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Biomedical subjects

B Jahn

Publications and source records attributed to B Jahn.

At least 19 recordsLinked to original sources
PubMed2003

cAMP signaling in Aspergillus fumigatus is involved in the regulation of the virulence gene pksP and in defense against killing by macrophages.

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease and resulting in high mortality. In order to determine the importance of the cAMP signaling pathway for virulence, three genes encoding putative elements of the pathway have been cloned and characterized: the adenylate cyclase gene acyA, and gpaA and gpaB, both of which encode alpha subunits of heterotrimeric G proteins. The acyA and gpaB genes were each deleted in A. fumigatus. Both mutants showed reduced conidiation, with the deltaacyA mutant producing very few conidia. The growth rate of the deltaacyA mutant was also reduced, in contrast to that of the deltagpaB mutant. Addition of 10 mM dibutyryl-cAMP to the culture medium completely restored the wild-type phenotype in both mutant strains. To study the influence of GPAB on the expression of the gene pksP, which encodes a virulence factor that is involved in pathogenicity, a pksPp-lacZ gene fusion was generated and integrated as a single copy at the pyrG gene locus of both the parental strain and the deltagpaB mutant strain. The deltagpaB mutant showed reduced expression of the pksPp-lacZ reporter gene relative to that in the parental strain. In mycelia of both the parental strain and the deltagpaB mutant pksPp-lacZ expression was increased when isobutyl-methyl-xanthine, an inhibitor of intracellular phosphodiesterases, was added to the medium. The survival rate of conidia after ingestion by human monocyte-derived macrophages was also determined. The killing rate for conidia from deltaacyA and deltagpaB strains was significantly higher than that for wild-type conidia. Taken together, these findings suggest that cAMP triggers a system that protects A. fumigatus from the effects of immune effector cells of the host.

Amino Acid Sequence↗
PubMed2002

Collagen sandwich culture affects intracellular polyamine levels of human hepatocytes.

Extracellular matrices, like collagen layers, play an important role in preventing dedifferentiation of hepatocytes in long-term culture experiments. It has also been shown that polyamines are crucial for cell growth and liver differentiation - regeneration. Primary cultured hepatocytes with their low mitotic activity might be a valuable tool in studying the role of polyamines in differentiation. Here, our goal was to investigate whether an extracellular cell culture matrix can influence intracellular polyamine levels in human hepatocytes during long-term culture. Primary human hepatocytes were isolated from surgical tissue resections and were maintained either in single collagen (SG) or double collagen gel (DG) layer (sandwich) culture systems. Cell viability and function were examined and intracellular polyamine levels were measured using a highly sensitive high performance liquid chromatography (HPLC) method. Hepatocytes showed high viability in both culture systems used, but albumin secretion was diminished in SG cultured hepatocytes after 14 days. In general, total intracellular polyamine levels of hepatocytes decreased markedly in both SG and DG within the first days of culture, but remained constant until day 21 with a SG/DG ratio of about 1.4. Individual polyamines levels were dependent on the culture time and system, where spermine decreased and putrescine increased in both SG and DG over time (day 14), but spermidine increased only in DG. Our results suggest that polyamine levels, in particular putrescine, might be important regulators of hepatocyte specific function in vitro and therefore serve as a marker of differentiation for cultivated human hepatocytes.

Biogenic Polyamines↗
PubMed2001

A rapid assay for evaluation of antiviral activity against coxsackie virus B3, influenza virus A, and herpes simplex virus type 1.

In order to identify new potential antiviral drugs, small amounts of extracts or compounds have to be examined for cytotoxicity and antiviral activity in primary screening using a rapid, easy, inexpensive, and highly standardised test system. In this study, high-throughput cytopathic effect (CPE) inhibitory assays were established for coxsackie virus B3 on HeLa Ohio cells, influenza virus A on Madin-Darby canine kidney cells, and herpes simplex virus type 1 (HSV-1) on green monkey kidney cells that meet these requirements. The cytotoxic and the antiviral effects were quantified using a crystal violet uptake assay allowing automated handling of large numbers of candidate agents. To ensure comparable results with plaque reduction assays, the 50 and 90% plaque inhibitory concentrations of guanidine, amantadine, and phosphonoformic acid were used to standardise the anti-coxsackie virus B3, anti-influenza virus A, and anti-HSV-1 tests, respectively. The strong correlation between the antiviral activity determined by CPE-inhibitory assays and plaque reduction assay was further proved for other antivirals. In summary, low amounts of large numbers of compounds may be tested inexpensively and standardised within 24 h (coxsackie virus B3 and influenza virus A) or 48 h (herpes simplex virus type 1) post-infection using CPE inhibitory assays.

Amantadine↗
PubMed2001

Differential expression of the Aspergillus fumigatus pksP gene detected in vitro and in vivo with green fluorescent protein.

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. To be able to analyze the expression of putative virulence-associated genes of A. fumigatus, the use of the enhanced green fluorescent protein (EGFP) as a reporter was established. Two 5' sequences, containing the putative promoters of the pyrG gene, encoding orotidine-5'-phosphate decarboxylase, and the pksP gene, encoding a polyketide synthase involved in both pigment biosynthesis and virulence of A. fumigatus, were fused with the egfp gene. The PpksP-egfp construct was integrated via homologous recombination into the genomic pksP locus. EGFP production was analyzed by fluorescence spectrometry, Western blot analysis, and fluorescence microscopy. Differential gene expression in A. fumigatus was observed. Fluorescence derived from the PYRG-EGFP fusion protein was detected during all developmental stages of the fungus, i.e., during germination, during vegetative growth, in conidiophores, and weakly in conidia. In addition, it was also detected in germinating conidia when isolated from the lungs of immunocompromised mice. By contrast, PKSP-EGFP-derived fluorescence was not found in hyphae or stalks of conidiophores but was found in phialides and conidia in vitro when the fungus was grown under standard conditions, indicating a developmentally controlled expression of the gene. Interestingly, pksP-egfp expression was also detected in hyphae of germinating conidia isolated from the lungs of immunocompromised mice. This finding indicates that the pksP gene can also be expressed in hyphae under certain conditions and, furthermore, that the pksP gene might also contribute to invasive growth of the fungus.

Amino Acid Sequence↗
PubMed2000

Attachment of coxsackievirus B3 variants to various cell lines: mapping of phenotypic differences to capsid protein VP1.

The coxsackievirus B3 (CVB3) strain Nancy P establishes a persistent carrier-state infection without visible cytopathic effect in primary human fibroblasts (HuFi H), whereas the derivative variant PD induces a complete lysis of the cell monolayer. To define the molecular basis of this exceptional growth property, the complete genomes of both viruses were sequenced and compared to all published sequences of CVB3. As a result, six unique amino acid substitutions in the VP1 capsid protein were observed. Via hybrid virus construction, the lytic phenotype was transferred to a nonlytic cDNA-generated CVB3. Mapping experiments indicate that the presence of amino acid residues K78, A80, A91, and I92 in VP1 is sufficient to induce "lytic" infections in HuFi H cells. Binding assays demonstrate that CVB3 Nancy P preferentially binds to the human coxsackievirus-adenovirus receptor (CAR), while PD exhibits a very weak interaction with CAR but strong binding to the decay accelerating factor (DAF). These results suggest that the mutated amino acid residues in VP1 are involved in receptor recognition/binding. Moreover, the lytic replication of CVB3 PD and the hybrid virus in various nonpermissive rodent cell lines indicates that cell surface molecules other than CAR and DAF may be involved in attachment of this variant to cell surfaces.

Amino Acid Substitution↗
PubMed2000

Cobalt-deficiency-induced hyperhomocysteinaemia and oxidative status of cattle.

In ruminants, Co is required for the synthesis of vitamin B12, which in turn is needed for the resynthesis of methionine by methylation of homocysteine and thus, cobalamin deficiency may induce hyperhomocysteinaemia which is brought into context with perturbations of the antioxidative-prooxidative balance. The present study was conducted to explore whether Co deficiency in cattle is also associated with homocysteine-induced disturbances of oxidative status. Co deficiency was induced in cattle by feeding two groups of animals on either a basal maize-silage-based diet that was moderately low in Co (83 micrograms Co/kg DM), or the same diet supplemented with Co to a total of 200 micrograms Co/kg DM, for 43 weeks. Co deficiency was apparent from a reduced vitamin B12 status in serum and liver and an accumulation of homocysteine in plasma which was in excess of 4.8 times higher in Co-deprived cattle than in controls. The much increased level of circulating homocysteine did not indicate severe disturbances in antioxidant-prooxidant balance as measured by individual markers of lipid peroxidation, protein oxidation, and the antioxidative defence system. There were no quantitative difference in plasma thiol groups, nor were there significant changes in concentrations of alpha-tocopherol, microsomal thiobarbituric acid-reactive substances and carbonyl groups in liver. However, there was a trend toward increased plasma carbonyl levels indicating a slight degradation of plasma proteins in the hyperhomocysteinaemic cattle. Analysis of the hepatic catalase (EC 1.11.1.6) activity revealed an 11% reduction in Co-deficient cattle relative to the controls. These results indicate that long-term moderate Co deficiency may induce a severe accumulation of plasma homocysteine in cattle, but considerable abnormalities in oxidative status failed to appear.

Animals↗
PubMed2000

Interaction of human phagocytes with pigmentless Aspergillus conidia.

A defect in the pksP gene of Aspergillus fumigatus is associated with the loss of conidial pigmentation, a profound change of the conidial surface structure, and reduced virulence. The structural change of the conidial surface structure was not observed in similar A. nidulans wA mutants. Our data indicate that the pigment of both species is important for scavenging reactive oxygen species and for protection of conidia against oxidative damage.

Aspergillus fumigatus↗
PubMed2000

Comparative evaluation of three different genotyping methods for investigation of nosocomial outbreaks of Legionnaires' disease in hospitals.

The increased incidence of nosocomial Legionnaires' disease in two hospitals prompted investigation of possible environmental sources. In the search for an effective DNA-typing technique for use in hospital epidemiology, the performance and convenience of three methods-SfiI macrorestriction analysis (MRA), amplified fragment length polymorphism (AFLP), and arbitrarily primed PCR (AP-PCR)-were compared. Twenty-nine outbreak-associated and eight nonassociated strains of Legionella pneumophila with 13 MRA types and subtypes were investigated. These strains comprised isolates from bronchoalveolar lavages, from environmental, patient-related sources, and type strains. All three typing methods detected one predominant genotype associated with the outbreaks in both hospitals. All of them correctly assigned epidemiologically associated, environmental isolates to their respective patient specimens. AP-PCR was the least discriminating and least reproducible technique. In contrast, AFLP was demonstrated as being the method with the best interassay reproducibility (90%) and concordance (94%) in comparison to the genotyping standard of MRA and the epidemiological data. Analysis of AFLP fragments revealed 12 different types and subtypes. Because of its simplicity and reproducibility, AFLP proved to be the most effective technique in outbreak investigation.

Bacterial Typing Techniques↗
PubMed1999

Multiple renal aspergillus abscesses in an AIDS patient: contrast-enhanced helical CT and MRI findings.

Renal insufficiency or allergic reactions for X-ray contrast agents are frequent limitations in immunocompromised hosts such as neutropenic or AIDS patients. Due to a better tolerance of contrast agents in MRI, this technique is well suited for investigation of parenchymal organs. We demonstrate an allergic AIDS patient who presented with fever and flank pain. At sonography, anechoic renal lesions were supposed to be non-complicated cysts; however, on T2-weighted MRI, the center was of high signal. Dynamic contrast-enhanced MRI of the kidneys demonstrated an enhancing rim with ill-defined margins. The lesions were supposed to be multiple bilateral abscesses. Due to the multiple dynamic contrast series, a delayed enhancement of renal parenchyma was detectable adjacent to the lesion. This was suggested as accompanying local pyelonephritis and an infectious etiology became more reliable. Aspergillus fumigatus was identified by CT-guided biopsy as the underlying microorganism. The MR appearance of this manifestation has not been described previously.

AIDS-Related Opportunistic Infections↗
PubMed1998

Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence.

Aspergillus fumigatus is an important pathogen of the immunocompromised host causing pneumonia and invasive disseminated disease with high mortality. Previously, we identified a mutant strain (white, W) lacking conidial pigmentation and, in addition, the conidia showed a smooth surface morphology, whereas wild-type (WT) conidia are grey-green and have a typical ornamentation. W conidia appeared to be less protected against killing by the host defence, e.g., were more susceptible to oxidants in vitro and more efficiently damaged by human monocytes in vitro than WT conidia. When compared to the WT, the W mutant strain showed reduced virulence in a murine animal model. Genetic analysis suggested that the W mutant carried a single mutation which caused all of the observed phenotypes. Here. we report the construction of a genomic cosmid library of A. fumigatus and its use for complementation of the W mutant. Transformation of the W mutant was facilitated by co-transformation with plasmid pHELP1 carrying the autonomously replicating ama1 sequence of A. nidulans which also increased the transformation efficiency of A. fumigatus by a factor of 10. Using this cosmid library a putative polyketide synthase gene, designated pksP (polyketide synthase involved in pigment biosynthesis) was isolated. The pksP gene has a size of 6660 bp. pksP consists of five exons separated by short (47-73 bp) introns. Its deduced open reading frame is composed of 2146 amino acids. The pksP gene complemented both the white phenotype and the surface morphology of the W mutant conidia to wild type. Whereas W mutant conidia caused a strong reactive oxygen species (ROS) release by polymorphonuclear leukocytes, the ability of pksP-complemented W mutant conidia to stimulate ROS release was significantly reduced and comparable to that of WT conidia. In addition, the complemented strains showed restored virulence in a mouse model.

Amino Acid Sequence↗
PubMed1998

Accumulation of amphotericin B in human macrophages enhances activity against Aspergillus fumigatus conidia: quantification of conidial kill at the single-cell level.

A cytofluorometric assay that allowed assessment of damage to phagocytosed Aspergillus fumigatus conidia at the single-cell level was developed. After ingestion by monocyte-derived macrophages (MDMs), conidia were reisolated by treatment of the cells with streptolysin O, a pore-forming toxin with lytic properties on mammalian cells but not on fungi. The counts obtained by staining of damaged conidia with propidium iodide and quantification by cytofluorometry correlated with colony counts. By the use of this method, we demonstrate that MDMs differentiated in vitro by low-dose granulocyte-macrophage colony-stimulating factor and gamma interferon have only a limited capacity to damage Aspergillus conidia in vitro. The killing rate 12 h after phagocytosis was found to be only 10 to 15%. However, intracellular loading of the phagocytes with amphotericin B (AmB) dose dependently enhanced the anticonidial activity. Preincubation of macrophages with only 1 microg of AmB per ml resulted in an uptake of 18 fg of AmB/cell, leading to killing rates of 50 to 60%. The experimental protocol provides a new tool for the rapid quantification of anticonidial activity against A. fumigatus in vitro. Intracellular accumulation of AmB may represent an important factor underlying the efficacy of this antifungal drug in the prophylaxis and treatment of Aspergillus infections.

Amphotericin B↗
PubMed1997

Isolation and characterization of a pigmentless-conidium mutant of Aspergillus fumigatus with altered conidial surface and reduced virulence.

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. The factors contributing to the predominance of A. fumigatus as an opportunistic pathogen are largely unknown. Since the survival of conidia in the host is a prerequisite for establishing disease, we have been attempting to identify factors which are associated with conidia and, simultaneously, important for infection. Therefore, an A. fumigatus mutant strain (white [W]) lacking conidial pigmentation was isolated. Scanning electron microscopy revealed that conidia of the W mutant also differed in their surface morphology from those of the wild type (WT). Mutant (W) and WT conidia were compared with respect to their capacities to stimulate an oxidative response in human phagocytes, their intracellular survival in human monocytes, and virulence in a murine animal model. Luminol-dependent chemiluminescence was 10-fold higher when human neutrophils or monocytes were challenged with W conidia compared with WT conidia. Furthermore, mutant conidia were more susceptible to killing by oxidants in vitro and were more efficiently damaged by human monocytes in vitro than WT conidia. In a murine animal model, the W mutant strain showed reduced virulence compared with the WT. A reversion analysis of the W mutant demonstrated that all phenotypes associated with the W mutant, i.e., altered conidial surface, amount of reactive oxygen species release, susceptibility to hydrogen peroxide, and reduced virulence in an murine animal model, coreverted in revertants which had regained the ability to produce green spores. This finding strongly suggests that the A. fumigatus mutant described here carries a single mutation which caused all of the observed phenotypes. Our results suggest that the conidium pigment or a structural feature related to it contributes to fungal resistance against host defense mechanisms in A. fumigatus infections.

Animals↗
PubMed1996

A novel SP-1 site in the human interleukin-1 beta promoter confers preferential transcriptional activity in keratinocytes.

To investigate the mechanisms of transcriptional activation of interleukin-1beta (IL-1beta) in non-monocytic cells, we constructed a series of reporter plasmids with the bacterial chloramphenicol acetyltransferase gene linked to various parts of the human IL-1beta promoter and performed transient transfection experiments. We identified a promoter segment that activates transcription most efficiently in keratinocytes. Electrophoretic mobility shift assays (EMSA) with a 43-mer oligonucleotide derived from the functionally identified cis-acting element revealed specific complexes. By competition analysis with transcription factor consensus sequence oligonucleotides and by immunosupershift, transcription factor SP-1 or a closely related protein was shown to bind to this regulatory element. The closest match to the known SP-1 consensus sequence within the respective region is a TCCCCTCCCCT motif. Mutation of this motif almost completely, and specifically, abolished the binding of two low-mobility complexes and led to a 95% decrease of constitutive transcriptional activation of a reporter construct IL-1beta (-170/+108). Likewise, activation of this reporter construct by tumor necrosis factor-alpha depended on the SP-1 site. These observations suggest that a so-far-unrecognized SP-1 site in the human IL-1beta promoter may participate in the transcriptional regulation of this gene in keratinocytes.

Cell Line, Transformed↗
PubMed1996

Colorimetric susceptibility testing for Aspergillus fumigatus: comparison of menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and Alamar blue tests.

Two colorimetric methods that use Alamar Blue or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for assaying the in vitro activities of antifungal agents have been described. We report that both tests performed similarly when the antifungal activity of amphotericin B against Candida albicans was determined. However, only the MTT test generated interpretable data when Aspergillus fumigatus was used.

Amphotericin B↗
PubMed1995

Lactate dehydrogenase-release assay: a reliable, nonradioactive technique for analysis of cytotoxic lymphocyte-mediated lytic activity against blasts from acute myelocytic leukemia.

Treatment of patients in remission of acute myelocytic leukemia using immunotherapy with interleukin 2 is a new approach to prolonging remission duration in this disease. As an important mechanism for the pathophysiology of eradication of residual myelocytic blast populations, activation of cytotoxic effector lymphocytes has frequently been discussed. However, the associated immunological research has been complicated to some extent, because in conventional chromium 51-release assays, blast cells frequently fail to incorporate sufficient amounts of 51Cr and/or spontaneously release high amounts of 51Cr. Recently, we established a culture system which promotes the outgrowth of cytotoxic T lymphocytes in bone marrow-derived mononuclear cells cultured in IL-2. To study cytotoxicity and the responsible mechanisms of the obtained T-cell lines and clones, we modified a previously described cytotoxicity assay, based on the release of lactate dehydrogenase (LDH-release assay) for use in cryopreserved blasts obtained from the bone marrow of patients with acute myelocytic leukemia. Using this assay, we were able to detect cytotoxicity of IL-2-activated peripheral blood lymphocytes from three healthy controls against a number of blast samples obtained from the bone marrow of patients with AML (up to more than 40% lysis at an effector target cell ratio of 20:1). However, a minority of AML blasts seem to be resistant to lysis by IL-2-activated lymphocytes. In bone marrow-derived T-cell lines from patients with AML we detected lytic activity against autologous blasts in three of seven cases tested by LDH release, ranging from 29 to 63% at an effector target ratio of 10:1. Additionally, T-cell clones with different phenotypes were established which were able to mediate cytotoxicity against autologous blast cells. Thus, cytotoxicity against freshly isolated blasts from patients with acute myelocytic leukemia can be analyzed reliably, reproducibly, and without the use of isotopes by the LDH-release assay.

Blast Crisis↗
PubMed1995

Bone marrow-derived T-cell clones obtained from untreated acute myelocytic leukemia exhibit blast directed autologous cytotoxicity.

The eradication of minimal residual blast populations by activation of autologous cytotoxic cells with interleukin 2 (IL-2) is a new promising tool in the treatment of acute myelocytic leukemia (AML). However, the immunological effector cells are not yet clearly defined. The present study was designed to investigate the presence of cytotoxic precursor cells in active AML and to identify phenotypical and functional characteristics of autologous anti-leukemic cytotoxic effector cells. For this purpose, mononuclear cells (MNC) containing at least 70% leukemic blasts were isolated from bone marrow of untreated AML and cultured in the presence of 3000 IU/ml recombinant IL-2 (rIL-2) for 6-8 weeks. Under these conditions, T-cells were selected in the bone marrow cultures and overgrew the leukemic blasts. The resulting T-cell populations were cloned by limiting dilution and the clones obtained were characterized for their phenotypical and functional patterns. Totally, cloning resulted in 68 clones and a few cell lines. The clonality was verified by RT PCR analysis of TCR V beta gene expression. All clones obtained stained positive for CD2, CD3, DR and CD56. The vast majority (68%) of T-cell clones/lines was CD4+, a few clones expressed CD8 (19%) or CD4 and CD8, and four clones were of TCR gamma delta origin. Seven of 15 clones tested, including three CD4+, two CD8+ and two TCR gamma delta(+)-clones were found to be cytotoxic against autologous leukemic blast cells. All except one clone expressed oncolytic activities against allogeneic blasts too. One of the TCR gamma delta(+)-clones demonstrated NK activity by lysis of K562 targets. The majority of the T-cell-clones released IL-2, IL-8, TNF-alpha, GM-CSF but only a few IFN gamma and expressed high levels of mRNA for IL-2, TGF-beta and IL-10. None of the clones was found to produce IL-3, IL-4, IL-7 and TNF-beta. The data provide evidence of the existence of T-cell precursors in untreated AML bone marrow differentiating to cytotoxic cells with activity against autologous and allogeneic AML blast cells.

Bone Marrow Cells↗