Antigenic sites of poliovirus type 3 eliciting IgA monoclonal antibodies in orally immunized mice.

A panel of neutralizing IgA monoclonal antibodies was produced from mice orally inoculated with poliovirus type 3 Sabin and cholera toxin as adjuvant. Low levels of neutralizing antibodies were elicited in mice after several boosts, but only in the presence of cholera toxin. Characterization of IgA MAbs by neutralization-escape virus mutants showed that all but one neutralizing MAbs against type 3 poliovirus were directed to antigenic site N-AgIII, which was previously found by us to be the major target of mucosal immune response to Sabin 1 in the mouse. Our data indicate that residue 236 of VP3, not previously reported, is also involved in forming site N-AgIII in addition to formerly described VP3 (aa 58-59) and VP1 (aa 286-290) residues. Unlike poliovirus type 1 IgA MAbs, all IgA MAbs herein described neutralized the wild-type parental poliovirus.

[Flaccid paralysis surveillance in the Latium Region]. / La sorveglianza della paralisi flaccida nella Regione Lazio.

The goal of World Health Organization is to reach the global eradication of poliomyelitis during the first decade of the third millennium. To achieve the certification of the eradication of the disease the main strategy is the Acute Flaccid Paralysis (AFP) surveillance. In Italy the active AFP surveillance was performed at national level since 1997. In the Latium region the active surveillance was performed since January 1997 by the laboratory of virology of Institute of Hygiene G Sanarelli which established a regional hospital network. During the years of survey 7 cases were found in 1997 (0.87/100,000), 4 in 1998 (0.5/100,000), 2 in 1999 (0.25/100,000) and 2 in 2000. No wild polioviruses were detected.

Surveillance of acute flaccid paralysis in Italy: 1996-1997. AFP Study Group. Acute flaccid paralysis.

The last case of poliomyelitis due to transmission of indigenous wild poliovirus occurred in Italy in 1982. To achieve the certification of the eradication of poliomyelitis in Italy, an active surveillance of acute flaccid paralysis (AFP) in the population aged less than 15 years was set up following the World Health Organization (WHO) guidelines. The survey started in 1996 with a pilot study involving 4 out of 21 regions, and was gradually extended to a national level in 1997. The two-year survey identified five patients with diagnosis of vaccine associated paralytic poliomyelitis (VAPP). Polioviruses type 2 and 3 Sabin-like were isolated and characterized in three of them. In the remaining two cases, samples were collected late after the onset of symptoms, and poliovirus could not be isolated. No wild polioviruses were detected during the survey. The rate of non-polio AFP found in Italy in 1997 was 0.61 cases per 100,000, which is lower than the level of 1.0 case per 100,000 considered as acceptable by the WHO. This was mainly due to the delay in organizing the hospital network and starting the active search of AFP cases in the largest and most densely populated regions. Therefore, the overall rate of AFP found in Italy underestimates the global effectiveness of the program, which however will be better evaluated in the next few years. This study is the first systematic attempt to determine the rate of AFP in Italy.

Seroepidemiological evaluation of 1989-91 mass vaccination campaigns against measles, in Italy.

In 1989-91 anti-measles vaccination campaigns were conducted in several Italian regions to vaccinate all children aged between 13 months and 10-12 years without a history of measles or measles vaccination. This study was conducted to evaluate serological status after the mass vaccination campaigns. In 1994, capillary blood samples were collected from randomly selected children, aged 2-14 years, living in 13 local health units. Antibody titres were determined by ELISA. Blood spot samples were analysed for 4114 (75.6%) of 5440 selected children. Among the 835 that reported measles before 1990, 806 (96.5%) were immune and of the 2798 vaccinated, 2665 (95.2%) were immune. The Edmoston-Zagreb (E-Z) strain vaccine was associated with a lower level of immunity than the Schwarz (SW) strain. A history of measles identified almost all immune children. Vaccination with the SW strain conferred persistent immunity (at least 5 years) in 98% of vaccinees. The strategy was able to unite natural and induced immunity.

Measles vaccine in egg allergic children: poor immunogenicity of the Edmoston-Zagreb strain.

Despite the fact that a number of recent studies have shown that measles/ mumps/rubella vaccine is safe for egg allergic children, many pediatricians are still concerned about immunization in egg allergic children. In Europe, a measles vaccine with the Edmoston-Zagreb strain (EZMV) grown in human fibroblast culture has been developed and recommended for children with egg allergy. However, some doubt arises on the efficacy of this strain due to its weak immunogenicity. The aim of this study was to investigate the immunogenicity of the EZMV in comparison to the measles vaccine with the Schwarz strain (SWMV) grown in a chick embryo fibroblast culture. Thirty-nine children affected by severe immediate manifestations due to IgE mediated egg allergy were enrolled. The children received at random the SWMV (Morupar, Sclavo) or the EZMV (Triviraten, Berna) in one 0.5 ml subcutaneous injection, and were checked for any immediate allergic reactions in the following 4 hours. Blood samples were taken for the detection of specific antibody response 5 months after the immunization. In SWMV seroconverted children (18/19) the geometric mean antibody titer was 3 times higher than that observed in EZMV seroconverted children (17/20) (p < 0.01). No allergic reactions occurred following the immunization with the two different vaccines. This data confirms the safety of SWMV in egg allergic children. In addition, the present study provides further data on the lower immunogenicity of the EZMV in comparison to the SWMV.

Prevention of allergies of infants: breast-feeding and special formulas. Influence on the response to immunization.

Infant formulas containing partially hydrolyzed cow milk-proteins are used for the prevention of allergy when maternal milk is not available, and, in preterm infants, also for improving gastric emptying. The nutritional adequacy of such formulas has not yet been completely defined. As the type of feeding may influence the antibody response to immunization, the aim of the present study was to evaluate the antibody response to oral polio virus immunization in term infants and to acellular pertussis and hepatitis B immunization in preterm infants, exclusively fed a partially hydrolyzed cow-milk formula during the first 5 months of life, in comparison with exclusively breast-fed infants. Active immune response occurred in all the infants after the second dose of immunization and no significant difference in the antibody titres was found according to the type of milk. On the basis of these results, it seems that protein nutrition based exclusively on a partially hydrolyzed formula does not impair the response to immunization in both preterm and term infants.

Comparison of capillary blood versus venous blood samples in the assessment of immunity to measles.

Seroepidemiological investigations are essential for assessing the efficacy of measles vaccination programmes. However, when large-scale sampling is needed, a major difficulty is the problem of taking venous blood, especially in children. An alternative method is the collection of capillary blood samples spotted on filter papers. The eluted extract from these 'blood' spots can be used instead of serum samples for measles laboratory diagnosis or investigations. Measles antibody detection is readily carried out by ELISA on serum samples. The same technique can be used on eluates from capillary blood spots. Measles antibody titres determined on matched serum and blood spot samples from 27 children were compared. A strong correlation was found between the results obtained with the two methods of blood sampling.

Topoisomerase-II-mediated DNA cleavage within the human ribosomal genes.

The interaction of topoisomerase II (topo II) with human ribosomal DNA (rDNA) was investigated in vivo using the antitumoral drug VM26, a specific inhibitor of topo II, that stabilizes the transient cleavable complex. rDNA-protein complexes isolated from nucleoli of TG cells were analyzed for double strand breaks with probes that covered almost all intergenic transcribed spacer (IGS) and all transcribed sequences of tandem repeat ribosomal DNA genes. Preferential cleavage sites were present in only a part of nucleolar rDNA, i.e., the transcribed region. Proteins, purified from the same complexes, were analyzed by Western-blot and stained by an antiserum against both topo II forms, showing the presence of topo II beta.

Inhibition of topoisomerase II activity and its effect on nucleolar structure and function.

The relationship between topoisomerase II activity and ribosomal RNA synthesis was investigated using the antitumoral drug VM26, a specific inhibitor of topoisomerase II. For this purpose TG cells, a human tumor cell line, were cultured in the presence of 2.5 microM VM26 for 1 and 3 h; VM26 reduced the topoisomerase II activity, measured in whole cell extracts. In the presence of VM26 the [3H]uridine incorporation into ribosomal RNA was decreased; electron microscopy investigation of nucleoli showed a segregation of nucleolar components. Because VM26 stabilizes the cleavable complex and inhibits the resealing reaction, thus causing potential cleavage sites, we have analyzed the double-strand breaks caused by the drug treatment in the tandem repeat ribosomal DNA (rDNA) genes, by indirect labeling with two probes recognizing the 5' portion of ETS (BES) and the 3' portion of 28S (LS6BE) transcribed gene. In VM26-treated cells rDNA is fragmented and a topoisomerase II preferential cleavage site is present, localized at 1.85 kb in 28S region from 3' EcoRI site.

Effects of camptothecin, an inhibitor of DNA topoisomerase I on ribosomal gene structure and function in TG cells.

The effects of camptothecin treatment and topoisomerase I inhibition on ribosomal gene structure and function were investigated in TG cells, a human tumour cell line. 90- and 180-min treatments with 25 microM camptothecin resulted in an increased DNA fragmentation and decreased activity of topoisomerase I in cell extracts. After 180-min treatment, the incorporation of labelled uridine into total cell RNA was reduced to 39% and the ribosomal RNA synthesis to 10%, as compared to values of control cells. At the ultrastructural level, the nucleolar components appeared to be segregated; after selective DNA staining, with osmium-amine complex, a part of the nucleolar chromatin of treated cells showed the presence of thin, extended DNA filaments, superimposable to those present in control cells.

The Ag-NOR proteins and transcription and duplication of ribosomal genes in mammalian cell nucleoli.

The relationship between the Ag-NOR (silver-stained Nucleolar Organizer Region) proteins and the functional-structural organization of the nucleolar ribosomal chromatin was studied in regenerating and cortisol-stimulated rat hepatocytes. Statistical analysis of Ag-NOR proteins, carried out with an automated image analyzer, indicated that in regenerating rat hepatocytes the quantity of Ag-NOR proteins mainly increased between the 4th and 12th h of regeneration, reaching a level twice that of resting hepatocytes. Also the synthesis of pre-ribosomal RNA (pre-rRNA) was stimulated after the 4th h of regeneration. Cycloheximide administered to rats at a dose of 0.025 mg/100 g body weight (bw) prevented any increase in Ag-NOR proteins but did not hinder the stimulation of pre-rRNA synthesis. In 8 h cortisol-stimulated hepatocytes no significant change in amount of Ag-NOR protein was observed whereas pre-rRNA synthesis was highly increased as in 12 h regenerating hepatocytes. These results indicated that in rat hepatocytes Ag-NOR proteins and stimulation of pre-rRNA synthesis are not related. The relationship between the Ag-NOR proteins and the distribution of the completely extended intranucleolar ribosomal chromatin was also studied in regenerating rat hepatocytes. At 12 h after partial hepatectomy an increased amount of completely extended ribosomal chromatin was observed, contemporaneously with an increased quantity of Ag-NOR proteins. These ribosomal chromatin changes preceded the beginning of DNA synthesis and were prevented by cycloheximide-induced inhibition of protein synthesis.

Characterization by T1-oligonucleotide fingerprinting of three strains of human hepatitis A virus isolated in Italy.

Three human hepatitis A virus strains, all of them isolated in Italy but one acquired abroad, were analyzed by T1-RNAase oligonucleotide mapping and by monoclonal antibody neutralization. The variation among their genomes according to T1-maps was calculated to be about 9%, thus confirming the poor genomic variation assessed by nucleotide sequencing (1-10%). However T1-maps of these Italian isolates were different from those reported in the literature (Weitz and Siegl, 1985). Neutralization by monoclonal antibody caused a reduction in titres of 2-25 log10. This genomic stability, if confirmed, is important with a view to a valuable vaccine.

Variations of the 1H spin-lattice relaxation in a fatty liver model as compared to normal liver.

We report the results of an in vitro study on ethionine-injected rat liver (EI) and on normal rat liver (C) performed analyzing with iterative fitting procedures the 1H spin-lattice relaxation curves detected by IR pulse sequence at 20 MHz and at 37 degrees C on fresh excised samples. Single-exponential functions did not adequately describe the experimental curves both in EI and in C group. The analysis of the curves by two-exponential hypothesis showed a small portion of the signal characterizable by a time constant of about 80 ms, common both to EI and to C samples. A slowing of about 30% in the relaxation characterized the remaining portion of the curve (90-95%) in EI as compared to C samples. The hypothesis that the 1H of the triglycerides vacuoles present in EI livers had a relaxation curve additional to the remaining signal was checked by three-exponential analysis. The results were not in contrast with the known value of the triglycerides percentage content and with the spin-lattice relaxation time of the -CH2 group 1H obtained in different experimental conditions in the same fatty liver model. The negative results of the three-exponential analysis on normal liver curves as well as the favorable controls performed to test the analysis procedure supported further this hypothesis. The remaining signal after subtraction of the triglycerides contribution showed still the small fast portion and the increase of the relaxation time of the major portion (from approximately 300 ms up to approximately 400 ms) as compared to C samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Spatial redistribution of ribosomal chromatin in the fibrillar centres of human circulating lymphocytes after stimulation of transcription.

We have studied the distributional changes of the completely extended ribosomal chromatin present in the fibrillar centres of resting human lymphocytes after phytohemagglutinin (PHA) treatment. In thin sections of resting lymphocytes selectively stained for DNA, the extended non-nucleosomal chromatin was located in a solitary, large agglomerate which corresponds to the solitary, large fibrillar centre observed in uranium-lead-stained sections. At 20 h after PHA stimulation the ribosomal chromatin agglomerate appeared to be fragmented into smaller agglomerates which correspond to numerous fibrillar centres surrounded by a thick rim of dense fibrillar component. The mean area of ribosomal chromatin agglomerates from resting lymphocytes was found to be 0.772 mu 2 + 0.125 SD, whereas in stimulated lymphocytes it was found to be 0.184 mu 2 + 0.052 SD. At 20 h after PHA treatment ribosomal RNA (rRNA) synthesis was 8-fold greater than the control value, whereas DNA synthesis had not started. These results indicate that ribosomal chromatin of resting lymphocyte fibrillar centres contains transcribable sequences, temporally not expressed.

Paralytic poliomyelitis in Italy (1981-85).

Fifteen cases of presumptive poliomyelitis occurring in Italy between 1981-85 were studied in order to differentiate between paralysis caused by poliovirus and that of different etiology. Out of seven confirmed cases three were "temporally associated with vaccination". Three aspects are discussed: the need for a careful differential diagnosis of paralytic cases; the over concern about the problem of vaccine-associated cases: the risk connected with re-importation of wild poliovirus strains.

Structure of ribosomal genes of mammalian cells in situ.

The structural and functional organization of ribosomal genes was investigated in situ in human circulating lymphocytes and a human tumour cell line, TG cells, Stereo-pair electron micrographs revealed that this ribosomal chromatin is not structured into nucleosomes, but composed of completely extended filaments, 2-3 nm thick. Despite its homogeneous morphological structure only a small portion of ribosomal chromatin present in the dense fibrillar component is transcriptionally active. This was demonstrated in TG cells by exclusive autoradiographic labelling on serial sections of the dense fibrillar component with 3H-uridine and by the distribution of RNase-gold particles in all the ribonucleoprotein (RNP) structures but not in the fibrillar centres. The extended, non-nucleosomal configuration of both transcriptionally inactive and active ribosomal chromatin could be explained by the peculiar protein composition of this chromatin. Staining with the acrolein-silver-methenamine technique for basic proteins indicated that all the completely extended ribosomal chromatin is devoid of histones, even after inactivation of transcription by actinomycin D. Stereo-electron-microscopical visualisation of the Ag-NOR proteins revealed a thread-like structural organization of these proteins with a spatial distribution superimposable on that of the ribosomal chromatin filaments.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 2. Structural modification of 7-(substituted aryl)-3,5-dihydroxy-6-heptenoic acids and their lactone derivatives.

A series of 7-(substituted aryl)-3,5-dihydroxy-6-heptenoic (heptanoic) acids and their lactone derivatives have been prepared and tested for inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in vitro. A systematic exploration of the structure-activity relationships in this series led to the synthesis of (+)-trans-(E)-6-[2-[2,4-dichloro-6-[(4-fluorophenyl) methoxyl]phenyl]ethyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (66(+)), which has one-half of the inhibitory activity of compactin.