, and interpretation services. Specific critical issues were care and organizational processes oriented to the respect and specificity of the person and care of the relationship with the patient. Perceived humanization questionnaires revealed a lack of recreational facilities and mediation and translation services. As for specific features investigated by both tools, it was found that mediation and interpretation services were lacking in all facilities while patient perceptions and observer ratings for space, comfort, and orientation concurred only in the general hospital evaluations. CONCLUSIONS: Future humanization interventions to ensure child- and family-friendly hospital care call for careful preliminary assessments, tailored to each pediatric ward category, which should consider possible differences between perceived and verified characteristics.

Multiple gut-liver axis abnormalities in children with obesity with and without hepatic involvement.

BACKGROUND: Gut-liver axis (GLA) dysfunction appears to play a role in obesity and obesity-related hepatic complications. OBJECTIVES: This study sought to concurrently explore several GLA components in a paediatric obese population with/without liver disease. METHODS: Thirty-two children (mean age 11.2 years) were enrolled: nine controls with normal weight and 23 patients with obesity (OB+). Of the 23 patients OB(+), 12 had not steatosis (ST-), and 11 had steatosis (ST+) (associated [n = 8] or not [n = 3] with hypertransaminasaemia [ALT +/-]). Subjects were characterized by using auxologic, ultrasonographic and laboratory parameters. A glucose hydrogen breath test was performed to test for small intestinal bacterial overgrowth, a urinary lactulose/mannitol ratio (LMR) was obtained to assess intestinal permeability, and tests for transaminases, blood endogenous ethanol, endotoxin and faecal calprotectin were also conducted. RESULTS: Eleven out of 23 patients OB(+) (p < 0.05) exhibited pathological (>90th percentile of the control group values) LMR, with values paralleling the grade of liver involvement (normal weight < OB[+] < OB[+]ST[+]ALT[-] < OB[+)]ST[+]ALT[+] [p < 0.05]). LMR significantly correlated with ethanolaemia (r = 0.38, p = 0.05) and endotoxaemia (r = 0.48, p = 0.015) concentrations. Increased permeability was a risk factor for the development of steatosis (p < 0.002). SIBO was present only in patients with obesity. Faecal calprotectin concentrations were within normal limits in all subjects. CONCLUSIONS: Increased permeability, endogenous ethanol and systemic endotoxin concentrations reflect some GLA dysfunction in obesity and its hepatic complications. Pending further results to establish their potential causative roles, the modulation of the GLA appears to represent a possible target for the prevention and treatment of these conditions.

Randomised clinical trial: a Lactobacillus GG and micronutrient-containing mixture is effective in reducing nosocomial infections in children, vs. placebo.

BACKGROUND: Nosocomial infections are a major public health issue and preventative strategies using probiotics and micronutrients are being evaluated. AIM: To investigate the efficacy of a mixture of Lactobacillus GG and micronutrients in preventing nosocomial infections in children. METHODS: A randomised, double-blind, placebo-controlled trial was conducted in hospitalised children. Children (6 months to 5 years of age) received Lactobacillus GG (6 × 10(9) CFU/day) together with vitamins B and C and zinc or placebo, for 15 days, starting on the first day of hospitalisation. The incidence of gastrointestinal and respiratory nosocomial infections after discharge was determined by follow-up telephone call at 7 days. After 3 months, another telephone call estimated the incidence of further infections during follow-up. RESULTS: Ninety children completed the follow-up. Of 19/90 children with a nosocomial infection (20%), 4/45 children (9%) were in the treatment group and 15/45 (33%) in the placebo group (P = 0.016). Specifically, 2/45 (4%) children in the treatment group vs. 11/45 (24%) children in the placebo group (P = 0.007) presented with diarrhoea. The duration of hospitalisation was significantly shorter in the treatment group (3.9 days ± 1.7 vs. 4.9 ± 1.2; P = 0.003). At the follow-up, a total of 11/45 (24.4%) children in the treatment group had at least one episode of infection compared to 22/45 (48.9%) in the placebo group (P = 0.016). CONCLUSION: A mixture containing Lactobacillus GG and micronutrients may reduce the incidence of nosocomial infections, supporting the hypothesis that this may represent a valid strategy to prevent nosocomial infections.

Mitochondria-cytoskeleton associations in mammalian cytokinesis.

BACKGROUND: The role of the cytoskeleton in regulating mitochondrial distribution in dividing mammalian cells is poorly understood. We previously demonstrated that mitochondria are transported to the cleavage furrow during cytokinesis in a microtubule-dependent manner. However, the exact subset of spindle microtubules and molecular machinery involved remains unknown. METHODS: We employed quantitative imaging techniques and structured illumination microscopy to analyse the spatial and temporal relationship of mitochondria with microtubules and actin of the contractile ring during cytokinesis in HeLa cells. RESULTS: Superresolution microscopy revealed that mitochondria were associated with astral microtubules of the mitotic spindle in cytokinetic cells. Dominant-negative mutants of KIF5B, the heavy chain of kinesin-1 motor, and of Miro-1 disrupted mitochondrial transport to the furrow. Live imaging revealed that mitochondrial enrichment at the cell equator occurred simultaneously with the appearance of the contractile ring in cytokinesis. Inhibiting RhoA activity and contractile ring assembly with C3 transferase, caused mitochondrial mislocalisation during division. CONCLUSIONS: Taken together, the data suggest a model in which mitochondria are transported by a microtubule-mediated mechanism involving equatorial astral microtubules, Miro-1, and KIF5B to the nascent actomyosin contractile ring in cytokinesis.

14-3-3 protects against stress-induced apoptosis.

Expression of human Bax, a cardinal regulator of mitochondrial membrane permeabilization, causes death in yeast. We screened a human cDNA library for suppressors of Bax-mediated yeast death and identified human 14-3-3ß/α, a protein whose paralogs have numerous chaperone-like functions. Here, we show that, yeast cells expressing human 14-3-3ß/α are able to complement deletion of the endogenous yeast 14-3-3 and confer resistance to a variety of different stresses including cadmium and cycloheximide. The expression of 14-3-3ß/α also conferred resistance to death induced by the target of rapamycin inhibitor rapamycin and by starvation for the amino acid leucine, conditions that induce autophagy. Cell death in response to these autophagic stimuli was also observed in the macroautophagic-deficient atg1Δ and atg7Δ mutants. Furthermore, 14-3-3ß/α retained its ability to protect against the autophagic stimuli in these autophagic-deficient mutants arguing against so called 'autophagic death'. In line, analysis of cell death markers including the accumulation of reactive oxygen species, membrane integrity and cell surface exposure of phosphatidylserine indicated that 14-3-3ß/α serves as a specific inhibitor of apoptosis. Finally, we demonstrate functional conservation of these phenotypes using the yeast homolog of 14-3-3: Bmh1. In sum, cell death in response to multiple stresses can be counteracted by 14-3-3 proteins.

Growth retardation, developmental delay, distinctive face, multiple endocrine abnormalities, and adenylyl cyclase dysfunction: a new syndrome?

We report on a 17-year-old male with severe pre- and postnatal growth retardation, craniosynostosis, distinctive facial features, acanthosis nigricans, deafness, mental retardation and progressive multi-organ involvement, particularly of the endocrine system, including hypothyroidism, hypogonadism, transitory hypoparathyroidism, and insulin resistance. In order to find a common mechanism explaining these multiple abnormalities, we searched for a possible defect in the signal transduction pathways from membrane to nucleus involving G-protein coupled receptors (GPCR). Adenylyl cyclase activity was evaluated by assaying c-AMP in the patient's cultured fibroblasts stimulated with several drugs and toxins acting on different effectors upstream of adenylyl cyclase. The preliminary results indicate a reduced cAMP accumulation in the patient, neither caused by constitutive activation of Gi nor inhibition of Gs signaling, and probably resulting from an alteration in the adenylyl cyclase system. The differential diagnosis with syndromes showing common clinical features with our patient is discussed.

Contraction and polymerization cooperate to assemble and close actomyosin rings around Xenopus oocyte wounds.

Xenopus oocytes assemble an array of F-actin and myosin 2 around plasma membrane wounds. We analyzed this process in living oocytes using confocal time-lapse (four-dimensional) microscopy. Closure of wounds requires assembly and contraction of a classic "contractile ring" composed of F-actin and myosin 2. However, this ring works in concert with a 5-10-microm wide "zone" of localized actin and myosin 2 assembly. The zone forms before the ring and can be uncoupled from the ring by inhibition of cortical flow and contractility. However, contractility and the contractile ring are required for the stability and forward movement of the zone, as revealed by changes in zone dynamics after disruption of contractility and flow, or experimentally induced breakage of the contractile ring. We conclude that wound-induced contractile arrays are provided with their characteristic flexibility, speed, and strength by the combined input of two distinct components: a highly dynamic zone in which myosin 2 and actin preferentially assemble, and a stable contractile actomyosin ring.

Analysis of cortical flow models in vivo.

Cortical flow, the directed movement of cortical F-actin and cortical organelles, is a basic cellular motility process. Microtubules are thought to somehow direct cortical flow, but whether they do so by stimulating or inhibiting contraction of the cortical actin cytoskeleton is the subject of debate. Treatment of Xenopus oocytes with phorbol 12-myristate 13-acetate (PMA) triggers cortical flow toward the animal pole of the oocyte; this flow is suppressed by microtubules. To determine how this suppression occurs and whether it can control the direction of cortical flow, oocytes were subjected to localized manipulation of either the contractile stimulus (PMA) or microtubules. Localized PMA application resulted in redirection of cortical flow toward the site of application, as judged by movement of cortical pigment granules, cortical F-actin, and cortical myosin-2A. Such redirected flow was accelerated by microtubule depolymerization, showing that the suppression of cortical flow by microtubules is independent of the direction of flow. Direct observation of cortical F-actin by time-lapse confocal analysis in combination with photobleaching showed that cortical flow is driven by contraction of the cortical F-actin network and that microtubules suppress this contraction. The oocyte germinal vesicle serves as a microtubule organizing center in Xenopus oocytes; experimental displacement of the germinal vesicle toward the animal pole resulted in localized flow away from the animal pole. The results show that 1) cortical flow is directed toward areas of localized contraction of the cortical F-actin cytoskeleton; 2) microtubules suppress cortical flow by inhibiting contraction of the cortical F-actin cytoskeleton; and 3) localized, microtubule-dependent suppression of actomyosin-based contraction can control the direction of cortical flow. We discuss these findings in light of current models of cortical flow.

Evidence for direct membrane retrieval following cortical granule exocytosis in Xenopus oocytes and eggs.

Rapid exocytosis is typically followed by rapid resorption of exocytosed membrane; however, whether membrane retrieval occurs via indirect endocytosis of numerous small vesicles or direct resealing of the original, larger exocytotic vesicles is controversial. Here we show that cortical granule (CG) exocytosis in Xenopus oocytes and eggs is followed by rapid formation of endosomes as large as the CGs. Large endosomes are translucent, and their formation has the same developmental and pharmacological profile as CG exocytosis. Time course analyses show that large endosomes are not derived from small endosomes. Large endosome formation is triggered by stimuli that do not trigger increases in intracellular-free calcium and is insensitive to perturbation of microtubules by treatment with nocodazole. Perturbation of the f-actin cytoskeleton with latrunculin, however, sharply reduces large endosome formation. We conclude that CG membrane is directly retrieved in Xenopus oocytes and eggs and suggest that this retrieval is not directly dependent on an increase in intracellular-free calcium, but is dependent on the actin cytoskeleton.

Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes.

BACKGROUND: Both single cells and multicellular systems rapidly heal physical insults but are thought to do so by distinctly different mechanisms. Wounds in single cells heal by calcium-dependent membrane fusion, whereas multicellular wounds heal by a variety of different mechanisms, including circumferential contraction of an actomyosin 'purse string' that assembles around wound borders and is dependent upon the small GTPase Rho. RESULTS: We investigated healing of puncture wounds made in Xenopus oocytes, a single-cell system. Oocyte wounds rapidly assumed a circular morphology and constricted circumferentially, coincident with the recruitment of filamentous actin (F-actin) and myosin-II to the wound borders. Surprisingly, recruitment of myosin-II to wound borders occurred before that of F-actin. Further, experimental disruption of F-actin prevented healing but did not prevent myosin-II recruitment. Actomyosin purse-string assembly and closure was dependent on Rho GTPases and extracellular calcium. Wounding resulted in reorganization of microtubules into an array similar to that which forms during cytokinesis in Xenopus embryos. Experimental perturbation of oocyte microtubules before wounding inhibited actomyosin recruitment and wound closure, whereas depolymerization of microtubules after wounding accelerated wound closure. CONCLUSIONS: We conclude the following: actomyosin purse strings can close single-cell wounds; myosin-II is recruited to wound borders independently of F-actin; purse-string assembly is dependent on a Rho GTPase; and purse-string assembly and closure are controlled by microtubules. More generally, the results indicate that actomyosin purse strings have been co-opted through evolution to dispatch a broad variety of single-cell and multicellular processes, including wound healing, cytokinesis and morphogenesis.

Direct observation of microtubule-f-actin interaction in cell free lysates.

Coordinated interplay of the microtubule and actin cytoskeletons has long been known to be crucial for many cellular processes including cell migration and cytokinesis. However, interactions between these two systems have been difficult to document by conventional approaches, for a variety of technical reasons. Here the distribution of f-actin and microtubules were analyzed in the absence of fixation using Xenopus egg extracts as an in vitro source of microtubules and f-actin, demembranated Xenopus sperm to nucleate microtubule asters, fluorescent phalloidin as a probe for f-actin, and fluorescent tubulin as a probe for microtubules. F-actin consistently colocalized in a lengthwise manner with microtubules of asters subjected to extensive washing in flow chambers. F-actin-microtubule association was heterogenous within a given aster, such that f-actin is most abundant toward the distal (plus) ends of microtubules, and microtubules heavily labeled with f-actin are found in close proximity to microtubules devoid of f-actin. However, this distribution changed over time, in that 5 minute asters had more f-actin in their interiors than did 15 minute asters. Microtubule association with f-actin was correlated with microtubule bending and kinking, while elimination of f-actin resulted in straighter microtubules, indicating that the in vitro interaction between f-actin and microtubules is functionally significant. F-actin was also found to associate in a lengthwise fashion with microtubules in asters centrifuged through 30% sucrose, and microtubules alone (i.e. microtubules not seeded from demembranated sperm) centrifuged through sucrose, indicating that the association cannot be explained by flow-induced trapping and alignment of f-actin by aster microtubules. Further, cosedimentation analysis revealed that microtubule-f-actin association could be reconstituted from microtubules assembled from purified brain tubulin and f-actin assembled from purified muscle actin in the presence, but not the absence, of Xenopus oocyte microtubule binding proteins. The results provide direct evidence for an association between microtubules and f-actin in vitro, indicate that this interaction is mediated by one or more microtubule binding proteins, and suggest that this interaction may be responsible for the mutual regulation of the microtubule and actomyosin cytoskeletons observed in vivo.

The C677T mutation of the 5,10-methylenetetrahydrofolate reductase gene is a moderate risk factor for spina bifida in Italy.

OBJECTIVE: To estimate the risk for spina bifida associated with the common mutation C677T of the MTHFR gene in a country with a relatively low prevalence of NTDs. DESIGN: Case-control study. CASES: 203 living patients affected with spina bifida (173 myelomeningocele and 30 lipomeningocele); controls: 583 subjects (306 young adults and 277 unselected newborns) from northern and central-southern Italy. CASES: three spina bifida centres; young adult controls: DNA banks; newborn controls: regional neonatal screening centres. MAIN OUTCOME MEASURES: Prevalence of the C677T genotypes in cases and controls by place of birth; odds ratios for spina bifida and estimated attributable fraction. RESULTS: The prevalence of T/T, T/C, and C/C genotype was 16.6%, 53.7%, and 29.7% in controls and 25.6%, 43.8%, and 30.6% in cases, respectively. We found no differences between type of defect or place of birth. The odds ratio for spina bifida associated with the T/T genotype v C/C plus T/C was 1.73 (95% CI 1.15, 2.59) and the corresponding attributable fraction was 10.8%. No increased risk was found for heterozygous patients (OR=0.79, 95% CI 0.53-1.18). CONCLUSION: This study, as well as the meta-analysis we updated, shows that homozygosity for the MTHFR C677T mutation is a moderate risk factor in Europe, and even in Italy where there is a relatively low prevalence of spina bifida. The estimated attributable fraction associated with this risk factor explains only a small proportion of cases preventable by periconceptional folic acid supplementation. Thus, other genes involved in folate-homocysteine metabolism, their interaction, and the interaction between genetic and environmental factors should be investigated further.

The Effects of Eicosanoid Biosynthesis Inhibitors On Prophenoloxidase Activation, Phagocytosis and Cell Spreading in Galleria mellonella.

The invertebrate immune system produces melanotic nodules in response to bacterial infections and this has previously been shown to be mediated by eicosanoids. Nodulation occurs in two phases: the first involves hemocyte degranulation and activation of the prophenoloxidase cascade; the second involves formation of a cellular capsule by attachment and spreading of hemocytes. We demonstrate that inhibitors of eicosanoid biosynthesis affect both of these phases of nodulation in Galleria mellonella. The phospholipase A(2) inhibitor, dexamethasone, as well as the cyclooxygenase inhibitor, indomethacin, significantly inhibit phagocytosis in vitro and prophenoloxidase activation in vivo. The inhibitory effects of dexamethasone were abolished by the addition of exogenous arachidonic acid. Furthermore, 5,8,11,14- eicosatetraynoic acid, dexamethasone and indomethacin inhibit hemocyte spreading in vitro. The findings support the idea that eicosanoid derivatives mediate both phases of the nodulation response and are consistent with previous studies which attribute roles for eicosanoids in other species as modulators of cell activity.

Imaging electrophoretic gels with a scanning beam laser macroscope.

A scanning beam laser macroscope has been developed which scans an area of 7.5 x 7.5 cm in 5 s. This new imaging system is examined as a potential tool for scanning electrophoretic gels. A specially-designed telecentric, f* theta laser scan lens is used in the instrument to achieve a linear scan and a flat focal plane. The laser scan lens focuses the incoming beam from a laser to a 10 microns spot inside the gel. A raster scan is performed across the gel and the signal is detected with a photomultiplier, forming a 512 x 512 digital image stored as a computer file. Silver-stained protein polyacrylamide gels have been imaged in transmission and double-transmission, while DNA agarose gels (stained with ethidium bromide) have been imaged in fluorescence with better than 25 pg sensitivity. The macroscope has the advantage that it is not tied to the electrophoresis system as are end-of-line scanners, and the scan is rapid, so that several gels can be scanned in a very short time.