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E Ortona

Publications and source records attributed to E Ortona.

At least 37 records · Page 2Linked to original sources
PubMed1999

Cloning and expression of a cDNA encoding an elongation factor 1beta/delta protein from Echinococcus granulosus with immunogenic activity.

A cDNA clone (Eg EF-1beta/delta) of Echinococcus granulosus has been isolated by an expression library screened with immunoglobulin (Ig)E of sera from patients with cystic echinococcosis (CE). The Eg EF-1beta/delta was identified on the basis of sequence homology to the subunits beta or delta of the elongation factor-1. The amino acid sequence deduced from this open reading frame is 244 residues long with a predicted molecular mass of 31 kDa. In Southern blot under high stringent condition, Eg EF-1beta/delta hybridized to genomic DNA of E.granulosus at two bands of 4 and 2.5 Kb. In immunoblotting analysis, the Eg EF-1beta/delta protein shows immunological reactivity with sera from CE patients: 51.7% of sera contained IgE, 41.7% IgG and 18.3% IgG4 specific to the recombinant protein. We identify the Eg EF-1beta/delta by immunoblotting with specific monoclonal antibody both in protoscoleces and in sheep hydatid fluid. The higher percentage of humoral immune response against Eg EF-1beta/delta observed in CE patients with calcified cysts than in patients with active cysts indicates the possible release of the protein in the hydatid fluid after protoscoleces degeneration suggesting the possible use of this antigen in the immunosurveillance of CE. Overall, these findings seem to assign to Eg EF-1beta/delta a key role in the allergic disorders and in the complex host-parasite relationship in CE.

Allergens↗
PubMed1999

Immunochemical and molecular characterization of vertebral hydatid fluid.

This study describes a 42-year-old male born in Tunisia, who presented with vertebral hydatidosis which was initially misdiagnosed as Pott's disease. The correct diagnosis was finally made utilizing CT and MRI imaging and confirmation of the diagnosis was made possible by molecular and immunochemical characterization of the content of vertebral cyst fluid.

Adult↗
PubMed1998

Potential impact of Pneumocystis genetic diversity on the molecular detection of the parasite in human host.

Our aim was to evaluate if genetic diversity of Pneumocystis carinii could influence the detection by molecular techniques in bronchoalveolar lavage (BAL) fluids and in non-invasive specimens (induced sputum, oropharyngeal washing and serum/blood). P. carinii is morphologically similar in different hosts although several strains have been identified by biomolecular techniques. Variations of mt-LSU and ITSs sequences could determine a lack of hybridization of some clinical samples and could have diagnostic consequences with loss in sensitivity and specificity of available molecular tests, but at the moment no data support a significant impact of genetic diversity in these sequences on molecular detection of P. carinii for clinical purposes.

DNA Primers↗
PubMed1997

Biodiversity of Pneumocystis carinii hominis: typing with different DNA regions.

The purpose of this study was to identify the most useful gene for the detection of biodiversity of Pneumocystis carinii hominis isolates and to compare samples from French and Italian subjects. We studied 20 bronchoalveolar lavage fluid specimens from 20 human immunodeficiency virus-infected patients (10 French and 10 Italian patients) with Pneumocystis carinii pneumonia by DNA sequencing of the thymidylate synthase (TS), 5S rRNA, large-subunit mitochondrial rRNA (mt LSU rRNA), and internal transcribed spacer (ITS1 and ITS2) genes. Thirteen of the 20 sequenced samples had the prototype TS gene sequence. Fourteen of the 20 samples showed the prototype sequence of the 5S rRNA gene, and 6 had variant sequences of the 5S rRNA gene. The mt LSU rRNA gene was sequenced for 18 of the 20 samples; all sequences were different from the prototype sequence and were classified into four groups. Thirteen of the 20 ITS1 and ITS2 sequences were analyzed, and all the sequences were found to be different from the prototype sequence and were classified into 10 groups. The internal transcribed spacer regions thus appear to be the most discriminatory region of DNA for analysis of the biodiversity of P. carinii hominis isolates.

AIDS-Related Opportunistic Infections↗
PubMed1997

Detection of Pneumocystis carinii in respiratory specimens by PCR-solution hybridization enzyme-linked immunoassay.

By using a recently developed PCR-solution hybridization enzyme-linked assay (PCR-SHELA), we investigated Pneumocystis carinii in bronchoalveolar lavage fluid samples and induced sputa of patients with pneumocystosis. In detecting P. carinii, PCR-SHELA proved more sensitive than immunofluorescence staining or a single PCR and significantly more diagnostically specific than a nested PCR. Our data suggest that PCR-SHELA could be used to detect P. carinii organisms in respiratory samples, particularly in patients with uncertain diagnoses.

AIDS-Related Opportunistic Infections↗
PubMed1996

Non specific PCR products using rat-derived Pneumocystis carinii dihydrofolate reductase gene-specific primers in DNA amplification of human respiratory samples.

DNA amplification using dihydrofolate reductase (DHFR) primers in bronchoalveolar lavage fluids (BALFs) from patients with Pneumocystis carinii (PC) pneumonia yielded low sensitivity and specificity. Amplified products of BALFs from an AIDS patient without PC pneumonia and five patients with PC pneumonia were cloned and sequenced. All samples showed the same sequence without any homology with DHFR cDNA of rat PC, or with any DHFR sequences in databases at the DNA or amino acid level. The data demonstrate that these DHFR primers amplify a non-specific region of DNA with a sequence unrelated to the human PC DHFR gene both in PC positive and in PC negative samples. This finding precludes the use of these DHFR primers for the diagnosis of PC pneumonia in respiratory specimens.

Base Sequence↗
PubMed1996

Immunological responses to antigen B from Echinococcus granulosus cyst fluid in hydatid patients.

The immunological reactivity of Echinococcus granulosus antigen B was evaluated in 30 hydatid patients. Antigen B was purified from sheep hydatid cyst fluid by electroelution from a non-reducing SDS-PAGE gel (AgB). In ELISA and immunoblotting (IB), determining antibody production in sera from patients with hydatid disease and with other parasitic infections, purified AgB showed higher specificity than a partially purified antigen named pH5PPT (100% vs 83% in pH5PPT-ELISA and 58% in pH5PPT-IB). AgB-IB achieved higher sensitivity than AgB-ELISA (80% vs 63%). All AgB-IB positive sera recognized the 12 kDa subunit. Qualitative AgB-IB assessment of IgG isotype responses identified IgG4 as the predominant isotype (87%). The other isotypes showed a lower percentage of positive reactions: IgG1, 33%; IgG2, 21%; and IgG3, 17%. PBMC proliferative assay revealed a cellular response to AgB in 100% of patients' PBMC. These findings confirm antigen B, especially its smallest subunit, as a good diagnostic molecule.

Animals↗
PubMed1996

Imbalance between Pneumocystis carinii cysts and trophozoites in bronchoalveolar lavage fluid from patients with pneumocystosis receiving prophylaxis.

Detection and quantification of different Pneumocystis carinii (PC) life cycle forms were performed by polymerase chain reaction (PCR) and by morphological stains on bronchoalveolar lavage fluids (BALF) from HIV-infected patients with P. carinii pneumonia (PCP). The number of PC trophozoites was higher in patients with PCP who were receiving prophylaxis than in those not receiving prophylaxis. Also the cyst: trophozoite ratio was lower in the first group. No difference was observed between patients receiving different prophylactic medications. The imbalance between PC forms in BALF from patients with PCP receiving anti-PC prophylaxis may hamper the sensitivity of cyst stains. Multiple stains or PCR examination should be performed on BALF from patients with clinically suspected PCP who are receiving prophylaxis.

AIDS-Related Opportunistic Infections↗
PubMed1996

In vitro and in vivo immunomodulatory effects of anti-Pneumocystis carinii drugs.

The anti-Pneumocystis carinii drug effects on mitogen-, antigen-, and interleukin-2-induced proliferative responses and on natural killer (NK) cell-mediated activity were analyzed in vivo (rats) and in vitro (normal human peripheral blood mononuclear cells). Splenocytes derived from in vivo piritrexim- and clindamycin-treated rats showed a significant inhibition of mitogen-induced proliferative responses. In vitro exposure to clindamycin, piritrexim, and pyrimethamine caused an inhibition of human T lymphocyte proliferation in response to mitogen, antigen, and interleukin-2 stimulation. Rat NK cell-mediated cytotoxic activity was not affected by the drugs, and human NK cell activity was reduced only at the highest concentration (10 micrograms/ml) of the drugs. The potential immunotoxicity of the long-term administration of these agents in humans needs further investigation.

Adjuvants, Immunologic↗
PubMed1996

Genetic variability in Italian sheep isolates of Echinococcus granulosus.

The possible polymorphism of distinct Italian Echinococcus granulosus isolates was evaluated by RAPD-PCR using five arbitrarily chosen random primers. Sheep DNA was used as control in each RAPD-PCR to ascertain the host DNA contamination. Distinct amplification patterns were detected for each isolate revealing identical bands, with the same or different intensity, as well as distinct bands. The results of this study confirm the usefulness of RAPD-PCR technique in genetic typing of parasites and show the existence of genetic variability within the Italian E. granulosus sheep strain.

Animals↗
PubMed1995

Variable efficiency of three primer pairs for the diagnosis of Pneumocystis carinii pneumonia by the polymerase chain reaction.

The efficiency of three different primer pairs, complementary to different Pneumocystis carinii DNA regions, was compared in the polymerase chain reaction (PCR) for the diagnosis of Pneumocystis carinii pneumonia (PCP) on bronchoalveolar fluid (BALF) from patients with AIDS. PCR coupled with dot-blot hybridization (BLOT) using primers and probe from the mitochondrial 23SrDNA region showed the highest sensitivity, with a lower detection limit of 0.5-1 organisms microliter-1. When testing 47 BALF, PCR plus BLOT of the mitochondrial 23SrDNA region showed also the best diagnostic efficiency (97% sensitivity, 100% specificity). Sensitivity was significantly higher than with PCR and BLOT of the 5SrDNA region (81.5% sensitivity; P = 0.025, McNemar test); and of the dehydrofolate reductase (DHFR) gene region (75.6% sensitivity; P = 0.019). Sensitivity was also significantly higher than indirect immunofluorescence (75.8% sensitivity; P = 0.008). Using DHFR primers and probe, specificity was also reduced. The diagnostic sensitivity in clinical specimens paralleled the detection limit in the standard dilutions. The use of repeated DNA sequences of proven specificity as target of PCR amplification favourably influences sensitivity and specificity. This comparative study demonstrates that primer selection plays a significant role in the diagnosis of PCP by PCR.

AIDS-Related Opportunistic Infections↗
PubMed1995

Immunological markers indicating the effectiveness of pharmacological treatment in human hydatid disease.

The relation of interferon-gamma (IFN-gamma), IL-4, IL-10 production and specific IgE, total IgG, IgG subclass expression to the effectiveness of pharmacological treatment in human hydatid disease (Echinococcus granulosus infection) was evaluated in 27 hydatid patients divided into four clinical groups according to their response to albendazole/mebendazole therapy (full, partial, low and non-responders). After parasite antigen stimulation, peripheral blood mononuclear cells (PBMC) from full responders produced significantly more IFN-gamma (P = 0.038), significantly less IL-4 (P = 0.001) and less IL-10 than PBMC from non-responders. PBMC from partial and low responders produced intermediate cytokine concentrations. ELISA determining immunoglobulin production showed that sera from all non-responders had IgE and IgG4 antibodies, both regulated by IL-4. In contrast to IgG4, IgE decreased rapidly in full responders. Full responders also showed the highest percentage of IgG3 reactions. Qualitative analysis of total IgG responses in hydatid patients' sera determined by immunoblotting showed that binding profiles to hydatid cyst fluid antigen differed in the four groups of treated patients. Non-responders had the highest percentage of reactions to all subunits of antigens 5 and B, and full responders had the highest percentage of reactions to antigen 5 alone. The high IFN-gamma production associated with a lack of IL-4 and low IL-10 production in the full responders, and vice versa the high IL-4 and IL-10 production associated with lack of or low IFN-gamma production in the non-responders implies Th1 cell activation in protective immunity and Th2 cell activation in susceptibility to hydatid disease. IgE may be a useful marker of therapeutic success in hydatid patients with pretreatment specific IgE antibodies. IgG subclass responses and differential immunoglobulin subclass binding pattern to hydatid antigens may also be useful in the immunosurveillance of hydatid disease.

Adult↗
PubMed1995

In vitro production of cytokines by peripheral blood mononuclear cells from hydatid patients.

The role of cytokines in human hydatidosis (Echinococcus granulosus infection) was evaluated in immunoassays determining production of IL-4, IL-10 and interferon-gamma (IFN-gamma) in peripheral blood mononuclear cell (PBMC) cultures from 30 hydatid patients and 14 uninfected controls. In cell cultures from hydatid patients parasite and non-parasite antigen stimulation significantly increased IL-4 production (P < or 0.005). Spontaneous and mitogen-driven IL-4 production was similar in patients and controls. IL-10 and IFN-gamma production did not differ statistically in the two groups, even though some hydatid patients produced these cytokines in large amounts. Notably, antigen-driven IFN-gamma concentrations were invariably higher in patients than in uninfected controls. Data analysis showed a relationship between IgE and IgG4 responses and parasite-driven cytokine production. High IgE and IgG4 responders produced high IL-4 and IL-10 concentrations. High IgE responders showed decreased IFN-gamma production, but high IgG4 responders had IFN-gamma levels slightly higher than those of low responders. Cytokine response patterns did not relate to the clinical stage of disease. The significantly increased IL-4 and the high IL-10 concentrations found in PBMC from many hydatid patients in this study are consistent with Th2 cell activation in human hydatidosis. The presence of antigen-driven IFN-gamma production in patients with E. granulosus infection implies concurrent intervention of the Th1 or Th0 cell subset.

Antibodies, Helminth↗
PubMed1995

Genetic variability in different Pneumocystis isolates from man and rats.

To gain further insight into the genetic variability between different isolates of Pneumocystis carinii of rat and human origin, we sequenced three DNA regions from the mitochondrial rRNA gene, the 5S rRNA gene and the dihydrofolate reductase gene (DHFR) of microorganisms isolated from three different immunosuppressed rat sources and from one HIV + patient with P. carinii pneumonia. Some point mutations and deletions were found among rat isolates in two regions (mitochondrial rRNA gene and 5S rRNA gene). Nucleotide sequence variations in these conserved regions support the hypothesis of the existence of several Pneumocystis strains infecting the same host species.

Animals↗
PubMed1995

Use of a monoclonal antibody against the antigen B of Echinococcus granulosus for purification and detection of antigen B.

A monoclonal antibody directed against the 8 kDa subunit of antigen B of Echinococcus granulosus was raised. This antibody was used for purification of antigen B by affinity chromatography. SDS-PAGE and immunological analysis of the purified antigen B demonstrated that the 8 kDa antigen B subunit was purified to homogeneity. The purified antigen retained its strong immunoreactivity in ELISA using human hydatid sera. Furthermore, a sandwich ELISA was established for detection of antigen B from hydatid cysts. The usefulness of this test system was demonstrated by the detection of antigen B in human hydatid cyst fluids, thus confirming the diagnosis of an echinococcal disease.

Animals↗
PubMed1994

Cellular and humoral responses to antigenic subunits of Echinococcus granulosus cyst fluid in hydatid patients.

The hydatid fluid antigenic subunits which stimulate the proliferative response of T cells in PBMC isolated by hydatid patients were identified. In the same subjects, the serum IgG antibody profiles were determined by immunoblotting to compare T cell activation and IgG production. The study was carried out on 18 patients with clinically and serologically diagnosed hydatidosis and on ten healthy blood donors. To assess the proliferative response to the different antigenic subunits, sheep hydatid fluid was separated by the SDS-PAGE and the fractions were blotted onto nitrocellulose and solubilized. The comparison between immunoblotting and PBMC proliferation assay shows that the 55 and 65 kDa subunits of antigen 5 and the 12 kDa subunit of antigen B are the most reactive subunits in the two techniques. The 16 and 20 kDa subunits of antigen B are more reactive in PBMC proliferation assay than in immunoblotting, thus assigning a specific role to antigen B in cellular response.

Animals↗