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Biomedical subjects

G Jahn

Publications and source records attributed to G Jahn.

At least 37 records · Page 2Linked to original sources
PubMed2000

Tropism of human cytomegalovirus for endothelial cells is determined by a post-entry step dependent on efficient translocation to the nucleus.

Marked interstrain differences in the endothelial cell (EC) tropism of human cytomegalovirus (HCMV) isolates have been described. This study aimed to define the step during the replicative cycle of HCMV that determines this phenotype. The infection efficiency of various HCMV strains in EC versus fibroblasts was quantified by immunodetection of immediate early (IE), early and late viral antigens. Adsorption and penetration were analysed by radiolabelled virus binding assays and competitive HCMV-DNA-PCR. The translocation of penetrated viral DNA to the nucleus of infected cells was quantified by competitive HCMV-DNA-PCR in pure nuclear fractions. The intracytoplasmic translocation of capsids that had penetrated was followed by immunostaining of virus particles on a single particle level; this was correlated with the initiation of viral gene expression by simultaneous immunostaining of viral IE antigens. The infectivity of nonendotheliotropic HCMV strains in EC was found to be 100-1000-fold lower when compared to endotheliotropic strains. The manifestation of this phenotype at the level of IE gene expression indicated the importance of initial replication events. Surprisingly, no interstrain differences were detected during virus entry. However, dramatic interstrain differences were found regarding the nuclear translocation of penetrated viral DNA. With nonendotheliotropic strains, the content of viral DNA in the cell nucleus was 100-1000-fold lower in EC when compared to endotheliotropic strains, thereby reflecting the strain differences in IE gene expression. Simultaneous staining of viral particles and viral IE antigen revealed that interstrain differences in the transport of penetrated capsids towards the nucleus of endothelial cells determine the EC tropism of HCMV.

Active Transport, Cell Nucleus↗
PubMed2000

Monocyte-derived dendritic cells are permissive to the complete replicative cycle of human cytomegalovirus.

The susceptibility of monocyte-derived immature dendritic cells (DC) to infection by various strains of human cytomegalovirus (HCMV) was analysed. Immature DC were generated by incubation of peripheral blood monocytes with interleukin-4 and granulocyte-macrophage colony-stimulating factor for 7 days and were characterized by a CD1a+/CD40+/CD80+/CD86+/HLA-DR+/CD14- phenotype. Viral antigen expression and production of infectious progeny virus were analysed in infected immature DC cultures. Immature DC were 80-90 % susceptible to HCMV strains that had been propagated in endothelial cell culture, whereas the infection rate was negligible with fibroblast-adapted HCMV strains. Immature DC infection resulted in expression of viral immediate early, early and late genes. Productive infection was proven by the detection of infectious virus in single-step growth curves and in infectious centre assays. It is concluded that HCMV might interfere with the host immune reaction by permissive, lytic infection of immature DC.

Antigens, Viral↗
PubMed2000

Efficient lytic infection of human arterial endothelial cells by human cytomegalovirus strains.

Endothelial cells (EC) are common targets of permissive infection by human cytomegalovirus (HCMV) in vivo during acute disease. However, studies of HCMV-EC interactions in vitro have generated discordant results. While lytic infection of cultured venous EC has been well established, Fish et al. (K. N. Fish, C. Soderberg Naucler, L. K. Mills, S. Stenglein, and J. A. Nelson, J. Virol. 72:5661-5668) have reported noncytopathic persistence of the virus in cultured aortic EC. We propose that interstrain differences in viral host cell tropism rather than the vascular bed of origin of infected EC might account for these discrepancies. In the present investigation we compared the responses of EC derived from human adult iliac artery, placental microvasculature, and umbilical vein to infection with various HCMV strains. Regardless of the vascular bed of origin, infection with EC-propagated HCMV strains induced 100% efficient cytopathic change progressing to complete lysis of inoculated monolayers. While fibroblast-propagated strains persisted at low titer in infected arterial EC cultures, they were also cytolytic for individual infected cells. The finding of cytopathic lytic infection of arterial EC by HCMV implicates a mechanism of vascular injury in the pathogenesis of HCMV infection.

Adult↗
PubMed2000

Transmission of cytomegalovirus infection through breast milk in term and preterm infants. The role of cell free milk whey and milk cells.

We investigated the reactivation of cytomegalovirus during lactation and analysed the role of human milk whey and milk cells in mother-to-child-transmission. In contrast to term infants, preterm infants may be infected symptomatically by breastfeeding. Human milk whey is the material of choice for detection of maternal DNAlactia and virolactia, whereas milk cells not necessarily have to be infected in transmitters.

Cytomegalovirus↗
PubMed1999

A simple and rapid method for preparation of viral DNA from cell associated cytomegalovirus.

In the field of human cytomegalovirus pathogenesis there is growing interest in analyzing recent clinical isolates rather than cell culture adapted laboratory strains. However, true low passage isolates are strictly cell associated prior to cell culture adaptation and only a minor fraction of cells are infected at low passage number. Both conditions hinder the preparation of pure viral DNA. To date, genetic analyses had been carried out mostly with supernatant associated cytomegalovirus. A rapid and simple method is described for preparation of viral DNA from low passage cell associated isolates with little cytopathogenic effect. The protocol is based on a combination of Triton X-100 lysis, nuclease treatment, and subsequent phenol chloroform extraction. Cellular background was reduced significantly to enable clear detection of all viral DNA fragments in restriction fragment length analysis. The method yielded DNA which was suitable for downstream applications like cloning of viral DNA fragments or transfection of genomic viral DNA. This method may facilitate genomic analyses of pathogenic cell associated recent cytomegalovirus isolates.

Cells, Cultured↗
PubMed1999

Successful treatment of human herpesvirus-6 encephalitis after bone marrow transplantation.

We report two cases of human herpesvirus-6 (HHV-6)-associated encephalitis in patients after BMT. Both patients reported distinct neurological symptoms with disorientation, sleepiness and loss of short-term memory. Diagnosis was based on PCR analysis of the cerebrospinal fluid (CSF) positive for HHV-6 variant B-DNA. After institution of therapy with foscarnet in both cases, neurological symptoms improved and in one patient clearance of HHV-6-DNA from CSF was demonstrated. These cases show that HHV-6 infection has to be considered in patients with neurological symptoms following BMT and effective treatment of HHV-6 encephalitis is possible if instituted early.

Adult↗
PubMed1999

Comprehensive restriction analysis of the UL97 region allows early detection of ganciclovir-resistant human cytomegalovirus in an immunocompromised child.

Children with innate immunodeficiencies may be at high risk for early development of ganciclovir-resistant human cytomegalovirus (HCMV) infection after bone marrow transplantation (BMT). For early and frequent monitoring of the occurrence of ganciclovir resistance-associated mutations in codons of the UL97 gene, a panel of previously described restriction assays was expanded for use on codons 591, 592, and 603. This technique enabled detection of suddenly emerging ganciclovir-resistant HCMV after BMT in a 7-year-old child with a T cell defect. Resistance emerged among the isolation of a ganciclovir-sensitive HCMV strain 32 days after transplantation, the first detection of genotypical resistance at day 44, and the isolation of resistant HCMV (ID50>12 microM) at day 54. Simple and yet comprehensive methods for therapy surveillance may be important in this patient group, in which the restriction assays proved useful.

Antiviral Agents↗
PubMed1999

Hepatocytes are permissive for human cytomegalovirus infection in human liver cell culture and In vivo.

The cytopathic potential of human cytomegalovirus (HCMV) in human liver cells was analyzed in cell culture and in tissue sections from patients with HCMV hepatitis. Liver cell cultures, consisting of hepatocytes, bile duct epithelial cells, and stromal cells were infected by various HCMV strains. Cytopathic effects, viral gene expression, and virus production were detected. Infected cell types were identified by immunocytochemical double labeling. Hepatocytes were the predominant target cells of HCMV infection in liver tissues and in cell culture. Late-stage infected cultured hepatocytes produced infectious progeny virus, and infectious virus was propagated from liver tissue specimens. HCMV infection in cultured liver cells closely resembled in vivo infection of the liver with regard to the target cell spectrum and the permissive course of infection. It is concluded that HCMV can cause direct liver parenchyma damage by efficient cytolytic infection of hepatocytes.

Antigens, Viral↗
PubMed1999

Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome.

Following extensive propagation in fibroblasts, human cytomegalovirus (HCMV) loses tropism for a number of otherwise natural host cells, in particular, endothelial cells. In this study, the hypothesis was tested that loss of endothelial tropism is associated with the appearance of genomic variants. Initial quantitative focus expansion assays on endothelial monolayers demonstrated that, while the laboratory strains AD169 and Towne failed to form detectable foci, 29 out of 30 recent clinical HCMV isolates had the potential to expand in endothelial cell culture. By long-term adaptation in fibroblast cultures, nonendotheliotropic strains could be selected from clinical HCMV isolates, while long-term endothelial-adapted strains of the same isolates retained both fibroblast tropism and endothelial tropism. Such differentially adapted isolate pairs always displayed genomic differences in restriction fragment length analyses. Coinfection of endothelial cells by two nonendotheliotropic HCMV strains yielded an endotheliotropic recombinant HCMV variant combining portions of the genomes of both parental viruses. When DNA purified from various isolates was transfected into fibroblasts, progeny virus retained the specific tropism of parental virus from which the DNA was isolated. These findings demonstrate that endothelial tropism is an inherent property of most clinical HCMV isolates and is determined by the viral genome. Although the specific determinants of HCMV cell tropism are still unknown, this study provides the first evidence for a genetic contribution.

Cells, Cultured↗
PubMed1999

Human cytomegalovirus infection of immature dendritic cells and macrophages.

A central aspect of human cytomegalovirus (HCMV) pathogenesis is the interaction of the virus with different antigen-presenting cell (APC) types of the host. In principle, a number of various cell types have the potential of antigen presentation when MHC II expression is induced by appropriate stimuli. The most potent antigen presenters are monocytes/macrophages and dendritic cells (DCs), therefore called professional APCs. Interestingly, these cells seem to be targets of productive HCMV infection. The susceptibility of the monocyte/macrophage system has been analyzed intensively during the past decade. Investigation of the role of DCs during HCMV infection, however, has begun only recently.

Antigen-Presenting Cells↗
PubMed1998

Discordant detection of human cytomegalovirus DNA from peripheral blood mononuclear cells, granulocytes and plasma: correlation to viremia and HCMV infection.

BACKGROUND: There exist only few data about the HCMV infection of single positive leukocyte subtypes in immunosuppressed patients. Most reports describe HCMV coinfection of cells of the myelomonocytic line or even T- and B-cell populations. Correlation of positive PCR findings from two major leukocyte fractions and plasma to viremia and HCMV infection in general should contribute to select suitable sources of HCMV DNA for diagnostic purposes. OBJECTIVE: The diagnostic value of qualitative leukoDNAemia of simultaneously isolated peripheral blood mononuclear cells (PBMC), granulocytes as well as plasmaDNAemia was evaluated by comparing the positive results of nested PCR from blood with virus isolation either from leukocytes or from any other sources, with serology and the clinical status of immunosuppressed patients. STUDY DESIGN: PBMC, granulocytes and plasma were prepared of a total of 220 blood samples of 75 immunosuppressed patients with clinically suspected primary or recurrent HCMV infection. In a collective of 35 patients consisting mainly of recipients of marrow or solid organ transplants positive results of leuko- or plasmaDNAemia were correlated with data from HCMV screening and the clinical status. For standardization, HCMV IE Exon 4 DNA was amplified from 100 ng cellular DNA of each leukocyte population. Cross contamination can be excluded. DNA from plasma was extracted by phenol/chloroform. Using this experimental design, HCMV DNA was not detectable in PBMC, granulocytes and plasma of 23 healthy HCMV seropositive blood donors. RESULTS: Leukocyte separation in a collective of 30 patients with positive leukoDNAemia revealed in only 12 cases (40%) double infection of PBMC and granulocytes. In the majority of cases (18 patients, 60%) however, HCMV DNA was detectable in only one leukocyte fraction, either in PBMC or granulocytes. LeukoDNAemia did not correlate to viremia. HCMV DNA amplified from plasma was shown to be cell free. Infectious virus from plasma was not isolated. The predictive value of qualitative nested PCR from blood to detect HCMV infection was high for plasma and decreased in the following sequence: plasma (0.92) > PBMC (0.83) > granulocytes (0.65). CONCLUSIONS: Qualitative nPCR from plasma and PBMC seems to be sufficient to detect (an ongoing) HCMV infection of immunosuppressed patients. However, the rate of single positive leukocyte fractions is approximately 60%. Therefore, viral leukoDNAemia in 40% of cases seems to be restricted to either PBMC or granulocytes. For diagnostic purposes the whole leukocyte population should be used for PCR analysis.

Bone Marrow Transplantation↗
PubMed1998

A simplified assay for screening of drug resistance of cell-associated cytomegalovirus strains.

BACKGROUND: Conventional phenotypic drug resistance determination of cell-free clinical human cytomegalovirus (HCMV) isolates is usually very laborious and may take 8-12 weeks, since serially passages of slowly growing viral isolates in tissue cultures are required to obtain a sufficient viral titer for an appropriate inoculum. Rapid screening of a large number of samples would therefore only be possible if simplified, less work-intensive methods are employed. OBJECTIVE: The aim of this work was to develop an assay which speeds up the whole procedure of phenotypic drug resistance determination. Steps of the classical plaque reduction assay should be simplified or omitted, but on the other hand, the assay should be reliable and reproducible. STUDY DESIGN: Twenty-six clinical HCMV isolates from 20 immunocompromised patients (ten pre-treatment and 16 post-treatment with ganciclovir) were tested for drug susceptibility with the simplified plaque reduction assay. Most isolates were tested at least twice in independent assays. Virus titration could be avoided by using four different doses of cell-associated virus from the secondary culture for coculture susceptibility testing. Drug susceptibility values were determined by plaque titration and Probit analysis. RESULTS: All clinical HCMV isolates tested showed a mean ganciclovir ID50 value of 1.98 microM, (range 0.2-12.2; median 0.95) and a mean foscarnet ID50 value of 92.4 microM (range 35.7-181; median 81). All except one isolate were classified ganciclovir sensitive when compared to ID50 values of two ganciclovir resistant control stains (53.7 +/- 6.4 and 12.7 +/- 0.9 microM) and the sensitive laboratory strain Towne (2.1 +/- 0.8 microM). Repeated tests of individual isolates were reproducible, although the infectivity of the inoculum has not been determined prior of the assay. The mean time which elapsed between receipt of the clinical specimen and read-out of the assay was circa 4 weeks. CONCLUSIONS: Phenotypic resistance testing of HCMV isolates following to this protocol drastically reduces expenditure of time and work. The assay allows reliably the discrimination of HCMV isolates as drug resistant or sensitive according to the recent classification criteria of the AIDS Clinical Trials Group (ACTG). The simple handling and uncomplicated calibration of this assay facilitates the screening of large specimen numbers and renders drug susceptibility determination of HCMV more accessible to diagnostic routine use.

Antiviral Agents↗
PubMed1998

Evaluation of restriction fragment length polymorphism analysis of the UL10-UL13 genomic region for rapid identification of human cytomegalovirus strains.

A sensitive semi-nested polymerase chain reaction (PCR) was established which allows rapid identification of human cytomegalovirus strains directly on clinical specimens, thereby permitting virus isolation and propagation on cell cultures to be avoided. The assay is based on restriction analysis of PCR products derived from the polymorphic UL10-UL13 region of the human cytomegalovirus genome. The method was evaluated using clinical samples from 23 subjects comprising 16 breast-feeding mothers and seven bone marrow transplantation recipients. For eight mothers, postnatal virus transmission to their offspring via breast milk was studied. Interestingly, for one mother-infant pair, a double infection with two distinct human cytomegalovirus strains could be demonstrated. Stepwise digestion with different restriction enzymes raised the possibility of detecting different strains almost twofold compared to analysis with only one enzyme. This assay is a practical tool for monitoring human cytomegalovirus transmission in various clinical settings.

Adult↗
PubMed1998

Detection of cytomegaloviral DNA in human milk cells and cell free milk whey by nested PCR.

Human cytomegalovirus (HCMV) DNA can be detected in different compartments of human milk. A protocol for the preparation of milk whey free of fat and cells for the detection of human cytomegalovirus (HCMV) by nested PCR is presented. This is based upon the experience of the separation of more than 200 milk specimens of healthy seropositive breast feeding mothers. HCMV DNA could be detected in freshly centrifuged and filtrated milk whey specimens without contamination by cellular DNA. In limiting dilution experiments using HCMV plasmid DNA, the effect of different DNA extraction procedures from native milk and milk whey on the detection limit of cytomegaloviral DNA was demonstrated. About 200 viral genome equivalents/ml in milk whey or native milk were detectable by classical organic phenol/chloroform extraction or a spin column method, respectively. The detection of viral DNA in milk cells depended on a minimum number of milk cells (10(5)-2 x 10(5)) available for DNA extraction. In contrast to the findings of cytomegaloviral DNA in native sera or plasma of immunosuppressed patients we failed to amplify low level viral DNA from native breast milk by nested PCR due to an inhibition of Taq polymerase by lipid components. Finally, the course of cell associated and cell free DNAlactia was monitored. Analyzing sequential milk specimens, in some cases the presence of HCMV DNA in colostrum could be demonstrated. DNAlactia of milk cells and whey was partially discordant. Onset (week 1-4 after delivery) and duration (2 weeks up to more than 3 months) of DNAlactia showed distinct individual patterns. The methods described, allow further analysis of the mechanisms involved in the postnatal HCMV transmission by breast feeding seropositive mothers.

Cell-Free System↗