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Biomedical subjects

S Venturoli

Publications and source records attributed to S Venturoli.

At least 73 records · Page 4Linked to original sources
PubMed1996

Detection of parvovirus B19 DNA in bone marrow cells by chemiluminescence in situ hybridization.

A chemiluminescence in situ hybridization method was developed for the search of B19 parvovirus DNA in bone marrow cells, employing digoxigenin-labeled B19 DNA probes, immunoenzymatically detected with a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. The light emitted from the in situ-hybridized probe was analyzed and measured by a high-performance luminograph connected to an optical microscope and to a personal computer for the quantification of the photon fluxes from the single cells and for image analysis. The chemiluminescence in situ hybridization was applied to bone marrow cell smears of patients with aplastic crisis or hypoplastic anemia, who had been previously tested by in situ hybridization with colorimetric detection, dot blot hybridization, and nested PCR. The chemiluminescent assay provided an objective estimation of the data, proved specific, and showed an increased sensitivity in detecting B19 DNA compared with in situ hybridization with colorimetric detection.

Anemia, Aplastic↗
PubMed1996

Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA.

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxigenin-labeled probes, and the spatial morphological resolution and localization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes constructed in our laboratory that were immunoenzymatically visualized employing anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal from the hybrid formation was detected, analyzed, and measured with a high performance, low light level imaging luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell, corresponding to the presence of hybridized CMV DNA, could be found in infected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive. The chemiluminescent in situ hybridization assay developed in this work can be a useful tool for a sensitive and specific diagnosis of viral infection and can be easily adapted to detect and study any specific gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.

Cell Line↗
PubMed1995

Persistent B19 parvovirus infections in haemophilic HIV-1 infected patients.

B19 infection can be acquired by transmission with blood factors in patients with congenital bleeding disorders, requiring clotting factor concentrates. In immunodeficient patients, the failure of immunity to clear B19 virus may produce persistent infections. The presence of B19 DNA in blood samples from seven haemophilic patients with concomitant HIV-1 infection was studied over a period of three-to-four years. Dot blot hybridization assays with DNA and RNA probes were used to detect medium high viremias, and polymerase chain reaction (PCR) to detect very low viremic titres. Three patients were negative for B19 DNA in all the blood samples, while four patients were persistently positive for B19 DNA. Viral persistence, which in one patient was detected throughout the study period (40 months), occurred at low titre in all four positive patients with some recurrent increases in viral titre. In the four patients persistently positive for B19 DNA, acute or chronic clinical symptoms and signs that could be associated with B19 were not noted when virus was present at low titre (B19 DNA detectable only by PCR). When patients had a higher viral titre (B19 DNA detectable by dot blot hybridization) acute manifestations (aplastic crisis, Fifth disease, fevers, pneumonitis) were found.

Adolescent↗
PubMed1995

Extent of sequence variability in a genomic region coding for capsid proteins of B19 parvovirus.

Genomic variability in human parvovirus B19 was analysed by direct partial sequencing of different isolates collected in Italy between 1989 and 1994. DNA was purified from viremic serum samples and the region of viral genome coding for structural proteins was amplified by polymerase chain reaction and partially sequenced (nt. 2400-3400). Data were compared to reference isolates Wi (U.K., 1973) and Au (U.S.A., 1982). The average relative distance between isolates was estimated at the low level of 0.61%, comparable with the distance to reference isolates. Out of 22 nucleotide substitutions found, 9 resulted in amino acid changes. From our results, this region of human parvovirus B19 genome appears to be stably conserved.

Amino Acid Sequence↗
PubMed1995

Automated detection of digoxigenin-labelled B19 parvovirus amplicons by a capture hybridization assay.

An automated method to identify B19 amplicons, directly labelled with digoxigenin during amplification reaction was developed. The labelled amplicons were hybridized with a biotinylated B19 oligo-probe and captured on commercially available test tubes coated with streptavidin. The hybridized amplicons labelled with digoxigenin were detected using anti-digoxigenin Fab fragments conjugated to peroxidase and the colourimetric reaction automatically evaluated as an immunoenzymatic assay. Fifty serum samples were tested by the assay and the results were in accordance with those obtained by Southern blot analysis of amplified products. Due to the high sensitivity, specificity and reproducibility shown, the assay seems to be a practical and reliable test for the diagnosis of B19 infection and can be easily adapted to identify any digoxigenin-labelled amplified product of viral genomes.

Bacterial Proteins↗
PubMed1995

Parvovirus B19 clearance from peripheral blood after acute infection.

The clearance of B19 parvovirus from peripheral blood was followed after acute infection to determine how long the virus is present in blood, even at low titer. The presence of B19 DNA in serum was investigated with dot blot hybridization during an epidemic. Fourteen patients positive for B19 DNA were followed for up to 1 year for its presence in samples taken monthly, using both dot blot hybridization and nested polymerase chain reaction (PCR). All patients examined showed medium to high viremic titers (detectable with hybridization assay) only at the beginning of observation; later the virus titer decreased and was detectable only by nested PCR. Of the 14 patients followed, 13 were positive for B19 DNA by nested PCR for 2-6 months; 1 patient showed a persistent infection associated with chronic arthritis and was positive for B19 DNA for 1 year without clearance of the virus.

Acute Disease↗
PubMed1995

Longitudinal change of sonographic ovarian aspects and endocrine parameters in irregular cycles of adolescence.

We longitudinally studied clinical endocrine and ultrasound parameters of the ovaries in 73 healthy adolescents having persistent menstrual irregularities. After the first examination, they were reexamined after a variable period ranging from 2 to 7 y. During the first examination, three basic features of the ovaries were observed: homogeneous (36%), multifollicular (23%), and polycystic (41%). Polycystic ovaries were most frequent, and they generally exceeded the normal adult range. During the last examination, in the entire group of irregular adolescents, homogeneous ovaries decreased (-14%), polycystic ovaries increased (+18%), and a further higher number of subjects exceeded the normal adult range (+10%). The subjects with enlarged ovaries had the highest values of LH, testosterone, and androstenedione. Fourteen subjects out of 46 (30%), with normal ovarian volume in the first examination, registered an ovarian enlargement in the last examination, exceeding the normal range. Moreover, a change from the homogeneous or multifollicular structure to the polycystic one was observed. Twenty-one subjects out of 27 (78%) with enlarged ovaries in the first examination confirmed the high ovarian volume and the unchanged structure in the last examination, whereas six subjects (22%) showed ovaries within the normal adult range; the polycystic structure was substantially confirmed. These results indicate the following. 1) Homogeneous, multifollicular, and polycystic ovaries can usually be found in the postmenarcheal period. 2) Enlarged ovaries, polycystic structure, hyperandrogenemia, and high LH values are strongly linked, and they are frequent in irregular cycles even in the absence of signs of hyperandrogenism. These characteristics may all persist or in various aggregations become a permanent feature. 3) Only a few subjects may lose ovarian enlargement and show a change in the polycystic structure; however, they frequently maintain hyperandrogenemia. 4) During the postmenarcheal period, normal ovarian characteristics may suddenly change, and the ovaries may take on a polycystic structure and increase in volume. Moreover, some endocrine parameters may reach pathologic levels.

Adolescent↗
PubMed1995

Application of a low-light imaging device and chemiluminescent substrates for quantitative detection of viral DNA in hybridization reactions.

In this quantitative dot-blot hybridization assay for detecting B19 parvovirus DNA, we used three different chemiluminescent substrates [adamantyl-1,2-dioxetane phenyl phosphates (PPD and the new PPD-Plus) and the chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) plus Emerald enhancer] and a high-performance, low-intensity-light imaging luminograph apparatus. The hybridization test uses digoxigenin-labeled DNA probes, which are immunoenzymatically revealed by anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. All the detection systems with the various chemiluminescent substrates gave sensitive and reproducible results for calibrators and positive or negative reference clinical samples, with high reproducibility (CV 4-17%). The signal was measured after 45 min of incubation. The luminograph apparatus could detect 10 fg of homologous DNA with the PPD-Plus substrate, whereas the detection limit with the CSPD and PPD substrates was 20 fg and 20-50 fg, respectively. Analysis of 26 samples with the three substrates showed good sensitivity and specificity for viral detection.

DNA, Viral↗
PubMed1994

Skeletal maturation and hormonal levels after the menarche.

Skeletal maturity was studied in 82 healthy adolescent girls age between 12 and 19 who had reached the menarche from 0.5 to 5 years previously. 56 subjects (68.2%) had closed epiphyses while they were open in 26 (31.8%). The incidence of open epiphyses was 61% in the first year after the menarche and progressively decreased showing negative correlation with time elapsed since the menarche (r = 0.98, P < 0.0001). The mean +/- SE height increased from 158.7 +/- 1.3 cm in the first year after the menarche, to 162.7 +/- 1.3 cm in the fifth year after the menarche. No significant differences were seen in estradiol, estrone, testosterone, dihydrotestosterone and androstenedione levels between subjects with open or closed epiphyses while dehydroepiandrosterone and its sulfate levels were higher in the closed epiphyses group. Girls with open epiphyses at first evaluation grew about 3.5 cm in the next three years.

Adolescent↗
PubMed1994

Frequency and treatment of ovarian cysts in adolescence.

Ultrasound examination of the ovaries was performed monthly in the follicular phase of 139 healthy adolescents for one year. In 17 girls (12.2%) ovarian cysts spontaneously disappeared within 3 months of first observation. In 6 subjects, treatment with the combined estrogen/progestogen pill was given. The treatment was effective in 4 girls after 2-6 months while 2 subjects required surgical intervention for a cystic teratoma and a simple follicular cyst respectively. A spell of serial sonographic observation of ovarian cysts would seem appropriate in adolescents.

Adolescent↗
PubMed1994

Body fat distribution has weight-independent effects on clinical, hormonal, and metabolic features of women with polycystic ovary syndrome.

This study was performed to investigate whether different patterns of body fat distribution may have distinct effects on the clinical, hormonal, and metabolic features of women with clinical hyperandrogenism such as polycystic ovary syndrome (PCOS). Ninety-seven consecutive women with PCOS were included in the study after assessment of gynecological and obesity history and careful clinical examination. Women were divided into three tertile groups based on the waist to hip ratio (WHR). Those with peripheral body fat distribution (P-BFD) had a WHR of less than 0.80, those with intermediate body fat distribution (I-BFD) had a WHR of 0.81 to 0.90, and those with abdominal body fat distribution (A-BFD) had a WHR exceeding 0.90. Baseline blood and urine samples were obtained for several hormone and lipid determinations, and the response of glucose, insulin, and C-peptide to a glucose oral challenge (75 g) was investigated. In the PCOS group, WHR values were higher than those used to define P-BFD and A-BFD in the normal female population. As WHR values increased, a significantly greater prevalence of obesity and acanthosis nigricans and a lower prevalence of acne was present. No significant differences were present in any of the other clinical features between the three groups. Ovarian morphology and volumes were similar in all groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1994

Polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex and still poorly understood process. This review examines current theories regarding the development of PCOS and focuses on the physiologic processes involved in the pubertal and adolescent period and their relationship with pathogenesis of PCOS.

Adolescent↗
PubMed1994

Rapid diagnosis of cytomegalovirus encephalitis in patients with AIDS using in situ hybridisation.

AIMS: To evaluate the presence of cytomegalovirus (CMV) DNA in the cerebrospinal fluid of patients with AIDS and suspected viral encephalitis using an in situ hybridisation assay with digoxigenin labelled CMV DNA probes. METHODS: The presence of CMV DNA was evaluated in cerebrospinal fluid cells of 10 patients with AIDS using in situ hybridisation. The positivity of CMV DNA was confirmed by the presence of CMV induced antigens in the same specimens. The presence of CMV DNA and CMV induced antigens was also analysed in peripheral blood leucocytes. The time required to perform the in situ hybridisation assay was about eight hours. RESULTS: The in situ hybridisation assay was sensitive, specific, and provided good resolution. Six patients proved positive for the presence of CMV DNA in CSF cells and all six also proved positive for CMV DNA in blood leucocytes. Of the six CMV positive patients, five were treated with specific antiviral drugs: of these, one died during the treatment while four clinically recovered after one month of treatment. CONCLUSIONS: The in situ hybridisation assay using digoxigenin labelled CMV DNA probes can be used as a valuable diagnostic test for the detection of CMV DNA in the cerebrospinal fluid cells of patients with suspected CMV encephalitis and can therefore prompt adequate antiviral therapeutic intervention.

AIDS-Related Opportunistic Infections↗
PubMed1993

Evaluation of strand-specific RNA probes visualized by colorimetric and chemiluminescent reactions for the detection of B19 parvovirus DNA.

A dot-blot hybridization assay was developed to detect B19 DNA using strand-specific RNA probes labelled with digoxigenin. The sensitivity of the assays was evaluated either using 'plus' and 'minus' sense RNA probes in two different hybridization assays, or in two successive reactions of the same assay. The hybridized probes were revealed immunoenzymatically using anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. The enzyme was visualized by colorimetric reaction. Since 'minus' sense RNA probe gave the best results in the dot-blot procedures, we increased the sensitivity of the hybridization assay visualizing the 'minus' sense digoxigenin-labelled RNA probe by chemiluminescent reaction. In these experimental conditions up to 20 fg of target B19 DNA could be visualized. In the search for B19 DNA, 4656 serum samples were analyzed by chemiluminescent reaction of 'minus' sense digoxigenin-labelled RNA probe and for comparison with the digoxigenin-labelled DNA probe. Positive results were confirmed by Southern blotting. Out of 4656 serum samples analyzed, 4648 gave negative results, 1 resulted positive to all the hybridization assays, 6 only using RNA probe and 1 only by DNA probe.

Adult↗