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Johannes Löwer

Publications and source records attributed to Johannes Löwer.

15 recordsLinked to original sources
PubMed2006

APOBEC3 proteins inhibit human LINE-1 retrotransposition.

The human cytidine deaminase family APOBEC3 represents a novel group of proteins in the field of innate defense mechanisms that has been shown to be active against a variety of retroviruses. Here we examined whether members of the APO-BEC3 family have an impact on retrotransposition of human long interspersed nuclear elements (LINE-1s or L1s). Using a retrotransposition reporter assay in HeLa cells, we demonstrate that in the presence of transiently transfected APOBEC3A, L1 retrotransposition frequency was reduced by up to 85%. Although APOBEC3G and -3H did not influence L1 retrotransposition notably, expression of APOBEC3B, -3C, and -3F inhibited transposition by approximately 75%. Although reverse transcription of L1s occurs in the nucleus and APOBEC3 proteins are believed to act via DNA deamination during reverse transcription, activity against L1 retrotransposition was not correlated with nuclear localization of APOBEC3s. We demonstrate that APOBEC3C and APOBEC3B were endogenously expressed in HeLa cells. Accordingly, down-regulation of APOBEC3C by RNA interference enhanced L1 retrotransposition by approximately 78%. Sequence analyses of de novo L1 retrotransposition events that occurred in the presence of overexpressed APOBEC3 proteins as well as the analyses of pre-existing endogenous L1 elements did not reveal an enhanced rate of G-to-A transitions, pointing to a mechanism independent of DNA deamination. This study presents evidence for a role of host-encoded APOBEC3 proteins in the regulation of L1 retrotransposition.

APOBEC Deaminases↗
PubMed2006

Expression of the human endogenous retrovirus-K transmembrane envelope, Rec and Np9 proteins in melanomas and melanoma cell lines.

The human endogenous retrovirus-K encodes two potential tumor proteins, Rec and Np9. Rec is related to the Rev protein of HIV-1 and has been shown to be associated with tumor development in nude mice. Having shown the expression of human endogenous retrovirus-K in human melanomas and melanoma cell lines, tools were developed to allow the expression of the transmembrane envelope, Rec and Np9 mRNA and proteins to be studied in more detail. The expression of spliced env, rec and np9 was investigated by reverse transcriptase-polymerase chain reaction using a set of primers developed to discriminate between full-length and spliced mRNA. Env-specific, Rec-specific and Np9-specific antisera were produced, characterized and used to study protein expression in melanomas and melanoma cell lines by immunohistochemistry, immunofluorescence and Western blot analyses. Existence of human endogenous retrovirus-K Rec and Np9-specific antibodies in the sera of melanoma patients were analyzed by Western blot of immunofluorescence studies. The expression of both spliced env and rec mRNA was detected in 39% of the melanomas and in 40% of the melanoma cell lines and np9 mRNA was detected in 29 and 21%, respectively. In normal neonatal melanocytes, spliced rec mRNA was detected in the absence of spliced env mRNA. Using antisera specific for Rec and Np9, Rec protein was found in 14% of the melanomas but Np9 in none. In addition, cell surface expression of the putatively immunosuppressive transmembrane envelope protein and release of virus particles were shown. Antibodies specific for neither Rec nor Np9 were detected. The transmembrane envelope protein, Rec and Np9 proteins are expressed in melanoma cells with a pattern similar to that seen in teratocarcinoma cell lines. Additional experiments are needed to determine their involvement, if any, in cell proliferation and tumor progression.

Amino Acid Sequence↗
PubMed2006

Neopterin levels during the early phase of human immunodeficiency virus, hepatitis C virus, or hepatitis B virus infection.

BACKGROUND: A study was conducted to assess the diagnostic sensitivity of neopterin screening of blood donors with regard to the detection of window-phase specimens of human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) infection. STUDY DESIGN AND METHODS: In total, 1002 diagnostic window-phase specimens from 98 seroconversion panels (29 HIV-1, 52 HCV, and 17 HBV) were analyzed with viral antigen detection, viral nucleic acid amplification testing (NAT), and neopterin quantitation assays. The study was completed by the analysis of 92 anti-hepatitis B core antigen (HBc)-reactive and 103 alanine aminotransferase (ALT)-elevated blood donor specimens. RESULTS: A significant association between elevated neopterin concentrations and the very early phase of HIV-1 infection was found. No significant correlation could be observed between neopterin levels and the early phase of HCV or HBV infection. Neopterin concentration was not increased in specimens from blood donors with anti-HBc reactivity or ALT elevation. CONCLUSIONS: Neopterin screening of blood donors may identify window-phase cases of HIV, but not of HCV or HBV infection. The diagnostic sensitivity of neopterin screening during the HIV window phase is similar to that of the p24 antigen test. With the introduction of viral NATs in blood screening, there is no additional benefit of neopterin screening with regard to the three blood-borne viruses HIV, HCV, and HBV. Acute phases of other infectious agents, however, have been reported to be detected by neopterin enzyme-linked immunosorbent assays.

Alanine Transaminase↗
PubMed2005

Analysis of 5' junctions of human LINE-1 and Alu retrotransposons suggests an alternative model for 5'-end attachment requiring microhomology-mediated end-joining.

Insertion of the human non-LTR retrotransposon LINE-1 (L1) into chromosomal DNA is thought to be initiated by a mechanism called target-primed reverse transcription (TPRT). This mechanism readily accounts for the attachment of the 3'-end of an L1 copy to the genomic target, but the subsequent integration steps leading to the attachment of the 5'-end to the chromosomal DNA are still cause for speculation. By applying bioinformatics to analyze the 5' junctions of recent L1 insertions in the human genome, we provide evidence that L1 uses at least two distinct mechanisms to link the 5'-end of the nascent L1 copy to its genomic target. While 5'-truncated L1 elements show a statistically significant preference for short patches of overlapping nucleotides between their target site and the point of truncation, full-length insertions display no distinct bias for such microhomologies at their 5'-ends. In a second genome-wide approach, we analyzed Alu elements to examine whether these nonautonomous retrotransposons, which are thought to be mobilized through L1 proteins, show similar characteristics. We found that Alu elements appear to be predominantly integrated via a pathway not involving overlapping nucleotides. The results indicate that a cellular nonhomologous DNA end-joining pathway may resolve intermediates from incomplete L1 retrotransposition events and thus lead to 5' truncations.

Alu Elements↗
PubMed2005

Characterization of Parvovirus B19 genotype 2 in KU812Ep6 cells.

An infectious parvovirus B19 (B19V) genotype 2 variant was identified as a high-titer contaminant in a human plasma donation. Genome analysis revealed a 138-bp insertion within the p6 promoter. The inserted sequence was represented by an additional 30 bp from the end of the inverted terminal repeat adjacent to a 108-bp element found also, in inverted orientation, at the extreme right end of the unique sequence of the genome. However, despite the profound variations in the promoter region, the pattern of gene expression and DNA replication did not differ between genotype 1 and genotype 2 in permissive erythroid KU812Ep6 cells. Capsid proteins of both genotypes differ in their amino acid sequences. However, equivalent kinetics of virus inactivation at 56 degrees C or pH 4 indicated a comparable physicochemical stability of virus capsids. Sera from six individuals infected by B19V genotype 1 were investigated on cross-neutralization of B19V genotype 2 in vitro. Similar neutralization of both B19V genotypes was observed in sera from three individuals, while the sera from three other individuals showed weaker cross-neutralization for genotype 2. In conclusion, the in vitro replication characteristics and physical stability of B19V capsids are very similar between human parvovirus B19 genotypes 1 and 2, and cross-neutralization indicates a close antigenic relation of genotypes 1 and 2.

Base Sequence↗
PubMed2004

Leptin receptor isoform 219.1: an example of protein evolution by LINE-1-mediated human-specific retrotransposition of a coding SVA element.

Phylogenetically new insertions of repetitive sequences may contribute to genome evolution by altering the function of preexisting proteins. One example is the SVA sequence, which forms the C-terminal coding exon of the human leptin receptor isoform 219.1. Here, we report that the SVA insertion into the LEPR locus has occurred after divergence of humans and chimpanzees. The SVA element was inserted into a Hal-1/LINE element present in all monkeys and apes tested. Structural features point toward an integration event that was mediated by the L1 protein machinery acting in trans. Thus, our findings add evidence to the hypothesis that retrotransposition events are a driving force in genomic evolution and that the presence or absence of specific retroelements are one distinguishing feature that separates humans from chimpanzees.

Amino Acid Sequence↗
PubMed2004

Different susceptibility of B19 virus and mice minute virus to low pH treatment.

BACKGROUND: Parvoviridae are small nonenveloped viruses that are known to be highly resistant to physico-chemical treatments. Because low pH is frequently applied to process intermediates or final products, the impact of such conditions on the human erythrovirus B19 (B19V) and the mouse parvovirus (mice minute virus, MMV) was assessed, which is often used as a model for B19V. Owing to the lack of a suitable cultivation and/or detection system for B19V no such data exist so far. STUDY DESIGN AND METHODS: Virus inactivation was monitored by decrease of infectivity and loss of capsid integrity. Infectious B19V was quantified by detection of virus-specific messenger RNA from Ku812Ep6 cells. To measure capsid integrity, endonucleases were added after exposure to low pH and the encapsidated (endonuclease-protected) virus DNA was quantified by real-time PCR. RESULTS: B19V was inactivated greater than 5 log after 2 hours at pH 4, whereas MMV was resistant over 9 hours. Infectivity data strongly correlated with data obtained by the endonuclease assay. Capsid disintegration was observed in immunoglobulin G as well as in different albumin solutions. Temperature and pH showed concerted impact on B19V capsid disintegration. CONCLUSION: Our data show that B19V is much more vulnerable toward low pH conditions than MMV. Together with the previously reported susceptibility of B19V toward wet heat conditions, low pH is the second treatment where erythrovirus B19V is less resistant than viruses from the parvovirus genus.

Animals↗
PubMed2002

Hepatitis A virus infection in tamarins: experimental transmission via contaminated factor VIII concentrates.

An experimental hepatitis A virus (HAV) transmission study was performed in 3 tamarins, using a factor VIII concentrate linked to a recent outbreak of HAV infections in German hemophiliacs. The typical indicators for HAV infection were investigated in feces and serum samples. One tamarin showed a classical HAV infection with seroconversion. HAV antigen and HAV RNA were detected in feces of a second animal, but no seroconversion was observed until 19 weeks after inoculation. The HAV sequences from the reverse-transcription- and polymerase chain reaction-positive samples of the 2 animals were identical to the deduced HAV sequences of the chain of infection (from plasma pool to final product to patients). The study results provide conclusive evidence of the presence of infectious HAV in coagulation factor concentrate. Because a number of HAV transmission episodes have been described for solely solvent/detergent-treated factor VIII preparations, continued use of these agents seems questionable.

Alanine Transaminase↗
PubMed2002

Comparable virus inactivation by bovine or vegetable derived Tween 80 during solvent/detergent treatment.

A mixture of Tri-n-butyl phosphate (TNBP) and Polysorbate 80 (Tween 80) is often used for virus inactivation during the manufacture of medicinal products derived from human plasma. This procedure, known as solvent/detergent treatment, is of high effectiveness for inactivation of enveloped viruses. Tween 80 can be manufactured from bovine tallow or from vegetable material. As the bovine-derived Tween 80 is normally used for the solvent/detergent treatment, the question has been raised whether vegetable-derived Tween 80 can be applied as an alternative substance for the solvent/detergent treatment. Comparable inactivation studies were therefore performed using Vesicular Stomatitis Virus (VSV), Pseudorabiesvirus (PRV), Semliki Forest Virus (SFV) and Bovine Diarrhoea Virus (BVDV). In principle, no differences were observed in the effectiveness of the solvent/detergent treatment when bovine or vegetable-derived Tween 80 was used. The comparability in the efficiency of both detergents for virus inactivation was shown to be independent of solvent/detergent concentration, of temperature (16 degrees C and 6 degrees C vs. 27 degrees C and 25 degrees C) and protein concentration (10% and 5% human albumin). In summary, vegetable-derived Tween 80 is of the same effectiveness as bovine-derived Tween 80, when used for virus inactivation by the solvent/detergent treatment.

Animals↗
PubMed2002

Inactivation of parvovirus B19 during pasteurization of human serum albumin.

BACKGROUND: It has been shown that HSA may be contaminated with parvovirus B19 (B19) DNA. However, the presence of B19 DNA does not necessarily indicate infectious virus. HSA is pasteurized at 60 degrees C for 10 hours and it remains unclear whether this procedure inactivates B19. Studies with animal parvoviruses indicate considerable heat resistance at 60 degrees C. However, due to the lack of a suitable cell culture system, the pasteurization process has not been investigated in the past. STUDY DESIGN AND METHODS: The recently described cell clone KU812Ep6 was used to establish a system for investigation of B19 inactivation during pasteurization. Virus-infected cells were detected by immunofluorescent staining of viral capsid antigen and by RT-PCR assay of virus-specific capsid mRNA. RESULTS: B19 was inactivated after 10 minutes at 60 degrees C for > or = 4 log. In contrast, porcine parvovirus was widely resistant at 60 degrees C. Inactivation of B19 was independent of the analyzed albumin products (5, 20, and 25% albumin from three manufacturers) and from the specific virus source used for the inactivation experiments. Degradation of B19 DNA by deoxyribonuclease I treatment after pasteurization indicated that the virus capsid is destroyed during heat treatment. CONCLUSION: Heat resistance of B19 markedly differs from heat resistance of animal parvoviruses. While animal parvoviruses widely withstand pasteurization of albumin, B19 was rapidly inactivated. These results confirm the safety of pasteurized albumin and are in line with its good clinical safety record with respect to B19 infection. However, conclusions regarding the safety of other blood-derived medicinal products should not be derived from B19 inactivation in albumin, because different processes or different composition of product intermediates may significantly influence B19 stability during heat treatment.

Hot Temperature↗
PubMed2002

Sensitivity of HCV core antigen and HCV RNA detection in the early infection phase.

BACKGROUND: Various countries have introduced HCV NAT to exclude infectious donations collected during the preseroconversion window phase (PWP). For the same purpose, an ELISA has also been developed to detect HCV core antigen (cAg). STUDY DESIGN AND METHODS: Using sequential samples from regular plasma donors with very recent HCV infections, a total of 494 samples from 52 anti-HCV-negative donors were collected. These panels were used for direct comparison of the performance of PCR and ELISA in detecting viral markers (RNA and cAg) during the PWP of HCV infection. The panels were genotyped, and each sample was analyzed by qualitative and quantitative HCV PCR and by cAg ELISA. The HCV RNA doubling time was calculated from quantitation of viral RNA in consecutive samples during the earliest outbreak of viremia. RESULTS: Concurrent detection of HCV RNA and cAg in 218 and nondetection in 185 samples yielded 81.6-percent concordance in the results of 494 samples. Unidirectional discrepancy of results (i.e., PCR positive and cAg negative) was seen in 91 of 494 (18.4%) samples, which was consistent with 65 specimens with RNA concentrations ranging between 300 and 100,000 IU per mL and 26 specimens with less than 300 IU per mL (limit of quantitative PCR). Individual genotyped panels had different kinetics and courses of viremia. The mean doubling time in the early PWP at the onset of viremia was derived to be 10.8 (range, 5.8-21.0) hours. CONCLUSION: A majority of HCV RNA-positive samples were also cAg-positive during the PWP. The current cAg detection corresponds to 100,000 IU per mL of HCV RNA. Since low-titer samples would be identified only by single-donation NAT, which is often affordable only in developed countries, the cAg ELISA could offer a practical alternative for some countries. The doubling time for HCV RNA at the onset of viremia corresponds to a calculated mean delay of cAg detection during the virus burst phase of 2 or 5 days, when compared with minipool (5000 IU/mL) or single-donation NAT (50 IU/mL), respectively.

Enzyme-Linked Immunosorbent Assay↗
PubMed2002

Parvovirus B19 transmission by heat-treated clotting factor concentrates.

BACKGROUND: Human parvovirus B19 (B19) DNA can be frequently detected in plasma-derived coagulation factor concentrates. The production of some clotting factor products includes heat treatment steps for virus inactivation, but the effectiveness of such steps for B19 inactivation is unclear. Moreover, detailed transmission case reports including DNA sequence analysis and quantification of B19 DNA from contaminated heat-treated blood components have not been provided so far. Therefore, the correlation between B19 DNA in blood components and infectivity remains unclear. STUDY DESIGN AND METHODS: Asymptomatic B19 infections of two patients with hemophilia A were detected by anti-B19 seroconversion after administration of B19-contaminated heat-treated clotting factors. The suitability of nucleic acid sequence analysis for confirmation of B19 transmission was investigated. Furthermore, the B19 DNA level in blood components was determined and the drug administration was reviewed to calculate the amount of inoculated B19 DNA. RESULTS: Both B19 transmissions from clotting factor products could be confirmed by identical nucleic acid sequences of virus DNA from patients and blood components while sequences from unrelated controls could be differentiated. One patient received, for 4 days, a total of 180 mL vapor heat-treated prothrombin complex concentrate containing 8.6 x 10(6) genome equivalents per mL of B19 DNA. The other patient received 966 mL of low-contamination (4.0 x 10(3) genome equivalents/mL) dry heat-treated FVIII concentrate over a period of 52 days. CONCLUSION: B19 transmissions can be confirmed by nucleic acid sequencing. However, due to the low variability of the B19 genome, a large part of the B19 genome must be analyzed. The transmissions show that the applied heat treatment procedures were not sufficient to inactivate B19 completely.

Child, Preschool↗
PubMed1998

[Development of an alternative method to the monkey neurovirulence test for oral poliovirus vaccines]

Each oral poliovirus vaccine lot contains attenuated and small amounts of wildtype viruses. Vaccines containing more than a certain limit of wildtype viruses may cause a vaccine associated poliomyelitis. To provide safe vaccines for humans, each newly manufactured vaccine lot is tested in the monkey neurovirulence test. A certain point mutation on the poliovirus genome has been shown to be responsible for the attenuated phenotype of the vaccine virus. We developed a quantitative PCR method to determine the wildtype proportion at position 472 of poliovirus type 3 genome. This method possibly can be used as an alternative for the monkey neurovirulence test.

Journal Article↗