Search PubMed⌕ Search

Biomedical subjects

M Steinert

Publications and source records attributed to M Steinert.

At least 55 records · Page 3Linked to original sources
PubMed1997

Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii.

Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a non-replicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model.

Acanthamoeba↗
PubMed1997

[Amebic colitis and amebic liver abscess--epidemiology and personal case report].

The large intestine reacts relatively monomorphically to different stimuli. From this differential-diagnostic problems may result. The history of a patient is described which could be pursued clinically over 12 weeks and during the course of which the correction of the diagnosis ulcerative colitis into amoebic colitis was necessary. It is concluded that in every symptomatology of colitis bacterial and parasitologic examinations of the faeces should be performed primarily specially if there is a history of overseas travel. In these cases it must be also thought of spontaneous amoebic infections.

Abdomen, Acute↗
PubMed1996

Dissection of antigenic and irritative effects of epicutaneously applied haptens in mice. Evidence that not the antigenic component but nonspecific proinflammatory effects of haptens determine the concentration-dependent elicitation of allergic contact dermatitis.

Allergic contact dermatitis differs from most other immune reactions by its strict dose dependence during the elicitation phase. Moreover, almost all known contact allergens can also induce dose-dependent irritative dermatitis and in general only elicit allergic contact dermatitis in sensitized individuals when applied within a narrow dose range. Therefore, we hypothesized that elicitation of contact hypersensitivity (CHS) may require two signals, antigen-specific effector cell activation and a non-antigen-specific proinflammatory signal, both of which are provided by application of a sufficient dose of hapten. To dissociate these putative two signals, oxazolone-sensitized mice were ear challenged with a dose of the specific hapten which was too low to elicit CHS. At the same time, an unrelated hapten was applied in a conventional concentration to the same skin site. Whereas neither treatment alone elicited a significant CHS response, application of both compounds together resulted in a strong CHS response that was indistinguishable from that elicited by the full dose of the specific hapten. Upon coadministration of the irrelevant hapten, allergic contact dermatitis could be elicited even when the dose of the specific hapten was further reduced by a factor of 10(3). In contrast, a dose reduction of the irrelevant hapten by a factor of two resulted in the loss of the CRS response. These data indicate that non-antigen-specific effects of epicutaneously applied haptens significantly contribute to the elicitation of CHS responses and that the capacity of the hapten to evoke this proinflammatory stimulus rather than its antigenicity is responsible for the strict concentration dependence.

Administration, Cutaneous↗
PubMed1995

Removal of the majority of epidermal Langerhans cells by topical or systemic steroid application enhances the effector phase of murine contact hypersensitivity.

Whereas epidermal Langerhans cells (LC) are thought to be the principal APCs for initiation of contact hypersensitivity (CHS) responses, their role as APC in the effector phase of CHS is still unclear. It is currently thought that LC elicit the CHS response by presenting Ag to trafficking Ag-specific T cells within the skin. To test this hypothesis, we removed the majority of resident LC at the site of CHS challenge by topical application of various steroid creams to one ear in BALB/c mice (> 85% LC depletion). Either 2 days before or 4 days after steroid treatment of the ear, mice were sensitized on the abdomen with the hapten trinitrophenyl and challenged 10 days later at the steroid-pretreated ear. At that time point, direct anti-inflammatory effects of the steroid were no longer present. Surprisingly, CHS responses were markedly stronger at the sites of prior steroid application when compared with vehicle-treated controls, indicating that depletion of most of the resident LC not only fails to impair, but enhances, the expression of CHS significantly. UV irradiation or application of croton oil at the challenge site, as well as systemic steroid application, all of which are alternative methods of diminishing the number of epidermal LC, also significantly up-regulated CHS. In contrast, irritant dermatitis and sensitization or elicitation of CHS in steroid-treated mice at distant sites, as well as delayed-type hypersensitivity responses against the same hapten, were unaffected by topical steroid pretreatment. In conclusion, our data suggest that resident LC are not the relevant APC in the effector phase of CHS and that they may even provide down-regulatory, rather than stimulatory, signals during elicitation of CHS.

Administration, Topical↗
PubMed1995

The Lly protein protects Legionella pneumophila from light but does not directly influence its intracellular survival in Hartmannella vermiformis.

The lly locus (legiolysin) mediates the browning of the culture medium of Legionella pneumophila in the late stationary growth phase, presumably as a result of synthesis of homogentisic acid. Mutagenesis of the lly gene of the L. pneumophila Philadelphia I derivative JR32 did not affect intracellular replication in the natural host Hartmannella vermiformis. The Lly-negative mutant, however, showed a markedly decreased resistance to ordinary light. The cloned lly gene conferred an increased resistance to light in recombinant L. pneumophila and Escherichia coli K-12, indicating a contribution of the Lly protein to ecological adaptation of Legionella species.

Animals↗
PubMed1995

Influence of site specifically altered Mip proteins on intracellular survival of Legionella pneumophila in eukaryotic cells.

Legionella pneumophila, the causative agent of Legionnaires' disease, is able to survive intracellularly in eukaryotic cells such as monocytes, macrophages, and protozoan organisms. The Mip (macrophage infectivity potentiator) protein represents a factor of L. pneumophila necessary for optimal intracellular survival. Interestingly, Mip belongs to the substance class of FK 506-binding proteins and exhibits peptidyl-prolyl cis/trans isomerase (PPIase) activity that can be inhibited by the immunosuppressant FK506. In order to identify amino acids most likely to be involved in the enzymatic activity of Mip, site-directed mutagenized Mip proteins were constructed and characterized. It was shown that an Asp-142 to Leu-142 mutation and a Tyr-185 to Ala-185 substitution resulted in strongly reduced PPIase activity of the recombinant Mip proteins (5.3 and 0.6% of the activity of the wild-type Mip, respectively). Genes coding for the wild-type and for site-directed-mutagenized Mip proteins were used to complement three different Mip-negative mutants of the L. pneumophila Corby, Philadelphia I, and Wadsworth. While Mip protein expression could be restored in the corresponding complementants, significant Mip-specific PPIase activity could be detected only in Mip mutants complemented with wild-type mip genes. To investigate the influence of the PPIase activity of Mip on intracellular survival of L. pneumophila, invasion assays were performed using the macrophage-like cell line U937, human blood monocytes, and Acanthamoeba castellanii. The Mip-negative mutants were approximately 50- to 100-fold less infective for A. castellanii and for human mononuclear phagocytes in vitro compared with their isogenic Mip-positive parental strains. The wild-type invasion rate could be restored by introducing an intact copy of the mip gene into Mip-negative strains. In addition, no differences in intracellular survival were observed between the wild-type isolates and the Legionella strains exhibiting strongly reduced PPIase activity. These data indicated that the enzymatic activity of Mip does not contribute to intracellular survival of L. pneumophila.

Amino Acid Isomerases↗
PubMed1994

V beta gene repertoires in T cells expanded in local self-healing and lethal systemic murine cutaneous leishmaniasis.

Inbred mice infected with Leishmania major promastigotes display two different courses of leishmaniasis: resistant strains develop self-healing local sores, while susceptible strains show progressive systemic disease with lethal outcome. Resistance predominantly correlates with the production of T helper type 1 (TH1) lymphokines and susceptibility with production of TH2-type lymphokines. Here, we analyzed whether this TH phenotype difference correlates with expression of particular T cell receptor V beta chains. Our results show that T cells expand strongly during infection in all groups of mice and invariantly express the same V beta gene families as prior to infection. Our data indicate that TH1 and TH2 cells use similar V beta gene families, and argue against the engagement of a restricted set of V beta by dominant determinants associated with L. major.

Animals↗
PubMed1994

Sequence determination and mutational analysis of the lly locus of Legionella pneumophila.

The lly (legiolysin) locus codes for a 39-kDa protein which confers hemolysis, pigment production, and fluorescence on recombinant Escherichia coli K-12 clones carrying the lly gene. The nucleotide sequences of the lly genes from two Legionella pneumophila isolates were determined. The lly loci exhibited identical nucleotide sequences. They contained open reading frames of 348 amino acid residues, encoding proteins with a deduced molecular mass of 38.9 kDa. N-terminal amino acid sequencing further confirmed that the Lly protein corresponds to the open reading frame sequenced. The amino acid sequence of the Lly protein exhibits a high degree of homology with the sequences of the MelA protein responsible for melanin production in the freshwater bacterium Shewanella colwelliana and the 4-hydroxyphenylpyruvate dioxygenase of Pseudomonas spp. 4-Hydroxyphenylpyruvate dioxygenase is involved in the degradation of aromatic amino acids in various organisms. An Lly-negative mutant of L. pneumophila Philadelphia I derivative JR32 and an Lly-positive transcomplementant were constructed. The Lly-negative mutant lost the ability to produce brown pigment and to confer fluorescence but retained hemolysis. Introduction of a plasmid carrying the lly locus restored pigment production and fluorescence. Intracellular survival of L. pneumophila in U937 macrophage-like cells and in Acanthamoeba castellanii was not affected by mutagenization of the lly locus.

Amino Acid Sequence↗
PubMed1991

High homology between variant surface glycoprotein gene expression sites of Trypanosoma brucei and Trypanosoma gambiense.

The AnTat 11.17 variant surface glycoprotein (VSG) is synthesized in both metacyclic and bloodstream forms of Trypanosoma gambiense. We have characterized the AnTat 11.17 gene, and analyzed its expression site (ES) in the bloodstream form by Southern and Northern blotting with probes from the Trypanosoma brucei AnTat 1.3A VSG ES, and by run-on transcription. The AnTat 11.17 ES is located at the end of a 700-kb chromosome. It appears to contain all the genes (ESAGs, for Expression Site-Associated Genes) present in the AnTat 1.3A VSG ES, with the possible exception of ESAG 1. Limited nucleotide sequence analysis of ESAG cDNAs from the AnTat 11.17 ES shows considerable conservation with ESAGs of T. brucei. The transcription promoter of the AnTat 11.17 VSG ES, localized by virtue of the specific accumulation of promoter-proximal transcripts which occurs following UV irradiation, was found to be at the same relative position to the first ESAG (ESAG 7) as in AnTat 1.3A.

Amino Acid Sequence↗
PubMed1991

Ultraviolet irradiation inhibits RNA decay and modifies ribosomal RNA processing in Trypanosoma brucei.

A known effect of ultraviolet radiation on transcription is the arrest of RNA elongation. In Trypanosoma brucei, we show that UV also inhibits RNA decay, leading to specific accumulation of transcripts from the beginning of several transcription units. In addition, UV irradiation changes the pattern of ribosomal RNA processing, probably by altering the order in which the non-coding spacers are excised. These effects are still observed on RNA synthesized more than 2 h after irradiation, and do not depend on protein synthesis.

Animals↗
PubMed1990

Trypanosoma brucei: constitutive activity of the VSG and procyclin gene promoters.

The variant surface glycoprotein (VSG) and procyclin are the major surface proteins of the bloodstream and procyclic stages, respectively, of Trypanosoma brucei. The promoter regions of the VSG and procyclin gene transcription units could be mapped thanks to the specific enrichment of initial transcripts that occurs following UV irradiation. Whereas the VSG gene is 45 kb distant from its promoter, procyclin genes are located immediately downstream. We show, by run-on assays on isolated nuclei and by cDNA analysis, that transcription occurs from both promoters in bloodstream as well as in procyclic forms. It is inferred that the control of the stage-specific expression of VSG and procyclin genes is not effected at the level of transcription initiation, but most probably by interfering with the elongation and stability of the specific transcripts.

Animals↗
PubMed1990

Telomere interactions may condition the programming of antigen expression in Trypanosoma brucei.

The AnTat 1.1 antigen type typically occurs late in a chronic infection by the EATRO 1125 stock of Trypanosoma brucei. The AnTat 1.1 gene, which is located 24 kb from a chromosome end, seems exclusively expressed by acting as a donor in gene conversion events targeted to the telomeric expression site. We report that this gene is sufficiently provided with the homology blocks required for recombination with the expression site, and is not interrupted by stop codons up to the 3' block of homology. A possible reason for its low probability of activation is an inverse orientation with respect to the proximal chromosome end, since, if correctly positioned, it is readily expressed at an early stage of infection, following gene conversion. This suggests that interactions between chromosome ends may precede and favour the rearrangements leading to antigenic variation.

Animals↗
PubMed1990

Evidence for kinetoplast and nuclear DNA homogeneity in Trypanosoma evansi isolates.

The kinetoplast DNA minicircles from 13 stocks of trypanosomes designated as Trypanosoma evansi were digested with various restriction enzymes. We also examined the distribution of restriction site polymorphisms in the nuclear DNA of 9 of these stocks, using 7 different variable surface glycoprotein (VSG) and non-VSG probes. Restricted kinetoplast DNA (kDNA) fragments of some of these strains were cloned into M13 or PUC 18 vectors and sequenced. The restriction and sequence mapping showed that most of T. evansi isolates belonged to the A1 and A2 types of Borst and to two new closely related types A3 and A4. A notable exception was RoTat 4/1 derived from a Sudanese stock which was found to display a characteristic brucei-like minicircle heterogeneity. The T. evansi minicircles analysed are not only homogeneous in sequence but also the region similar to the conserved region in Trypanosoma brucei and Trypanosoma equiperdum is flanked on its 5' end by a palindromic repeat of part of the conserved region. The highly conserved sequence GGGCGGT which appears to correspond to the initiation of synthesis of one of the Okazaki fragments contains an additional G and is located as in T. brucei and T. equiperdum about 73 bp 5' from the ORI. The nuclear DNA analysis confirms the kDNA study in that all the T. evansi stocks are members of a very homogeneous group in terms of sequence divergence. Moreover, our analysis also confirms that T. evansi is more closely related to the West African T. b. brucei and T. b. gambiense than to other African trypanosomes.

Animals↗
PubMed1990

Differential expression of a family of putative adenylate/guanylate cyclase genes in Trypanosoma brucei.

The expression site for the variant surface glycoprotein (VSG) gene of Trypanosoma brucei contains several genes of unknown function (ESAGs, for expression site-associated genes). Among these, ESAG 4 shows homology to eukaryotic adenylate/guanylate cyclase genes, in the region encoding the presumptive enzyme catalytic domain. This gene belongs to a family of related sequences, and hybridizes to the genomic DNA of other trypanosomatids, such as Trypanosoma congolense, Trypanosoma vivax and Trypanosoma mega. While ESAG 4 is transcribed only in bloodstream forms by a RNA polymerase resistant to alpha-amanitin, at least three other members of this family are transcribed in both bloodstream and procyclic forms, by a RNA polymerase sensitive to the drug. These genes encode different putative transmembrane proteins showing high sequence conservation in the region corresponding to the adenylate/guanylate cyclase catalytic domain.

Adenylyl Cyclases↗
PubMed1990

8-Methoxypsoralen and ultraviolet A therapy for cutaneous manifestations of graft-versus-host disease.

The potentially beneficial effect of 8-methoxypsoralen and ultraviolet A (PUVA) irradiation for treatment of drug-resistant cutaneous manifestations of graft-versus-host disease led us to investigate the effect of this therapy in a larger series of patients with GvHD. To date, 11 patients with histologically demonstrated cutaneous GvHD (acute GvHD grade III-IV in 4 patients, extensive lichenoid chronic GvHD in 6 patients, sclerodermatous chronic GvHD in 1 patient) have received PUVA treatment for 2-24 weeks. All patients have been on CsA for GvH prophylaxis; 5 with mismatched grafts had additionally received methotrexate or monoclonal antibody campath-1 after bone marrow transplantation. Seven patients were on CsA and prednisolone; 2 patients on CsA, prednisolone, and azathioprine; 1 patient on azathioprine and prednisolone; and 1 patient had no immunosuppressive treatment for the duration of PUVA treatment. The 8-methoxypsoralen (0.6 mg/kg bw) was given as photosensitizer before each ultraviolet A irradiation (0.3-8.5 joules/cm2). The only observed adverse reaction was mild nausea. In all patients improvement of cutaneous lesions could be observed with complete response in 5 patients and partial response in 6 patients. Immunosuppressive drugs could be withdrawn in 2 patients and reduced in 8 patients after initiation of PUVA treatment. These findings suggest that PUVA therapy may be a useful adjunct to conventional therapy for cutaneous GvHD.

Adolescent↗