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Biomedical subjects

W Bohne

Publications and source records attributed to W Bohne.

At least 37 records · Page 2Linked to original sources
PubMed1995

Cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1) of Toxoplasma gondii, related to genes encoding small heat-shock proteins of plants.

Stage conversion between the tachyzoite and bradyzoite forms of the protozoan parasite Toxoplasma gondii is an important aspect in the pathogenesis of toxoplasmosis. In an initial investigation of molecular regulation of stage conversion in T. gondii, we describe the cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1). Bradyzoite formation was induced in cell culture by alkaline pH, and this was followed by purification of this parasitic stage using magnetic cell sorting. A bradyzoite cDNA library was constructed by random amplification using the polymerase chain reaction. Screening with a bradyzoite-specific monoclonal antibody identified a reactive clone. The amino acid sequence derived from the 687 bp open reading frame showed similarity to the conserved C-terminal region of small heat-shock proteins from plants. Stage-specific expression of the naturally occurring 30 kDa antigen in bradyzoites was confirmed by polyclonal antisera generated against the recombinant antigen. Immunoelectron microscopy indicated a cytosolic location of this antigen in bradyzoites. The expression of HSP30/BAG1 seems to be regulated at the mRNA level, since reverse polymerase chain reaction using bradyzoite-specific primers amplified transcripts in bradyzoites only, not in tachyzoites.

Amino Acid Sequence↗
PubMed1995

Monoclonal rat antibodies directed against Toxoplasma gondii suitable for studying tachyzoite-bradyzoite interconversion in vivo.

We previously reported the in vitro analysis of stage differentiation of Toxoplasma gondii in murine bone marrow-derived macrophages. The purpose of this study was to generate monoclonal rat antibodies that might be suitable for investigating tachyzoite-bradyzoite interconversion in vivo with the murine model. Immunization of Fischer rats with cysts of T. gondii NTE resulted in the generation of seven monoclonal antibodies of the immunoglobulin G2a, G2b, or M isotype, which were further characterized by the immunoblot technique, immunofluorescence assay, immunohistology, and immunoelectron microscopy. Immunoblots demonstrated specific reactivity of five monoclonal antibodies with proteins with molecular masses of 40, 52, 55, 60, 64, 65, and 115 kDa. One antibody (CC2) appeared to recognize a differently expressed antigen depending on the parasite stage, reacting with a 40-kDa molecule in tachyzoites and a 115-kDa antigen in bradyzoites and oocysts. Several other monoclonal antibodies were shown to be stage specific and to react in immunofluorescence assays or in immunoblots with either tachyzoites or bradyzoites. Kinetics of stage conversion in vitro could be monitored by immunofluorescence with two of these monoclonal antibodies. Preliminary immunohistological investigations of tissue sections from infected mice demonstrated the possible usefulness of these monoclonal antibodies for future in vivo studies on stage differentiation of T. gondii in the murine system.

Animals↗
PubMed1994

Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: a possible role for nitric oxide in triggering stage conversion.

Stage conversion between tachyzoites and bradyzoites of Toxoplasma gondii was investigated in vitro by using murine bone marrow-derived macrophages (BMMs) as host cells. Following infection of untreated BMMs with tachyzoites, spontaneous expression of bradyzoite-specific antigens (Bsa) occurred at low frequency with Toxoplasma strain-dependent ratios from 0.03 to 2%. As previously described for peritoneal macrophages, activation of tachyzoite-infected BMMs with gamma interferon (IFN-gamma) or lipopolysaccharide resulted in the induction of Bsa. When bradyzoites were used for infection, prolonged expression of Bsa could be observed in IFN-gamma-activated BMMs. The induction of Bsa expression seemed to be closely linked to parasite multiplication and increased to maximal values of 50 to 70% in intermediately activated macrophages with nitric oxide (NO) levels that allowed reduced parasite replication. Identical results in stage conversion were obtained when sodium nitroprusside was used as a source of exogenous NO, indicating that NO might be a molecular trigger of stage conversion. NO is reactive with iron-sulfur centers in proteins, thereby inhibiting proteins involved in the mitochondrial respiratory chain. Using oligomycin and antimycin A as inhibitors of mitochondrial function, growth inhibition of parasites and induction of Bsa were obtained. Since microglia are the functional correlates of macrophages in the central nervous system and inhibit T. gondii upon activation with IFN-gamma, a similar mechanism might be involved during cyst development in the brain.

Animals↗
PubMed1993

Coexistence of heterogeneous populations of Toxoplasma gondii parasites within parasitophorous vacuoles of murine macrophages as revealed by a bradyzoite-specific monoclonal antibody.

The expression of bradyzoite-specific antigens (Bsa) of Toxoplasma gondii was studied in murine bone-marrow-derived macrophages that had previously been infected with tachyzoites. Growth conditions that allowed only restricted replication of Toxoplasma gondii resulted in heterogeneous populations; (1) Bsa-positive and Bsa-negative parasites could be observed within one parasitophorous vacuole (heterogeneous vacuole community), and (2) homogeneous Bsa-positive and homogeneous Bsa-negative vacuole communities coexisted within one macrophage host cell. These observations suggest that stage conversion does not seem to be a synchronous event for a vacuole community.

Animals↗
PubMed1993

Comparison of a commercial enzyme immunoassay and an immunoblot technique for detection of immunoglobulin A antibodies to Toxoplasma gondii.

A commercial enzyme immunoassay (Platelia-Toxo IgA) and an immunoblot technique were compared with regard to their ability to detect IgA antibodies to the major surface protein P30(SAG1) of Toxoplasma gondii in 105 serum samples from patients with suspected or proven acquired toxoplasmosis. Comparison of the IgA-EIA with the immunoblot technique showed a concordance of 81.0%, with a sensitivity of 92.6% and a specificity of 78.4%. Due to its high sensitivity the IgA-EIA might detect IgA antibodies against Toxoplasma gondii at an early stage of infection, although excessive sensitivity could lead to detection of IgA antibodies for an extended period of time following the onset of infection.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Induction of bradyzoite-specific Toxoplasma gondii antigens in gamma interferon-treated mouse macrophages.

By using stage-specific monoclonal antibodies, an in vitro model has been developed to analyze the kinetics of expression of stage-specific antigens during the conversion process between tachyzoites and bradyzoites of Toxoplasma gondii. Following infection of murine macrophages with bradyzoites, the expression of bradyzoite-specific antigens declined, whereas the expression of tachyzoite-specific antigens increased during the first 72 h postinfection. Conversely, in gamma interferon-treated murine macrophages infected with tachyzoites, the inhibitory effect of gamma interferon on replication of parasites was accompanied by the induction of bradyzoite-specific antigens.

Animals↗
PubMed1993

Differentiation between mouse-virulent and -avirulent strains of Toxoplasma gondii by a monoclonal antibody recognizing a 27-kilodalton antigen.

Using a murine monoclonal antibody, we were able to differentiate between mouse-virulent and -avirulent strains of Toxoplasma gondii. Monoclonal antibody TB6G5 was reactive with eight clinical mouse-avirulent isolates but not with mouse-virulent laboratory strains RH and BK. The antibody-reactive antigen was identified by indirect immunofluorescence and immunoblot as a 27-kDa cytoplasmic protein expressed by tachyzoites as well as by bradyzoites.

Animals↗
PubMed1993

High residual arylsulfatase A (ARSA) activity in a patient with late-infantile metachromatic leukodystrophy.

We identified a patient suffering from late-infantile metachromatic leukodystrophy (MLD) who has a residual arylsulfatase A (ARSA) activity of about 10%. Fibroblasts of the patient show significant sulfatide degradation activity exceeding that of adult MLD patients. Analysis of the ARSA gene in this patient revealed heterozygosity for two new mutant alleles: in one allele, deletion of C 447 in exon 2 leads to a frameshift and to a premature stop codon at amino acid position 105; in the second allele, a G-->A transition in exon 5 causes a Gly309-->Ser substitution. Transient expression of the mutant Ser309-ARSA resulted in only 13% enzyme activity of that observed in cells expressing normal ARSA. The mutant ARSA is correctly targeted to the lysosomes but is unstable. These findings are in contrast to previous results showing that the late-infantile type of MLD is always associated with the complete absence of ARSA activity. The expression of the mutant ARSA protein may be influenced by particular features of oligodendrocytes, such that the level of mutant enzyme is lower in these cells than in others.

Alleles↗
PubMed1992

[New views on the pathogenesis and diagnosis of toxoplasmosis].

T. gondii is one of the most occurring human pathogenic parasites in Europe. While the majority of immunocompetent individuals with T. gondii infection do not present clinical symptoms, congenital toxoplasmosis and reactivation of a latent infection in immunocompromised patients (i. e. patients with AIDS) are of high clinical relevance. A classification of T. gondii isolates is not available so far, although it was possible to demonstrate strain differences by the use of several methods, i. e. by using monoclonal antibodies. T. gondii seems to be able to infect any mammalian cell. Host cell-derived as well as parasite-derived factors seem to be important for the contact between host cell and parasite and the subsequent internalization. Following invasion, T. gondii is located within a parasitophorous vacuole that does not fuse with lysosomes. The multiplication rate of these obligately intracellular growing parasites decreases during conversion from the tachyzoite stage to the bradyzoite stage. Finally, the bradyzoites-harbouring cysts persist for the lifetime of the host. Reconversion from bradyzoites to tachyzoites may occur in immunocompromised patients. Probably, IFN-gamma is involved in this process. In addition to serological methods, direct detection of T. gondii using PCR or demonstration of circulating antigens might be routinely used as diagnostical tools in the future. Determination of specific IgA antibodies, which can be evaluated using the immunoblot technique, seem to be important for early serological diagnosis. The use of recombinant antigens might be helpful in future diagnosis to circumvent discrepancies between serological test results which could have resulted from strain-specific differences.

Animals↗
PubMed1991

An 11-bp deletion in the arylsulfatase A gene of a patient with late infantile metachromatic leukodystrophy.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. Examination of the arylsulfatase A gene in a patient suffering from late infantile metachromatic leukodystrophy revealed an 11-bp deletion in exon 8. Although this allele produces normal amounts of ASA mRNA, no arylsulfatase A cross-reacting material could be detected in cultured fibroblasts from the patient. The patient was found to be a compound heterozygote, the other allele is also known to generate no ASA polypeptides. This patient is another example where absence of ASA polypeptides correlates with the severe late infantile form of metachromatic leukodystrophy.

Amino Acid Sequence↗
PubMed1991

Two new arylsulfatase A (ARSA) mutations in a juvenile metachromatic leukodystrophy (MLD) patient.

Fragments of the arylsulfatase A (ARSA) gene from a patient with juvenile-onset metachromatic leukodystrophy (MLD) were amplified by PCR and ligated into MP13 cloning vectors. Clones hybridizing with cDNA for human ARSA were selected, examined for appropriate size inserts, and used to prepare single-stranded phage DNA. Examination of the entire coding and most of the intronic sequence revealed two putative disease-related mutations. One, a point mutation in exon 3, resulted in the substitution of isoleucine by serine. Introduction of this alteration into the normal ARSA cDNA sequence resulted in a substantial decrease in ARSA activity on transient expression in cultured baby hamster kidney cells. About 5% of the control expression was observed, suggesting a small residual activity in the mutated ARSA. The second mutation, a G-to-A transition, occurred in the other allele and resulted in an altered splice-recognition sequence between exon 7 and the following intron. The mutation also resulted in the loss of a restriction site. Apparently normal levels of mRNA were generated from this allele, but no ARSA activity or immuno-cross-reactive material could be detected. A collection of DNA samples from known or suspected MLD patients, members of their families, and normal controls was screened for these mutations. Four additional individuals carrying each of the mutations were found among the nearly 100 MLD patients in the sample. Gene segregation in the original patient's family was consistent with available clinical and biochemical data. No individuals homozygous for either of these two mutations were identified. However, combinations with other MLD mutations suggest that the point mutation in exon 3 does result in some residual enzyme activity and is associated with late-onset forms of the disease. The splice-site mutation following exon 7 produces late-infantile MLD when combined with other enzyme-null mutations, implying that it is completely silent enzymatically.

Adolescent↗
PubMed1990

Light and ultrastructural studies of human chronic periapical lesions.

Forty clinically chronic periapical granulomas and cysts and their corresponding teeth were removed. Diagnosis was by light and transmission electron microscopy. Ultrastructural features of the root surfaces concerned and the corresponding soft tissue were detected by scanning electron microscopy. Chronic periapical inflammation had caused root resorption which affected the cementum and/or the dentin. There were no ultrastructural differences between granuloma- and cyst-induced root resorption. Resorption lacunae were devoid of epithelial and connective tissue attachment. However, root surface exposed to granulomas and cysts also indicated spontaneous cementum repair. Moreover, at their periphery, cellular debris, cells defined as fibroblasts, and cell projections and fibrils could be seen which were continuations of the periodontal ligament.

Adolescent↗
PubMed1989

[Epidemiologic investigations on caries prevalence among schoolchildren in Brittany (France)].

1421 randomly selected 6- to 15-year-old Breton children were examined in 1987 in order to assess the prevalence of caries in permanent teeth. Methods proposed by Turlot & Cahen (1989) were used. At 6, 85.9% of all children were caries-free and at 15, only 5.3%. At 14, DMFT and DMFS rates reached 6.46 and 12.36, respectively. The differences of scores stated between boys and girls and between children from urban and rural regions were not statistically significant. Pits and fissures were more frequently affected than approximal and smooth surfaces. Since 1986, fluoridated domestic salt is available in France. Thus, the present investigation will serve to study the effects of this recently adopted preventive measure.

Adolescent↗
PubMed1989

[Structural, ultrastructural and microanalytical study of multiple enamel pearls].

Numerous enamel drops and compound enamel pearls were found on the radicular proximal faces of maxillary molars and mandibular third molars of a young woman. Enamel and dentin of compound pearls as well as cementum next to drops and pearls presented the same structure and ultrastructure as enamel, dentin and cementum of the corresponding teeth. Microanalysis did not reveal differences between enamel of the mother tooth and enamel of drops and pearls. The enamel drops had no incremental growth lines. Cementum next to enamel drops and compound enamel pearls was acellular and covered occasionally with a thick layer of cellular cementum. Only enamel drops were partially covered by acellular cementum. Close to the enamel drops and at their surface, numerous fusing globular calcifications were observed. Formation of enamel drops and compound enamel pearls on dental root surfaces is rare. The simultaneous presence of numerous enamel drops and some compound enamel pearls on several roots of molars in the same denture seems to be an exceptional phenomenon. The involved factors inducing enamel formation remain still unknown. The multitude of both enamel drops and compound enamel pearl might be due to constitutional predisposition.

Adult↗
PubMed1988

[The oro-dental status of children 6-15-years of age in Pays de la Loire, Vienne and Deux Sèvres].

This survey was conducted on a representative sample of schoolchildren aged 6 to 15 years from Pays de la Loire, Vienne and Deux-Sèvres. The results showed that the DMFT and DMFS indices increased with age. The girls had a higher caries experience. The children from rural zones had a higher caries prevalence than those of urban areas. However the differences were not statistically significant. Relatively low values were recorded between 6 and 15 years for the plaque index, the calculus index and the gingival index.

Adolescent↗