Randomised clinical trial: The beneficial effects of VSL#3 in obese children with non-alcoholic steatohepatitis.

BACKGROUND: Gut microbiota modifiers may have beneficial effects of non-alcoholic fatty liver disease (NAFLD) but randomised controlled trials (RCT) are lacking in children. AIM: To perform a double-blind RCT of VSL#3 vs. placebo in obese children with biopsy-proven NAFLD. METHODS: Of 48 randomised children, 44 (22 VSL#3 and 22 placebo) completed the study. The main outcome was the change in fatty liver severity at 4 months as detected by ultrasonography. Secondary outcomes were the changes in triglycerides, insulin resistance as detected by the homoeostasis model assessment (HOMA), alanine transaminase (ALT), body mass index (BMI), glucagon-like peptide 1 (GLP-1) and activated GLP-1 (aGLP-1). Ordinal and linear models with cluster confidence intervals were used to evaluate the efficacy of VSL#3 vs. placebo at 4 months. RESULTS: At baseline, moderate and severe NAFLD were present in 64% and 36% of PLA children and in 55% and 45% of VSL#3 children. The probability that children supplemented with VSL#3 had none, light, moderate or severe FL at the end of the study was 21%, 70%, 9% and 0% respectively with corresponding values of 0%, 7%, 76% and 17% for the placebo group (P < 0.001). No between-group differences were detected in triglycerides, HOMA and ALT while BMI decreased and GLP-1 and aGLP1 increased in the VSL#3 group (P < 0.001 for all comparisons). CONCLUSIONS: A 4-month supplement of VSL#3 significantly improves NAFLD in children. The VSL#3-dependent GLP-1 increase could be responsible for these beneficial effects. Trial identifier: NCT01650025 (www.clinicaltrial.gov).

Amphiphysin 1 binds the cyclin-dependent kinase (cdk) 5 regulatory subunit p35 and is phosphorylated by cdk5 and cdc2.

Amphiphysin 1 is a phosphoprotein expressed at high levels in neurons, where it participates in synaptic vesicle endocytosis and neurite outgrowth. It is a substrate for cyclin-dependent kinase (cdk) 5, a member of the cyclin-dependent protein kinase family, which has been functionally linked to neuronal migration and neurite outgrowth via its action on the actin cytoskeleton. The yeast homologue of amphiphysin, Rvs167, functions in endocytosis and actin dynamics, is phosphorylated by the cdk5 homologue Pho85, and binds the Pho85 regulatory subunit Pcl2. We show here that amphiphysin 1 interacts with the cdk5-activating subunit p35 and that this interaction is mediated by the conserved NH2-terminal region of amphiphysin. Amphiphysin 1 colocalizes with p35 in the growth cones of neurons and at actin-rich peripheral lamellipodia in transfected fibroblasts. Amphiphysin is phosphorylated by cdk5 in a region including serines 272, 276, and 285. Amphiphysin 1 is also phosphorylated by the cdc2/cyclin B kinase complex in the same region and undergoes mitotic phosphorylation in dividing cells. These data indicate that phosphorylation by members of the cyclin-dependent kinase family is a conserved property of amphiphysin and suggest that this phosphorylation may play an important physiological role both in mitosis and in differentiated cells.

Overhead electricity power lines and childhood leukemia: a registry-based, case-control study.

AIMS AND BACKGROUND: To evaluate the role of exposure to low-frequency electromagnetic fields generated by overhead power lines on the risk of childhood leukemia, we carried out a case-control study in the area (Varese province) covered by the Lombardy Cancer Registry. METHODS AND STUDY DESIGN: Exposure to magnetic fields was estimated using line load data and the distance between subjects' homes and the nearest power line. A total of 101 cases and 412 controls were investigated. RESULTS: Twenty subjects (9 cases and 11 controls) were considered exposed. A significant fourfold increase in risk for leukemia in exposed subjects and a dose-response relationship were found. The risk was higher than that reported by other studies. Potential biases related to the representativity of controls and validity of exposure assessment do not seem to have influenced the risk estimates. CONCLUSIONS: We suggest that measures to remedy residential exposure should be taken wherever practicable.

KIF3C and KIF3A form a novel neuronal heteromeric kinesin that associates with membrane vesicles.

We have cloned from rat brain the cDNA encoding an 89,828-Da kinesin-related polypeptide KIF3C that is enriched in brain, retina, and lung. Immunocytochemistry of hippocampal neurons in culture shows that KIF3C is localized to cell bodies, dendrites, and, in lesser amounts, to axons. In subcellular fractionation experiments, KIF3C cofractionates with a distinct population of membrane vesicles. Native KIF3C binds to microtubules in a kinesin-like, nucleotide-dependent manner. KIF3C is most similar to mouse KIF3B and KIF3A, two closely related kinesins that are normally present as a heteromer. In sucrose density gradients, KIF3C sediments at two distinct densities, suggesting that it may be part of two different multimolecular complexes. Immunoprecipitation experiments show that KIF3C is in part associated with KIF3A, but not with KIF3B. Unlike KIF3B, a significant portion of KIF3C is not associated with KIF3A. Consistent with these biochemical properties, the distribution of KIF3C in the CNS has both similarities and differences compared with KIF3A and KIF3B. These results suggest that KIF3C is a vesicle-associated motor that functions both independently and in association with KIF3A.

The role of cyclin-dependent kinase 5 and a novel regulatory subunit in regulating muscle differentiation and patterning.

Cyclin-dependent kinase 5, coupled with its activator p35, is required for normal neuronal differentiation and patterning. We have isolated a novel member of the p35 family, Xp35.1, from Xenopus embryos which can activate cdk5. Xp35.1 is expressed in both proliferating and differentiated neural and mesodermal cells and is particularly high in developing somites where cdk5 is also expressed. Using dominant-negative cdk5 (cdk5 DN), we show that cdk5 kinase activity is required for normal somitic muscle development; expression of cdk5 DN results in disruption of somitic muscle patterning, accompanied by stunting of the embryos. Using explants of animal pole tissue from blastula embryos, which will differentiate into mesoderm in response to activin, we show that blocking cdk5 kinase activity down-regulates the expression of the muscle marker muscle actin in response to activin, whereas the pan-mesodermal marker Xbra is unaffected. Expression of MyoD and MRF4 (master regulators of myogenesis) is suppressed in the presence of cdk5 DN, indicating that these myogenic genes may be a target for cdk5 regulation, whereas the related factor Myf5 is largely unaffected. In addition, overexpression of Xp35.1 disrupts muscle organization. Thus, we have demonstrated a novel role for cdk5 in regulating myogenesis in the early embryo.

Screening for coeliac disease: the meaning of low titers of anti-gliadin antibodies (AGA) in non-coeliac children.

Coeliac disease is diagnosed by means of jejunal biopsy, an invasive procedure. Anti-gliadin antibodies (AGA) have therefore been used in the first screening of the disease. On the other hand, low titers of AGA are widely detected also in normal subjects. In order to investigate if low levels of AGA could be correlated with laboratory and clinical data, we performed a study on 167 subjects with various illnesses, such as recurrent abdominal pain, failure to thrive, short stature, diarrhoea or constipation, cow-milk protein intolerance and/or food allergy, recurrent vomiting or previous gastroenteritis, all non coeliac conditions which have been associated with AGA presence. Seventy coeliac children, all biopsied, were selected as a control group. Among the 167 cases we found 60 subjects positive for AGA (35.9%), a high proportion as compared with the general population. Only 33/167 patients, all IgG and IgA AGA positive, fulfil our laboratory and clinical criteria to perform a 'confirming' biopsy. For the 134 residual cases (14 IgA, 13 only IgG AGA positive, 107 AGA negative) a diagnosis of coeliac disease has been excluded by clinical criteria (scoring). As a whole, the patients with coeliac disease had significantly higher levels of AGA of both IgG and IgA classes (p < 0.01). On the other hand, no significant difference emerged for all the anamnestic and laboratory parameters considered between AGA+ and AGA- non-coeliac subjects. However, laboratory parameters of IgG-AGA and/or IgA-AGA positive patients were similar to those of coeliac children for ion, Xylose, total IgA count. As no biopsied case showed mucosal atrophy, it is suggested that the presence of even low AGA levels in non-coeliac children may represent a highly sensitive index of intestinal alteration causing an increased permeability to macromolecules, but it is very unlikely that one could detect coeliac children by means of Ig-AGA among such illnesses and normal subjects. Strong clinical diagnosis and laboratory parameters are required to justify intestinal biopsies. In fact, the production of AGA seems to be a merely immunological phenomenon linked to an increased and probably transient permeability to macromolecules of the intestinal mucosa.

Cloning and characterization of PSF, a novel pre-mRNA splicing factor.

Previously, we characterized cDNAs encoding polypyrimidine tract-binding protein (PTB) and showed that a complex between PTB and a 100-kD protein was necessary for pre-mRNA splicing. In this paper we have used two different in vitro-binding assays to confirm and extend the interaction between these two proteins. Peptide sequence information was used to clone and sequence cDNAs encoding alternatively spliced forms of the 100-kD protein. It contains two consensus RNA-binding domains and an unusual amino terminus rich in proline and glutamine residues. The protein is highly basic and migrates anomalously on SDS gels. Owing to its interaction with PTB and its role in pre-mRNA splicing, we have termed the 100-kD protein PTB-associated splicing factor (PSF). The RNA-binding properties of PSF are apparently identical to those of PTB. Both proteins, together and independently, bind the polypyrimidine tract of mammalian introns. Biochemical complementation, antibody inhibition, and immunodepletion experiments demonstrate that PSF is an essential pre-mRNA splicing factor required early in spliceosome formation. Bacterially synthesized PSF is able to complement immunodepleted extracts and restore splicing activity. Despite association with PSF, complementary experiments with antibodies against PTB do not suggest an essential role for PTB in pre-mRNA splicing.

Early wheezing and breast feeding.

There is uncertainty as to whether breast feeding protects against subsequent illnesses; it has been suggested that breast feeding may have some protective effects on the severity of long-term outcome of bronchiolitis and in reducing morbidity. We have assessed the effects of breast feeding in 266 patients and 199 controls, all patients were early wheezers (i.e., under 2 years old). Between these groups we found differences in socioeconomic, environmental, and atopic conditions, but there were no significant differences in the numbers who had been breastfed. However, within the group who had had early wheezing we found that infants who had been breastfed for at least one month subsequently had less severe wheezing. These results suggest that breast feeding may be a protective factor for early wheezing only during the first month of life, and a delaying factor in the following months.

[Epidemiologic correlation between microhematuria in children and hypertension in their parents]. / Correlazione epidemiologica fra microematuria nei bambini ed ipertensione nei genitori.

The significance of asymptomatic microhematuria in children is unknown. In 1976 Dodge et al. found a "surprising" high prevalence of proteinuria and haematuria in 12,000 healthy schoolchildren and, in the absence of knowledge of the natural history, this finding prompted them to postpone urinary mass screening. Nonetheless, the progressive course of most chronic renal disease in adulthood argues for investigation of microhematuria in children to uncover any sub-groups "at risk" of kidney diseases. The sensitivity of screening for microhematuria could be increased by a questionnaire on family medical history? We have investigated 1554 boys and 1484 girls aged 3-12 years, from the school population of a rural district near Rome. A self-administered questionnaire on renal disease and related symptoms in families was distributed to the parents. Urinalyses were done on all the children except for those with diseases or symptoms related to the urinary apparatus and girls who were menstruating. Haematuria was tested for by dipstick ("Combur 7"; Boehringer), children with microhematuria were retested 10 day and 1 year later. On the initial 3038 dipstick tests 175 (5.76%) were positive, and 52 children (1.71%) had haematuria in all three specimens. The questionnaires were used only if they had been filled in properly (1821/3038). Of the 1821 valid questionnaires 121 (6.64%) revealed a family history of hypertension, but the frequency of such a family history was significantly higher for the 128 children with haematuria (14.8%, p < 0.005) and the 52 with persistent haematuria (23.0%; p < 0.001).

Atopy and environmental factors in upper respiratory infections: an epidemiological survey on 2304 school children.

Upper respiratory infections (URI) during the first years of life are mostly viral in origin. However, a number of observations suggest the influence of both predisposing and triggering factors. Atopy in particular seems to play an important role as do environmental factors. Many children with early symptoms such as blocked or runny nose are likely to become skin-positive later in life to antigens such as, e.g., D. pteronissinus. A standardized questionnaire was administered to 2304 schoolchildren in order to ascertain the URI frequency and to correlate it with family and environmental factors and with results of prick tests for main allergens in our climate (D. pteronissinus and Grasses). Results showed a wide overlapping of URI and lower respiratory illnesses (in particular, asthma), which are widely distributed in the families of patients. Passive smoking and the quality of housing are the main triggering environmental factors. In our sample, skin positivity for D. pteronissinus and Grasses largely exceeds the symptomatic portion of the whole population. It is therefore suggested that many asymptomatic children are "at risk" for allergic respiratory illness. The highest incidence of winter rhinitis in skin-negative subjects (71.7%) and the skin positivity for D. pteronissinus in patients with perennial symptoms, suggest the importance of both atopy and viral infections in the occurrence of URI. Nasal troubles are most frequent in asthmatic subjects and may be considered the actual additional symptom in asthma.

Bronchiolitis in the history of the asthmatic child.

In a retrospective analysis of the history of 1,700 asthmatic children, 167 (9.8%) were found to have had bronchiolitis during the first 2 years of life. These 167 cases with asthma and bronchiolitis were matched against 215 selected cases of asthma without bronchiolitis. Both groups attended our Ambulatory Care Unit for Asthma. Data were collected from the clinical cards of the children. Asthmatics who had bronchiolitis were found to have an earlier onset of asthma (p less than 0.001), earlier resolution of symptoms (p less than 0.05 total; p less than 0.01 females), and less evidence of atopy (p less than 0.005). It is suggested therefore that the bronchial hyperreactivity in asthma following bronchiolitis may have different underlying mechanisms than in the typical atopic case, without preceding bronchiolitis. Long-term follow-up studies in infants with bronchiolitis are still necessary in order to clarify the process by which bronchiolitis predisposes to bronchial hyperreactivity and asthma. The consequences of the viral infection itself may lead to pathophysiological processes that promote bronchial hyperreactivity. On the contrary, an underlying condition of bronchial hyperreactivity could be the "primum movens" of the bronchiolitis itself.

Scanning from an independently specified branch point defines the 3' splice site of mammalian introns.

During pre-messenger RNA splicing the lariat branch point in mammalian introns is specified independently of the 3' splice site by the sequence surrounding the branch point and by an adjacent downstream polypyrimidine tract. The 3' splice site is dispensable for spliceosome assembly and cleavage at the 5' splice site, and is itself determined by a scanning process that recognizes the first AG located 3' of the branch point/polypyrimidine tract, irrespective of distance or sequence environment.

Evaluating bronchial hyperreactivity tests in epidemiology.

The aim of this study is to assess the value of the most common tests which can be used in epidemiological surveys to identify asthmatic children. We reviewed anamnestic information from a self-administered questionnaire, bronchial reactivity by means of exercise-test, bronchodilation-test with trimethochinol, carbachol-test (PD20 FEV1 was reported), cutaneous reactivity monitored by prick-test. (Data from a polycentric epidemiological study). We conclude that, while these tests are very useful in the initial study and in the further follow-up of the asthmatic child, providing also information about the possibilities of management of bronchoconstriction and its pharmacologic prevention, they show a limited value in the absolute diagnosis of asthma.

First trimester studies of a fetus at risk for triose phosphate isomerase deficiency.

A first trimester prenatal diagnosis was offered to a mother whose child had died of haemolytic anaemia and multisystem disease caused by TPI deficiency. The deficiency state was characterized by greatly reduced TPI activity in both erythrocytes and peripheral lymphocytes. Specific activity of TPI in trophoblast homogenates from the index fetus was about 30 per cent less than in the controls, but the heat stability test showed overlap. These data were confirmed in uncultured and cultured amniotic cells, where glycolytic intermediate concentrations DHAP, GAP and FDP fell in the range of controls. These results suggested that the fetus was a TPI heterozygote. This prenatal prediction was confirmed by RBC and haematological studies at birth.

The fetal pathology of the XXXXY-syndrome.

A second case of fetal XXXXY-syndrome detected by prenatal chromosome analysis is presented. The pathological findings include a facial aspect featuring fetal Down's syndrome, hypogenitalism and hypogonadism with excessive reduction of germ cells and also skeletal abnormalities that may be interpreted as early changes, preceding phalangeal shortening V and radioulnar synostosis.

Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.

We cloned the beta-tubulin gene of Neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. The gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. The coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. Intron position comparisons between the N. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. The mutation responsible for benomyl resistance was determined; it caused a phenylalanine-to-tyrosine change at position 167. Codon usage in the beta-tubulin gene is biased, as has been observed for other abundantly expressed N. crassa genes such as am and the H3 and H4 histone genes. This bias results in pyrimidines in the third positions of 96% of the codons in codon families in which there is a choice between purines and pyrimidines in this position. Bias is also evident by the absence of 19 of the 61 sense codons. We demonstrated that benomyl resistance is due to the cloned beta-tubulin gene of strain Bml511(r)a and that this gene can be used as a dominant selectable marker in N. crassa transformation.

Comparative study of 15 lysosomal enzymes in chorionic villi and cultured amniotic fluid cells. Early prenatal diagnosis in seven pregnancies at risk for lysosomal storage diseases.

A large number of chorionic villi samples obtained from women undergoing elective first trimester termination of pregnancy was analysed by enzyme assays similar to those applied to cultured amniotic cells. The levels of 15 lysosomal enzymes were compared to those observed in tissue cultures of amniotic cells obtained through amniocentesis at 16-18 weeks of pregnancy and the results were discussed in order to assess the usefulness of trophoblast biopsy for first trimester diagnosis of hereditary lysosomal diseases. The data suggest the applicability of this source of fetal cells for prenatal diagnosis of fifteen respective genetically determined enzyme deficiencies with the probable exception of alpha-L-iduronidase deficiency. Enzyme determinations were performed on chorionic villi samples of two pregnancies at risk for Tay-Sachs disease, three pregnancies for GM1 gangliosidosis type 1, one for mucopolysaccharidosis type VI and one for Wolman's disease.

Aortic arch interruption: two-dimensional echocardiographic recognition in utero.

A case of aortic arch interruption detected in a 17-week-old fetus and confirmed after therapeutic abortion is reported. The potential usefulness of cross-sectional echocardiography in prenatal detection of aortic arch anomalies is discussed.