Expression and cellular localization of insulin-like growth factor-II protein and mRNA in Sparus aurata during development

The spatial localization of IGF-II protein and mRNA was investigated during larval and postlarval developmental stages of the gilthead sea bream (Sparus aurata) by immunohistochemistry and in situ hybridization, using specific antisera and riboprobes. Steady-state levels of IGF-II mRNA in larvae were determined by Northern blot analysis and were found to be increased. Immunoreactivity towards IGF-II was found in larval skin, muscle, gills, gut, olfactory epithelium and kidney. After metamorphosis, the strongest immunoreactivity was found in red skeletal muscle. Positive reaction with IGF-II antibodies was also found in the olfactory epithelium and in the epithelia of pharynx, oesophagus, stomach and kidney. In the adult, the most intense signal was observed in the red and pink musculature and in heart musculature. Immunostaining was also found in saccus vasculosus, thymus, spleen and ovary. IGF-II mRNA was detected by in situ hybridization in the brain, olfactory epithelium, eye, pharynx, skeletal musculature and liver. The spatial distribution of IGF-II shown in this study is consistent with previous findings on the cellular localization of IGF type 1 receptor in the sea bream and supports a role for IGF-II during development and growth of sea bream. Furthermore, these results suggest that IGF-II acts in an autocrine/paracrine manner.     

Identification of four novel mutations in the alpha glucosidase gene in five Italian patients with infantile onset glycogen storage disease type II

Glycogen storage disease type II (GSDII) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme acid alpha glucosidase. Four novel mutations (C670T, G989A, G2188T, and Delta 23 nt 828-850) were identified in five Italian patients with the infantile form of the disease. The C670T mutation was present in two unrelated patients in heterozygosity; the effect on enzyme activity was assessed by in vitro expression. COS-1 cells expressing the C670T allele had a twofold higher activity than the negative control cells. The G989A and G2188T point mutations lead to the introduction of premature stop signals that results in truncated forms of alpha glucosidase. The in vitro expression of G2188T allele demonstrated no increment in activity compared to negative control. The frame shifting deletion of nucleotides 828-850 was identified in one patient in heterozygosity. The shift in the reading frame introduces a stop codon 135 nucleotides downstream the deletion junction that results in a truncated protein without catalytic activity. Nested PCR screening showed that the mutation was carried by the mother and was absent in the other members of the family. The four novel severe mutations herein described concerned only infantile onset GSDII patients; the loss of enzyme activity is correlated with the severity of the disease.     

Pediatric sentinel surveillance of vaccine-preventable diseases in Italy

BACKGROUND: Planning and evaluating vaccination programs depend on reliable systems of monitoring disease incidence in the community. In Italy vaccine-preventable diseases are subject to statutory notification, but they are often unreported. In January, 2000, a pediatric sentinel network was launched, with the aim of monitoring in a timely and accurate way the geographic and temporal trends of vaccine-preventable diseases. METHODS: The network consists of National Health System primary care pediatricians; participation is voluntary. The diseases under surveillance include measles, mumps, rubella, pertussis and varicella. Case definitions are based on specific clinical criteria, and pediatricians report cases on a monthly basis. Incidence rates are estimated and compared with those obtained by statutory notifications. The proportion of vaccinated cases is also computed. RESULTS: In 2000 an average of 468 pediatricians participated each month of a total of 7276 pediatricians under contract for primary care by the National Health System. The population under surveillance consisted of 371 670 children younger than 15 years (of a national total of 8.347.804 children of the same age). The annual national incidence per 100.000 children was estimated at 5345 for varicella, 1972 for mumps, 279 for pertussis, 108 for rubella and 62 for measles, although wide variations were observed among geographic areas. The national estimates are 3 to 7 times higher than those obtained through statutory notifications. For all of the diseases the ratio between the two sources of data was significantly higher in southern Italy, compared with the rest of the country. The proportion of vaccinated cases was similar for measles and rubella (21 and 17%) but was approximately 3 times higher for mumps (59%). Most (74%) of the vaccinated mumps cases had received the Rubini vaccine strain. CONCLUSIONS: The sentinel surveillance system is considerably more sensitive than statutory notifications, particularly in southern Italy. The high percentage of mumps cases vaccinated with the Rubini strain indicates a reduced effectiveness of this vaccine. Although further improvements are needed, pediatrician-based sentinel surveillance is a useful tool for evaluating vaccine-preventable disease trends.     

Identification of Echinococcus granulosus eggs

OBJECTIVE: The purpose of this QMP-LS patterns-of-practice survey was to determine quality assurance (QA) processes used in Ontario for purchased and in-house culture media, and compliance with recommended practices. METHODS: QMP-LS required laboratories to submit copies of media QC records for August to October 1998. Target media were blood agar, chocolate agar, MacConkey agar, sorbitol MacConkey agar (SMAC), Campylobacter agar, and selective media for pathogenic Neisseria spp. Procedures for acquisition and maintenance of QC stock cultures, media-type specific QC strains, time and temperature of incubation, and the test inoculum were required. Data were captured and analysed using Microsoft Excel. RESULTS: Marked variability and inadequacy of media QC records of 124 participating laboratories was noted. 12/124 prepared media in-house, most did not comply with NCCLS QA recommendations. Those purchasing prepared media frequently did not comply with NCCLS recommendations. In-house QC was not performed by 9% for chocolate agar, 87% for MacConkey agar without crystal violet, and 53% for SMAC agar. Recommended ATCC strains were used by less than half of participants. Time and temperature of incubation of the QC plates were not always appropriate. Only 65/117 laboratories used a correct inoculum to test the sensitivity and selectivity of media for pathogenic Neisseriae. Physical characteristics of prepared media were rarely recorded. Most laboratories were not aware of their supplier's QC protocols. DISCUSSION: Few Ontario laboratories comply with the NCCLS recommendations for QA of culture media. Major compliance failures were not performing QC at all, inappropriate choice of QC bacterial strains, and inoculum used. Incomplete record keeping was common and methods for maintaining stock cultures were sub-optimal. External quality assessment of this important parameter of microbiology practice needs to be undertaken on an ongoing basis.     

Exacerbation of HIV viral load simultaneous with asymptomatic reactivation of chronic Chagas' disease

Chronic Trypanosoma cruzi infection can reactivate in patients with immunosuppression related to human immunodeficiency virus (HIV) infection, resulting in severe meningoencephalitis or myocarditis and high parasitemia. The effects of T. cruzi on HIV infection are unknown. We describe an HIV-infected patient with chronic Chagas' disease who experienced an asymptomatic T. cruzi reactivation characterized by the finding of the parasite in direct microscopic examination of blood. The patient's HIV viral load had increased simultaneously with the exacerbation of T. cruzi parasitemia and decreased to previous levels after successful antiparasitic treatment. This otherwise unexplained finding suggests that T. cruzi infection might up-regulate HIV replication, which may affect HIV disease progression. Asymptomatic reactivation of Chagas' disease has not been reported before. This could mean that the severe clinical manifestations related to the reactivation of trypanosomiasis are just the tip of the iceberg.     

Trisomy 3 in two paediatric post-transplant lymphomas

Few cytogenetic data are available concerning the chromosomal constitution of post-transplant lymphomas. We report two paediatric cases of trisomy 3, as a primary anomaly, in post-transplant lymphoproliferative disease (PTLD) associated with B immunophenotype. Using cytogenetic analysis and fluorescence in situ hybridization on chromosome preparations, we found trisomy 3 in both patients and an extra X chromosome in one. Clinical, histological and immunophenotypical data are presented. Trisomy 3 has been observed in different types of non-Hodgkin's lymphomas but it is relatively rare in B-cell lymphomas, with the exception of marginal zone lymphoma and mantle cell lymphoma. To our knowledge, trisomy 3 is an uncommon cytogenetic finding in PTLD. Further cytogenetic studies of these lymphoproliferative disorders might contribute to evaluate the role of these chromosomal anomalies in the pathogenesis of this disease.     

Aspirin prevents Escherichia coli lipopolysaccharide- and Staphylococcus aureus-induced downregulation of endothelial nitric oxide synthase expression in guinea pig pericardial tissue

The aim was to analyze whether pericardial tissue expresses endothelial NO synthase (eNOS) protein and to determine the presence of cytosolic proteins that bind to eNOS mRNA. The effect of aspirin on the above-mentioned parameters was also analyzed. eNOS protein was expressed in pericardial tissue from male guinea pigs. Escherichia coli lipopolysaccharide (LPS, 10 microgram/mL) and Staphylococcus aureus endotoxin (SA, 10 microgram/mL) reduced eNOS protein expression and shortened the half-life of the eNOS messenger. Under basal conditions, cytosolic extracts from pericardial samples bound to the 3'-untranslated region (3'-UTR) of eNOS mRNA, which was enhanced by LPS and SA. Proteinase K fully prevented the binding of cytosolic pericardial extracts to 3'-UTR of eNOS mRNA, suggesting the involvement of proteins that were further characterized as 60- and 51-kDa proteins. Aspirin (1 to 10 mmol/L) restored eNOS expression in either LPS- and SA-stimulated pericardial samples and reduced the binding activity of the pericardial cytosolic proteins to 3'-UTR of eNOS mRNA. Indomethacin also reduced the downregulation of eNOS by LPS and diminished the binding activity of the cytosolic proteins, although higher doses of indomethacin than of aspirin were needed to improve these parameters. In conclusion, eNOS protein is expressed in guinea pig pericardial tissue. LPS and SA stimulate the binding activity of pericardial cytosolic proteins to 3'-UTR of eNOS mRNA and reduce eNOS protein expression. High doses of aspirin and indomethacin protect eNOS protein expression and reduce the binding activity of the cytosolic proteins to 3'-UTR of eNOS mRNA, suggesting an inverse association between the presence of these cytosolic proteins and eNOS expression.