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R Cevenini

Publications and source records attributed to R Cevenini.

At least 55 records · Page 3Linked to original sources
PubMed1993

Expression of a plasmid gene of Chlamydia trachomatis encoding a novel 28 kDa antigen.

Plasmid pCT is present in essentially all isolates of Chlamydia trachomatis and may encode factors important for survival in the natural environment. However, no pCT-associated phenotype has been described so far. With the purpose of investigating the possibility of a role of pCT in C. trachomatis pathogenicity we examined the expression of an ORF (ORF3), potentially encoding a 28 kDa polypeptide (pgp3). Analysis of RNA extracted from chlamydia-infected Vero cells detected ORF3-specific transcripts, from 20 h post-infection onwards, mainly as discrete RNA species of 1390 nucleotides comprising the downstream ORF4 sequence. ORF3 DNA was cloned and expressed in Escherichia coli as a 39 kDa fusion protein (MS2/pgp3). Antibodies raised against purified MS2/pgp3, specifically recognized a 28 kDa protein on Western blots of protein from purified chlamydial elementary bodies (EBs). The same antibodies detected chlamydial inclusions in methanol-fixed infected cells by immunofluorescence. Western blot analysis of EBs extracted with 2% Sarkosyl, showed that a large proportion of the 28 kDa antigen is associated with the detergent-insoluble ('membrane') fraction. Antibodies recognizing pgp3 epitopes were detected in sera from patients with chlamydial infections, but not in sero-negative control sera. The finding support the hypothesis that pCT may provide a function related to chlamydial cell physiology.

Amino Acid Sequence↗
PubMed1993

Animal and human antibodies reactive with the outer surface protein A and B of Borrelia burgdorferi are borreliacidal, in vitro, in the presence of complement.

Polyspecific antibodies present in ascitic fluids of mice (pMIAFs) immunized with whole Borrelia burgdorferi cells exerted borreliacidal activity in vitro when tested with complement and homologous antigen but not with heterologous B. hermsii. Similarly, monospecific mouse antibodies obtained by immunizing mice with purified preparations of outer surface protein A and B of B. burgdorferi were borreliacidal. On the contrary, mouse monospecific antibodies raised against the 41-kDa flagellar protein of B. burgdorferi did not kill borreliae in the presence of complement. A complement-mediated, in vitro, borreliacidal activity was observed in human sera from patients with Lyme disease when antibodies against OspA and/or OspB were detectable in sera by the Western blotting technique. The in vitro borreliacidal activity of human sera was evident after 14 h incubation with live B. burgdorferi spirochaetes and complement, whereas antibodies present in mouse immune ascitic fluids killed borreliae after 1 h incubation.

Animals↗
PubMed1993

Acylated proteins in Borrelia hermsii, Borrelia parkeri, Borrelia anserina, and Borrelia coriaceae.

Borrelia hermsii, Borrelia parkeri, Borrelia anserina, and Borrelia coriaceae produced several lipoproteins identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of bacteria grown in [3H]palmitate. Five major acylated proteins were demonstrated by sequential alkaline and acid hydrolysis. High-pressure liquid chromatography of isolated proteins confirmed that covalently bound radioactivity was represented by fatty acids.

Acylation↗
PubMed1993

Adherence of Borrelia burgdorferi and Borrelia hermsii to mammalian cells in vitro.

This study investigated the ability of Borrelia burgdorferi and Borrelia hermsii to attach the surface of several types of in vitro-cultured mammalian cells. Borreliae showed different adhesion efficiencies depending on cell type and temperature. Temperatures both lower and higher than 33 degrees and 37 degrees C respectively, decreased the adhesion of borreliae which preferentially adhere to human fibroblast-like cells. The adhesion process, mediated by structures exposed onto the surface of the microorganisms, also proved to be sensitive to the treatment of mammalian cells with hyaluronidase and sialidase, confirming that carbohydrate receptors are involved in the adhesion of borreliae to eukaryotic cells.

Animals↗
PubMed1992

Complement-mediated in vitro bactericidal activity of monoclonal antibodies reactive with outer-surface-protein OspB of Borrelia burgdorferi.

Murine monoclonal antibodies (mAbs) were obtained against the outer-surface-protein OspA and OspB and against the 41-kDa flagellar antigen of Borrelia burgdorferi. The specificity of mAb was determined by the Western blotting technique and the surface association of the antigens was inferred by immunofluorescence of living bacteria. In an in vitro assay in the presence of complement, two mAbs reactive with the Ospa were able to kill borreliae, whereas several mAbs reactive with the OspA as well as with the 41-kDa flagellar protein were not.

Animals↗
PubMed1992

Detection of glycoproteins in Borrelia burgdorferi.

The presence of carbohydrates on proteins of Borrelia burgdorferi, the causative agent of Lyme disease, was investigated by using a digoxigenin labeling method together with Schiff staining and N-glycosidase F assay. The two major outer surface exposed proteins of 31 kDa and 34 kDa showed to be glycosylated and gel filtration high pressure liquid chromatography (HPLC) of proteins of B. burgdorferi metabolically labeled with 14C-N-acetylglucosamine revealed the incorporation of the carbohydrate into the glycosyl residue of these proteins.

Acetylglucosamine↗
PubMed1992

Surface immunofluorescence assay for diagnosis of Lyme disease.

A surface immunofluorescence assay (SIFA) was analyzed and compared with a conventional indirect immunofluorescence assay (IFA) and whole-cell enzyme-linked immunosorbent assay (ELISA) for detecting immunoglobulin G (IgG) antibodies to Borrelia burgdorferi in sera from patients with Lyme disease. Fifty-five patients with syphilis and 33 patients with rheumatoid arthritis were used as disease controls. The sensitivity of the SIFA was low during the acute phase of Lyme disease (sera from seven of nine patients presenting with erythema chronicum migrans were negative during the first 2 months of illness); later, seroconversion was observed in all patients at various times during convalescence. Sera from five patients with complicated Lyme disease were strongly positive. SIFA was found to be highly specific, since sera from all patients with secondary or latent syphilis and patients with rheumatoid arthritis did not react in the test. Strong cross-reactivity occurred when these sera were tested in conventional IFA and ELISA; sera from 38 (69%) patients with syphilis were positive by IFA and sera from 51 (93%) patients were positive by ELISA, whereas 7 (21%) and 12 (36%) of the serum samples from patients with rheumatoid arthritis were positive by IFA and ELISA, respectively. Immunoblot analysis of SIFA-positive sera showed that the 31- and 34-kDa outer surface proteins (proteins A and B, respectively) of B. burgdorferi were the major reactive antigens involved in the test. The results support a role for SIFA in the investigation of complicated Lyme disease as well as in the differentiation of Lyme disease from other diseases associated with B. burgdorferi cross-reactive antibodies.

Animals↗
PubMed1992

Incorporation of cysteine by Borrelia burgdorferi and Borrelia hermsii.

The growth rate of Borrelia burgdorferi and Borrelia hermsii in BSK II medium prepared with cysteine-free or cysteine-containing (0.185-5.92 mM) CMRL 1066 medium was studied. In media with cysteine-free CMRL 1066, growth of borreliae was detectable, although it was reduced by approximately 80%. Bacterial growth was maximal when the concentration of cysteine in CMRL 1066 reached 1.48 mM, which represents the standard cysteine concentrations of the medium; higher concentrations inhibited the growth of borreliae. Cysteine incorporation, measured by the uptake of radiolabeled cysteine, showed that cysteine enters B. burgdorferi and B. hermsii cells by passive diffusion. Labeling studies of borreliae with [35S]cysteine indicated that B. burgdorferi has several cysteine-containing proteins, including ones at 22, 30 (OspA), and 34 kDa (OspB), whereas B. hermsii showed only two [35S]cysteine-incorporating proteins, at 22 and 24 kDa, which were exposed onto the outer cell surface. In addition, most of the cysteine-incorporating proteins could be biosynthetically radiolabeled when bacterial cells were grown in vitro with [3H]palmitate, and the differences in cysteine incorporation observed between B. burgdorferi and B. hermsii were found to be correlated with differences in lipoproteins.

Bacterial Proteins↗
PubMed1992

Differential cleavage of surface proteins of Borrelia burgdorferi by proteases.

The differential cleavage of surface proteins of Borrelia burgdorferi IRS strains by several proteases was examined. Proteinase K, trypsin, chymotrypsin and thermolysin all cleaved the outer surface protein B (OspB) to undetectable levels by Coomassie Brilliant Blue staining, whereas some residual protein was detected by immunoblotting with polyclonal and monoclonal antibodies. Not even antigenic fragments were detectable by immunoblotting with 1A8 monoclonal antibody reactive with OspB. Less effective or ineffective was the cleavage of OspB by V8 protease and proteinase A, respectively. The outer surface protein A was cleaved only by proteinase K. The effect of trypsin on borreliae viability and adhesion to cultured cells was also studied. The trypsin treatment of borreliae did not impair the viability of organisms which continued to synthesize the cleaved OspB. The attachment of B. burgdorferi to HEp-2 cells was reduced by 41% after treatment with trypsin, whereas preincubation of borreliae with monoclonal antibody 1A8 and guinea pig immune serum reduced the adhesion of borreliae to the cells by 32% and 87%, respectively.

Antibodies, Monoclonal↗
PubMed1991

Development of transplantable ascites tumours which continuously produce polyclonal antibodies in pristane primed BALB/c mice immunized with bacterial antigens and complete Freund's adjuvant.

Bacterial immunogens (whole cells of Borrelia burgdorferi, elementary bodies of Chlamydia trachomatis and purified proteins of 22 and 24 kDa of Borrelia hermsii) were emulsified with an excess of complete Freund's adjuvant and injected (i.p.) on days 0, 7, 14 and 21, into BALB/c mice treated with pristane on day 6. This procedure induced the development of antibody-producing ascites tumours which could be serially transplanted in pristane-conditioned mice. Ascites tumours continued to yield a consistent amount of specific polyclonal antibody after ten serial transplants. The method described appears to be particularly useful for the production of a large amount of antibody when only small amounts of immunogen are available.

Animals↗
PubMed1991

Susceptibility of iron-loaded Borrelia burgdorferi to killing by hydrogen peroxide and human polymorphonuclear leucocytes.

Borrelia burgdorferi grew more slowly in iron-depleted than in iron-sufficient media. The addition of increasing concentrations of iron stimulated borrelial growth and resulted in the intracellular accumulation of this element. Compared with iron-starved borrelia, iron-enriched organisms showed enhanced sensitivity to hydrogen peroxide. Intracellular iron-content did not, however, influence susceptibility to killing by human polymorphonuclear leucocytes [corrected].

Blood Bactericidal Activity↗
PubMed1991

Partial characterization of an 89-kDa highly immunoreactive protein from Chlamydia psittaci A/22 causing ovine abortion.

An 89-kDa immunogen from Chlamydia psittaci A/22 causing ovine abortion was partially characterized. The 89-kDa protein, localized on the outer membrane complex of chlamydiae, was synthesized relatively early in the developmental cycle. The protein contained cysteine but was not extensively cross-linked by disulfide bonds. Treatment with proteases apparently did not cleave the protein. The infectivity of strain A/22 was partially (60%) reduced by treatment of chlamydial elementary bodies with monoclonal antibody BS/89 specifically reacting with the 89-kDa antigen. Species-specific as well as strain-specific antigenic determinants were present on the 89-kDa protein.

Abortion, Veterinary↗
PubMed1991

Immunological characterization of a low molecular mass polypeptidic antigen of Borrelia burgdorferi.

The presence of a low molecular mass polypeptidic antigen in Borrelia burgdorferi was described. The protein was exposed at the bacterial surface since it was clearly identified by mAb 3H4 using the immunofluorescence test performed with living bacteria. This antigen was cleaved by proteinase K treatment, whereas it was resistant to the action of chymotrypsin, trypsin and thermolysin. Western blotting analysis of the immunological reactivity of this antigenic structure performed using monoclonal antibody, mouse-immune ascitic fluids raised against B. burgdorferi and other spirochetes, sera from patients with Lyme disease and other infirmities in which false positive results in serological tests for B. burgdorferi have been described, demonstrated that this protein expresses only species-specific epitopes which may be recognized during human B. burgdorferi infections.

Animals↗
PubMed1991

Detection of Chlamydia trachomatis DNA in patients with non-gonococcal urethritis using the polymerase chain reaction.

A practical protocol using the polymerase chain reaction (PCR) was designed for detecting Chlamydia trachomatis in clinical samples. DNA was extracted from material collected on urethral swabs and used as substrate for the PCR. The target was a 600 basepair DNA segment of the multicopy plasmid that is common to all strains of the bacterium. Negative samples were checked for loss of DNA or presence of polymerase inhibitors by a second PCR, targeted to a conserved segment of the human genome. The whole procedure was tested on 216 men with non-gonococcal urethritis (NGU). All patients were independently assessed by tissue culture isolation (60 positive samples) and a commercial immunoenzymatic assay. The PCR protocol, while sufficiently simple for routine application, was reliable and, for the diagnosis of urethritis, at least as good as tissue culture isolation.

Adolescent↗
PubMed1991

In-vitro activity of roxithromycin against Chlamydia trachomatis.

Ten Chlamydia trachomatis isolates were tested for their sensitivity to roxithromycin, in comparison with erythromycin and tetracycline. The minimum inhibitory and minimum bactericidal concentrations of roxithromycin ranged from 0.03-0.12 and 0.25-1 microg/ml, respectively.

Anti-Bacterial Agents↗
PubMed1991

Comparative ability of various detergents to extract proteins of Borrelia burgdorferi and Borrelia hermsii.

Borrelia burgdorferi and Borrelia hermsii were treated with the following detergents: sodium dodecyl sulphate (SDS), N-lauryl sarcosine (Sarkosyl) and Triton X-100, and the soluble and insoluble fractions obtained after each detergent treatment were examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Sarkosyl as well as SDS solubilized all Borrelia proteins, whereas Triton X-100 treatment selectively solubilized the majority of other borrelial proteins, leaving two proteins of 41 kDa and 66 kDa in Borrelia burgdorferi and one protein of 39 kDa in Borrelia hermsii associated with the Triton X-100 insoluble fraction.

Bacterial Proteins↗
PubMed1990

Diversity of the Chlamydia trachomatis common plasmid in biovars with different pathogenicity.

The 7.5-kb plasmid of Chlamydia trachomatis (CT) is believed to encode essential genes and might have a role in CT pathogenicity. Accordingly, analysis of plasmid-linked mutations in isolates from biovars with different pathogenic properties should help in identifying which plasmid-encoded genes, if any, may be involved in modulating virulence. For this purpose, the plasmid present in a low-virulence isolate (trachoma biovar, serotype D) was cloned and sequenced. Nucleotide changes were experimentally checked against the sequence of the plasmid variant from the highly virulent strain L2/434/Bu (LGV biovar). By aligning our data with two published sequences of different trachoma and LGV variants a general consensus structure was determined, comprising eight major open reading frames (ORF) and a number of points where there is consensus only between isolates of the same biovar (biovar-specific mutations). The degree of variation between different isolates is less than 1%. In particular, comparison of serotype-D and -L2 plasmids shows mutations which are generally silent or lead to few (one to four), often conservative, amino acid changes in ORFs 1, 2, 4, 5, 6, and 7. The protein encoded by ORF8 is completely conserved. In contrast, the polypeptide variants encoded by ORF3 show nine amino acid changes, seven of which are due to biovar-specific mutations.

Amino Acid Sequence↗