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Biomedical subjects

W Bohne

Publications and source records attributed to W Bohne.

At least 19 recordsLinked to original sources
PubMed2003

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture.

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.

Animals↗
PubMed2001

Transcriptional profile of Toxoplasma gondii-infected human fibroblasts as revealed by gene-array hybridization.

To investigate the host-cell response to infection with the obligate intracellular pathogen Toxoplasma gondii, the transcriptional profiles of infected and uninfected human fibroblasts (HFF) were determined by hybridization to gene arrays representing nearly 600 genes. Transcripts that displayed a greater than five-fold increase in level relative to uninfected controls were also examined by RT-PCR and Northern analysis, resulting in the identification of 13 genes that were strongly up-regulated after infection with T. gondii. Comparisons with the transcriptional profiles of fibroblasts infected with Salmonella typhimurium and Chlamydia trachomatis allowed the identification of genes which are specifically induced in T. gondii-infected cells. While most of the up-regulated genes were induced on infection with all three pathogens, the genes for the transferrin receptor and MacMARCKS were up-regulated in Toxoplasma-infected fibroblasts only. Expression of the transferrin receptor protein was examined by Western analysis and found to be specifically elevated in Toxoplasma-infected fibroblasts. Genes which are specifically induced in T. gondii-infected cells are particularly interesting for further studies, since they might be used to dissect specific interactions of this pathogenic parasite with its host cell.

Animals↗
PubMed2001

Toxoplasmosis: a persisting challenge.

The sixth biennial International Congress on Toxoplasmosis, organized by Uwe Gross (University of Göttingen, Germany), was held on 21-25 May 2001 in Freising, Germany. The first meeting of this kind in 1990 was attended by only 26 investigators and this year there were 115 participants covering various research topics including the immunology, epidemiology, cellular and molecular biology of Toxoplasma gondii.

Animals↗
PubMed2000

Developmental expression of a tandemly repeated, glycine- and serine-rich spore wall protein in the microsporidian pathogen Encephalitozoon cuniculi.

Microsporidia are intracellular organisms of increasing importance as opportunistic pathogens in immunocompromised patients. Host cells are infected by the extrusion and injection of polar tubes located within spores. The spore is surrounded by a rigid spore wall which, in addition to providing mechanical resistance, might be involved in host cell recognition and initiation of the infection process. A 51-kDa outer spore wall protein was identified in Encephalitozoon cuniculi with the aid of a monoclonal antibody, and the corresponding gene, SWP1, was cloned by immunoscreening of a cDNA expression library. The cDNA encodes a protein of 450 amino acids which displays no significant similarities to known proteins in databases. The carboxy-terminal region consists of five tandemly arranged glycine- and serine-rich repetitive elements. SWP1 is a single-copy gene that is also present in the genomes of Encephalitozoon intestinalis and Encephalitozoon hellem as demonstrated by Southern analysis. Indirect immunofluorescence and immunoelectron microscopy revealed that SWP1 is differentially expressed during the infection cycle. The protein is absent in replicative meronts until 24 h postinfection, and its expression is first induced in early sporonts at a time when organisms translocate from the periphery to the center of the parasitophorous vacuole. Expression of SWP1 appears to be regulated at the mRNA level, as was shown by reverse transcriptase PCR analysis. Further identification and characterization of stage-specific genes might help to unravel the complex intracellular differentiation process of microsporidia.

Amino Acid Sequence↗
PubMed1999

Stage differentiation of the protozoan parasite Toxoplasma gondii.

The obligate intracellular parasite Toxoplasma gondii is able to persist lifelong in its hosts by differentiating from the replicative tachyzoite stage into cyst forming latent bradyzoites. Beside the clinical relevance of stage conversion and its importance for pathogenesis and prevention of toxoplasmic encephalitis, reversible stage differentiation in T. gondii is an interesting model system of protozoan differentiation in general. In recent years a variety of molecular techniques have been developed for T. gondii, including transfection systems and the development of many selectable markers. Together with tissue culture models in which stage differentiation from tachyzoites to bradyzoites can be induced these techniques provide the tools for a molecular dissection of the differentiation pathways. Three aspects of stage conversion are highlighted in this review, including the alteration of the parasite surface, alterations in parasite metabolism and the induction of genes associated with stress response.

Animals↗
PubMed1998

Targeted disruption of the bradyzoite-specific gene BAG1 does not prevent tissue cyst formation in Toxoplasma gondii.

Expression of the 30 kDa small heat shock protein BAG1 is restricted to the latent bradyzoite 'tissue cyst' form of Toxoplasma gondii, first appearing approximately 2-3 days after the initiation of bradyzoite differentiation. Although developmental expression of small heat shock proteins has been described for many species, their precise function is unclear. In order to examine the function of BAG1 in T. gondii bradyzoites and its role during parasite differentiation, we have used homologous recombination to produce a knock-out mutant in the cyst-forming strain P(LK), a clonal derivative of ME49. Under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), the mutant was found to express several bradyzoite-specific markers with the same kinetics and frequency as the parental strain. Neither enhanced nor decreased susceptibility to stress was observed for the BAG1-deficient mutant. In vivo studies revealed that tachyzoites of the bag1 knock-out mutant were fully able to establish a chronic infection in C57BL/6 mice, producing brain cysts of a size, morphology and frequency indistinguishable from cysts formed by the parental control strain. Brain cysts of the bag1 knock-out mutant contained viable parasites capable of establishing an acute infection after oral administration, demonstrating that conversion of bradyzoites to tachyzoites is also unimpaired. We conclude that BAG1 is not essential for normal function of bradyzoite containing tissue cysts, at least in intermediate host species. This clone of P(LK) was found to be unable to produce oocysts and is therefore unsuitable for studies in cats.

Amino Acid Sequence↗
PubMed1998

Ultrastructural organization and microanalysis studies of deciduous enamel crystallites in regional odontodysplasia (RO).

The ultrastructural organization and chemical components of enamel crystallites in two deciduous teeth affected by RO, an uncommon developmental condition giving the tooth a ghost-like appearance, were investigated by scanning and transmission electron microscopy, X-ray diffraction, electron probe microanalysis and Fourier transform improved microspectroscopy. Pathologic mineralizations involving prismatic structures, which affected the size, shape and stoichiometric structure of crystallites and led to enhanced Mg/Ca and Na/Ca ratios and crystal defects, were observed. Local circulatory disorders may have caused this developmental anomaly.

Child, Preschool↗
PubMed1998

Toxoplasma gondii bradyzoites form spontaneously during sporozoite-initiated development.

Tachyzoites (VEG strain) that emerge from host cells infected with Toxoplasma gondii sporozoites proliferate relatively fast and double their number every 6 h. This rate of growth is intrinsic, as neither the number of host cells invaded nor host cell type appears to influence emergent tachyzoite replication. Fast tachyzoite growth was not persistent, and following approximately 20 divisions, the population uniformly shifted to slower growth. Parasites 10 days post-sporozoite infection doubled only once every 15 h and, unlike emergent tachyzoites, they grew at this slower rate over several months of continuous cell culture. The spontaneous change in tachyzoite growth rate preceded the expression of the bradyzoite-specific marker, BAG1. Within 24 h of the growth shift, 2% of the population expressed BAG1, and by 15 days post-sporozoite infection, 50% of the parasites were positive for this marker. Spontaneous BAG1 expression was not observed in sporozoites or in tachyzoites during fast growth (through day 6 post-sporozoite inoculation), although these tachyzoites could be induced to express BAG1 earlier by culturing sporozoite-infected cells at pH 8.3. However, alkaline treatment also reduced the replication of emergent tachyzoites to the rate of growth-shifted parasites, supporting a link between reduced parasite growth and bradyzoite differentiation. The shift to slower growth was closely correlated with virulence in mice, as the initially fast-growing emergent tachyzoites were avirulent (100% lethal dose, >10(4) parasites), while a mutant VEG strain (MS-J) that is unable to growth shift caused 100% mortality in mice inoculated with 10 parasites. Parasites recovered from gamma interferon knockout mice inoculated with emergent tachyzoites grew at a slow rate and expressed BAG1, confirming that the replication switch occurs in animals and in the absence of a protective immune response.

Animals↗
PubMed1998

Toxoplasma gondii inhibits the in vitro induced apoptosis of HL-60 cells.

The integrity of the host cell may represent an important prerequisite for the intracellular survival and development of obligate intracellular pathogens. In the present study, we investigated the influence of infections with the protozoan parasite Toxoplasma gondii on the rate of apoptosis in the human leukemia cell line HL-60. After infection with T. gondii tachyzoites of the strain NTE and in uninfected controls, less than 2% of the host cells showed typical signs of apoptosis, i.e. condensation of chromatin after staining with Hoechst 33258 or internucleosomal DNA fragmentation after agarose gel electrophoresis of genomic DNA. After treatment with 0.1 to 0.5 microg/ml actinomycin D for up to 16 hours, HL-60 cells considerably underwent apoptosis. However, this actinomycin D-induced apoptosis was clearly reduced after concomitant infection with T. gondii as shown by staining with Hoechst 33258 and by DNA fragmentation assay. Inhibition of apoptosis by the intracellular pathogen T. gondii might be recognized as an evasion mechanism that enables intracellular survival and establishes long-lasting persistence.

Animals↗
PubMed1997

Tagging genes and trapping promoters in Toxoplasma gondii by insertional mutagenesis.

Plasmid vectors that incorporate sequence elements from the dehydrofolate reductase-thymidylate synthase (DHFR-TS) locus of Toxoplasma gondii integrate into the parasite genome with remarkably high frequency (>1% of transfected parasites). These vectors may-but need not-include mutant DHFR-TS alleles that confer pyrimethamine resistance to transgenic parasites. Large genomic constructs integrate at the endogenous locus by homologous recombination, but cDNA-derived sequences lacking long stretches of contiguous genomic DNA (due to intron excision) typically integrate into chromosomal DNA by nonhomologous recombination. Nonhomologous integration occurs effectively at random; and coupled with the high frequency of transformation, this allows a large fraction of the parasite genome to be tagged in a single electroporation cuvette. Genomic tagging permits insertional mutagenesis studies conceptually analogous to transposon mutagenesis in bacteria, yeast, Drosophila, etc. In theory (and, thus far, in practice), this allows identification of any gene whose inactivation is not lethal to the haploid tachyzoite form of T. gondii and for which a suitable selection or screen is available. Transformation vectors can be engineered to facilitate rescue of the tagged locus and to include a variety of reporters or selectable markers. Genetic strategies are also possible, using reporters whose function can be assayed by metabolic, visual, or immunological screens to "trap" genes that are activated (or inactivated) under various conditions of interest.

Animals↗
PubMed1997

Bradyzoite-specific gene expression in Toxoplasma gondii requires minimal genomic elements.

BAG1 is a small heat-shock protein of Toxoplasma gondii that is specifically expressed in the cyst-forming bradyzoite stage of the parasite. Upregulation of BAG1 mRNA occurs early during the differentiation pathway from tachyzoites to bradyzoites. In order define genomic elements involved in bradyzoite-specific gene regulation, chloramphenicol acetyltransferase (CAT)-reporter gene studies were performed with 5' flanking sequences of the BAG1 gene. Tachyzoites, transiently transfected with the BAG1/cat construct, exhibited very low CAT activity (200 fold less than in parasites transfected with a tubulin promoter/cat construct). After induction of bradyzoite differentiation by alkaline pH shift, however, CAT activity increased 50 fold, demonstrating bradyzoite-specific expression of the CAT reporter gene under control of 5' flanking sequences of BAG1. Stage-specific regulation of BAG1/CAT was independent of the 3'-flanking region, since constructs containing 3'-flanking sequences of the tachyzoite-specific SAG1 gene showed identical regulation to those containing the BAG1 3'-flanking region. The kinetics of BAG1/CAT induction in stably transfected parasites is similar to the kinetics of endogenous BAG1 expression: increased CAT activity was first detected on day 3 after alkaline pH shift (20 fold) and was dramatically upregulated 250 fold on day 4. A series of deletions in the BAG1 5'-flanking sequences demonstrated that a 324 nucleotide (nt) fragment, starting 60 nt upstream of the BAG1 transcription start, is sufficient to confer stage-specific regulation on the CAT reporter. These deletion analyses demonstrate that bradyzoite-specific expression of a heterologeous reporter gene requires only minimal genomic sequences.

Animals↗
PubMed1997

Stage-specific expression of a selectable marker in Toxoplasma gondii permits selective inhibition of either tachyzoites or bradyzoites.

The establishment of culture conditions suitable for inducing differentiation of Toxoplasma gondii tachyzoites into parasites resembling the latent bradyzoite form has opened this important developmental transition to experimental analysis. In order to develop a genetic marker suitable for positive and negative selection during parasite differentiation. the T. gondii HXGPRT gene was placed under control of 5' flanking sequences derived from two bradyzoite-specific genes: BAG1 and LDH2. Random transgene integration at undefined genomic loci resulted in modest regulation (approximately 5-6-fold induction) above relatively high background levels (approximately 4% of wild-type controls). Integration of transgenes at a defined genomic position was achieved by targeting the uracil phosphoribosyl transferase (UPRT) locus using flanking homologous sequences and fluorouracil selection. This strategy was found to provide the added advantage of enhancing bradyzoite induction frequencies under conditions of pyrimidine starvation (low CO2). Constructs integrated in the direction of normal UPRT transcription exhibited moderate levels of inducibility, but transgenes integrated in the opposite direction were dramatically induced under differentiation conditions: 50-100-fold above the very low levels observed in tachyzoites (< 1% control). Positive selection (using mycophenolic acid) was shown to inhibit tachyzoites but not bradyzoites, while negative selection (using 8-azahypoxanthine) inhibited bradyzoites only. Stage-specific regulation of the HXGPRT selectable marker should permit genetic selections for the identification of mutants in the bradyzoite differentiation process.

Animals↗
PubMed1997

Macrodystrophia lipomatosa of the foot: a report of three cases and literature review.

Macrodystrophia lipomatosa is a distinct clinical entity often misdiagnosed as other forms of macrodactyly. The most specific finding is an overabundance of fibrofatty tissue on the plantar aspect of the foot. Three cases, with the diagnoses made from tissue specimens, are presented in this article. The clinical, pathologic, and roentgenographic findings are discussed and a review of the literature is provided.

Adult↗
PubMed1997

Reactivation of chronic toxoplasmosis: is there a link to strain-specific differences in the parasite?

The protozoan parasite Toxoplasma gondii comprises three clonal lineages that are associated with the clinical outcome in infected individuals. Whereas group C strains are mainly found in animals, group A and B strains are associated with human disease (Howe and Sibley, 1995). An increased level of transcripts of the tachyzoite-specifically expressed gene SAG1 could be identified in group A T. gondii strains compared to group B strains. Since SAG1-mediated host-cell invasion seems to be important for parasite replication, the observed higher replication rate in group A T. gondii strains might explain the association with clinically overt symptoms at the acute stage in patients who are infected with this group of parasite strains. The presence of external stress factors, such as interferon-gamma (IFN-gamma)-mediated nitric oxide (NO) formation has been identified to stabilize the cyst stage, most likely by activation of promoter(s) which drive the expression of genes encoding bradyzoite-specific antigens. Reactivation of chronic toxoplasmosis thus might occur in the absence of external stress factors, as has been observed in AIDS patients with decreases levels of IFN-gamma. Since group B T. gondii strains might form more cysts in infected individuals due to an increased potential to convert into bradyzoites, reactivation with resulting toxoplasmic encephalitis could be a more common event in those AIDS patients who were infected with persistent cysts of this group of parasite strains.

Animals↗
PubMed1996

Developmental differentiation between tachyzoites and bradyzoites of Toxoplasma gondii.

An important event in the pathogenesis of toxoplasmosis is the interconversion between the bradyzoite and the tachyzoite stage of Toxoplasma gondii within the intermediate host. The factors that influence either cyst formation (bradyzoites) or reactivation (tachyzoites) are unknown. Uwe Gross, Wolfgang Bohne, Martine Soête and Jean François Dubremetz here describe current knowledge about the mechanisms that might lead to the induction of stage differentiation of this protozoan parasite.

Journal Article↗