PubMed Health⌕ Search

Biomedical subjects

K Boller

Publications and source records attributed to K Boller.

At least 19 recordsLinked to original sources
PubMed2002

Productive infection of a mink cell line with porcine endogenous retroviruses (PERVs) but lack of transmission to minks in vivo.

Porcine endogenous retroviruses (PERVs) are considered a special risk for xenotransplantation because they are an integral part of the porcine genome and are able to infect cells of numerous species including humans in vitro. Among these cells, the mink lung epithelial cell line Mv1Lu could be productively infected with PERV. Provirus integration was detected by PCR, expression of viral proteins was shown by immunostaining and reverse transcriptase was detected in cell supernatants. PERV produced from mink cells could infect both, uninfected mink Mv1Lu cells and uninfected human 293 cells, with considerably higher virus production by human cells. Typical type C retroviruses were observed in PERV-infected mink cells using electron microscopy together with numerous multivesicular body (MVB)-like structures containing small virus-like particles, not present in uninfected mink cells. These MVBs could be stained with PERV-specific serum. In an attempt to establish a small animal model, PERV grown on mink cells was inoculated into adult and newborn American minks. Neither antibody production against PERV nor integration of viral DNA or production of viral proteins in tissues of different organs could be detected 12 weeks post virus inoculation, indicating that PERV infection had not occurred.

Animals↗
PubMed2001

Porcine endogenous retroviruses (PERVs): generation of specific antibodies, development of an immunoperoxidase assay (IPA) and inhibition by AZT.

Xenotransplantation may be associated with the risk of transmission of microorganisms. In particular, the porcine endogenous retroviruses (PERV) have raised concerns as in vitro experiments show susceptibility of human cells for PERV infection. However, it remains unclear whether PERVs are able to infect transplant recipients in vivo and whether they are pathogenic. It is therefore essential that the risks are evaluated and for this purpose specific and sensitive screening methods for PERVs have to be developed. We generated specific antibodies against all major structural proteins of PERV and developed several assays which allow antibodies against PERV to be detected as indirect evidence of infection. For direct detection of PERV production, reverse transcriptase (RT) assays were used. PCR methods were used to detect provirus integration and the presence of viral mRNA. Here we present an immunoperoxidase assay (IPA), which would allow the detection of viral proteins in infected cells as well as antibodies against PERV in the serum of an infected host. The specificity of the sera used in the assay was determined by several methods, including immunoelectron microscopy, and the sensitivity of the assay was compared with other methods. This IPA was used to detect PERV infection in in vitro experiments for evaluation of the virus host range, for titrating the virus and for testing anti-viral properties of AZT. Using this method it was shown that AZT inhibits replication of PERV. This IPA may be very useful for the surveillance of preclinical and clinical xenotransplantations.

Amino Acid Sequence↗
PubMed2000

Establishment and characterization of molecular clones of porcine endogenous retroviruses replicating on human cells.

The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems overcoming immunological and physiological barriers, the existence of numerous porcine microorganisms poses the risk of initiating a xenozoonosis. Recently, different classes of type C porcine endogenous retoviruses (PERV) which are infectious for human cells in vitro have been partially described. We therefore examined whether completely intact proviruses exist that produce infectious and replication-competent virions. Several proviral PERV sequences were cloned and characterized. One molecular PERV class B clone, PERV-B(43), generated infectious particles after transfection into human 293 cells. A second clone, PERV-B(33), which was highly homologous to PERV-B(43), showed a G-to-A mutation in the first start codon (Met to Ile) of the env gene, preventing this provirus from replicating. However, a genetic recombinant, PERV-B(33)/ATG, carrying a restored env start codon, became infectious and could be serially passaged on 293 cells similar to virus clone PERV-B(43). PERV protein expression was detected 24 to 48 h posttransfection (p. t.) using cross-reacting antiserum, and reverse transcriptase activity was found at 12 to 14 days p.t. The transcriptional start and stop sites as well as the splice donor and splice acceptor sites of PERV mRNA were mapped, yielding a subgenomic env transcript of 3. 1 kb. PERV-B(33) and PERV-B(43) differ in the number of copies of a 39-bp segment in the U3 region of the long terminal repeat. Strategies to identify and to specifically suppress or eliminate those proviruses from the pig genome might help in the production of PERV-free animals.

Animals↗
PubMed1999

Reverse transcriptase activity and particle production in B lymphoblastoid cell lines established from lymphocytes of patients with multiple sclerosis.

We have established spontaneously formed B lymphoblastoid cell lines from long-term cultured peripheral blood mononuclear cells (PBMNCs) from multiple sclerosis (MS) patients. The MS cell lines actively produce retrovirus-like particles as well as Epstein-Barr virus (EBV). Using three different variations of the highly sensitive polymerase chain reaction (PCR)-based assays for the detection of reverse transcriptase (RT) activity, we have verified the retroviral origin of the retrovirus-like particles that are produced in very low amounts by the MS cell lines.

B-Lymphocytes↗
PubMed1999

Oligonucleotide and plasmid DNA packaging into polyoma VP1 virus-like particles expressed in Escherichia coli.

The drug delivery system described here is based on the properties of the capsoid or capsid-like structure resulting from the assembly of polyoma virus capsid protein VP1 expressed in Escherichia coli. The capsid protein VP1 was expressed as a fusion protein with a completely removable N-terminal His6 affinity tag. The pentameric morphology of the recombinant VP1 protein was confirmed by electron microscopy after affinity chromatography and factor Xa cleavage under conditions of low ionic strength. The self-assembly of VP1 capsoids can be induced from purified VP1 pentamers by increasing the ionic strength with (NH4)2SO4. These VP1 capsoid particles were packed in vitro with anti-sense oligonucleotides and plasmid DNA. The loading with DNA was pH-dependent. We observed the highest efficiency at pH 5. DNase I treatment of particles with encapsidated material showed that 37-55% of the bound oligonucleotides and fragments of 1.5-1.8 kb double-stranded DNA were protected against degradation.

Base Sequence↗
PubMed1997

Characterization of human endogenous retrovirus type K virus-like particles generated from recombinant baculoviruses.

The family of human endogenous retrovirus type K (HERV-K) comprises members with long open reading frames (ORF) for retroviral proteins. The existence of a biologically active provirus with replicative capacities has not yet been demonstrated. To confirm the assumption that HERV-K codes for the previously observed retrovirus-like particles (human teratocarcinoma-derived virus, HTDV) in human teratocarcinoma cells, we have constructed recombinant full-length HERV-K cDNA-based baculoviruses with gag, pro, pol, and env ORFs. Two viral constructs were used for infections of insect cells, one bearing 67 bp of the 5' untranslated region upstream of the 5' splice donor (SD) site and of the retroviral genes, the second omitting the SD sequence. For both recombinant viruses, indirect immunofluorescence and laser scan analyses revealed expression of HERV-K Gag protein. Electron microscopy studies demonstrated efficient production of virus-like particles (VLPs) at the cytoplasmic cell membranes. These VLPs are morphologically identical with the HTDV phenotype. In immunoelectron microscopy of ultrathin frozen sections, anti-HERV-K Gag antibodies specifically reacted with HERV-K VLPs. In Western blots, in addition to the 76-kDa precursor protein, the putative major core protein with an apparent molecular mass of 32 kDa exhibited predominant immunoreactivity with anti-Gag antiserum. In contrast, neither HERV-K Env nor cORF proteins could be detected due to inefficient mRNA splicing. Purified particles from insect cell culture supernatants tested in an ultrasensitive reverse transcriptase assay revealed weak polymerase activity. The data demonstrate that HERV-K codes for retroviral particles of the HTDV phenotype.

Animals↗
PubMed1997

Preexposure effects on infant learning and memory.

The effect of passively exposing infants to visual information in a sensory preconditioning paradigm was assessed in five experiments with seventy-eight 6-month-olds. In the basic paradigm, infants were simultaneously exposed to two contexts (S1 and S2), trained in one of them (S1), and tested in the other (S2). Infants learned an association between S1 and S2 and exhibited transfer to S2 after preexposures of 1 hr/day for 1 week but not after 2 min or 1 hr on a single occasion. When S1 was the focal cue, however, infants transferred responding after a single preexposure lasting only 2 min. Extended preexposure also produced learned irrelevance, biasing subsequent selective attention, but there was no evidence of latent inhibition. These findings demonstrate that young infants pick up fairly sophisticated information about specific stimuli in their visual surround and the relationships between them merely as a result of passive observation, without explicitly being trained to do so. Moreover, infants may not express this knowledge for several days, until finally given an opportunity to do so. Sensory preconditioning may be an instance of "unitization"-a special type of infantile learning that enables immature organisms to acquire some associations more rapidly than adults.

Adult↗
PubMed1997

Characterization of the antibody response specific for the human endogenous retrovirus HTDV/HERV-K.

Differentiated human teratocarcinoma cell lines produce the human teratocarcinoma-derived virus (HTDV) particles encoded by the human endogenous retrovirus sequence HERV-K. We screened almost 2,000 human sera for antibodies against this endogenous human retrovirus, HTDV/HERV-K. Specificity of the immunofluorescence reactions using particle producing teratocarcinoma cells was confirmed by immunoelectron microscopy of ultrathin frozen sections. Immunoblot analyses using lysates of HTDV-producing cells revealed a 80-kDa HERV-K Gag precursor and a 90-kDa putative viral Env protein after incubation with positive sera. No processed Gag protein could be observed. Virus-specific bands were not detected in lysates of nonproducing cells. High antibody titers were found in about 60% of male patients with germ cell tumors. Antibody reactivity declined after tumor removal. In healthy blood donors, anti-HTDV reactivity was found only at low titers in a small percentage (3.9%) of individuals. A slightly elevated but statistically significant percentage of HTDV positivity was also observed for sera of pregnant women, whereas human immunodeficiency virus-positive individuals exhibited no peculiarity compared to normal blood donors. Our results provide evidence that HTDV particles are expressed in vivo and that the immune reaction against HTDV/HERV-K is specific for defined viral proteins.

Antibodies, Viral↗
PubMed1996

Infants' memory for context: timing effects of postevent information.

How infants' memories of the context or place where an event occurred are selectively distorted was studied in 6-month-olds, who kicked to activate a mobile in a distinctive context for 2 days and then saw that mobile in a different context for 2 min either 1 or 6 days later. Infants whose exposure delay was 1 day failed to recognize the mobile in either the original or the exposed context but did so in a completely novel one. Infants whose exposure delay was 6 days--after the details of the original context were forgotten--recognized the mobile in the exposed context and no other, and the mobile could reactivate the forgotten memory only in the exposed context and no other. In effect, the details of the exposed context appeared to have been substituted in memory for the original details that had been forgotten. Thus, when components of an event are encountered later in a new context, that new context may be falsely remembered as being where the event had actually occurred, and the original context may be forgotten altogether. This error is more likely when a memory is older and may underlie some of the inaccuracies of childrens' and adults' recollections of their prior experiences.

Adult↗
PubMed1996

HERV-K: the biologically most active human endogenous retrovirus family.

The human genome contains a wide variety of endogenous retrovirus-like sequences. The human endogenous retrovirus type K (HERV-K) family comprises 30-50 members per haploid genome in humans and is highly conserved in Old World monkeys and apes. Some proviruses are displaying open reading frames (ORF) with coding capacity for viral particles. HERV-K sequences most likely code for the previously described human teratocarcinoma-derived virus (HTDV) and correlated expression of HERV-K Gag has been demonstrated by immunoelectron microscopy studies. Protease, but not yet reverse transcriptase (RT), enzymatic activity was demonstrated for recombinant HERV-K proteins. However, an ultrasensitive RT assay revealed specific polymerase activity associated with the HTDV particles. HERV-K transcription is specifically regulated by viral long terminal repeats and RNA is expressed at low steady-state levels in a variety of human tissues and tumours. In teratocarcinoma cell lines, HERV-K is highly expressed in a complex pattern showing full-length as well as subgenomic envelope (env) and two alternatively spliced small transcripts. The doubly spliced 1.8-kb mRNA codes for cORF protein which resembles Rev of HIV-1 and is located in the nucleolus. In addition, the cORF sequence acts as a leader and is essential for effective expression of glycosylated HERV-K Env protein. Although HERV-K sequences code for all necessary retroviral proteins, infectious particles could not yet be demonstrated. The putative implication of HERV sequences in pathophysiological processes, for example, testicular malignancies, remains to be elucidated.

Animals↗
PubMed1995

Effects of postevent information on infants' memory for a central target.

In three experiments with 78 infants, we explored the effect of introducing novel information about a central target after a short delay on 6-month-olds' recognition of the original target, the novel exposure target, and a completely novel one. In all experiments, infants learned to move a particular crib mobile (the central target) by kicking and then were exposed to a novel mobile (the conflicting postevent information) immediately after training was over. In Experiment 1, memory for the original mobile was unimpaired, but infants treated both the exposure mobile and a completely novel one as if they had actually been present originally. In Experiment 2, only a completely novel mobile was an effective remainder in a reactivation paradigm, indicating that the impact of postevent information was relatively long-lasting and that the failure of the other mobiles to recover the training memory resulted from trace competition at the time of retrieval. In Experiment 3, identical novel information did not proactively affect recognition. Thus, 6-month-olds' memory of a central target is resistant to impairment by conflicting postevent information after a short delay, but they are highly prone to source misattribution. We propose that postevent information effects are cognitively efficient.

Association Learning↗
PubMed1994

Contextual updating of infants' reactivated memories.

The ease with which a reactivated memory is updated has major implications for whether or not a prior memory is likely to be retrieved in the future. In three experiments, we explored this problem with nonverbal human infants, whose newly acquired memory of training in a specific context is readily updated by novel contextual information. In Experiment 1, exposing infants to a novel context immediately after a successful reactivation treatment neither impaired their retention in the original context nor facilitated it in the novel exposure one. In Experiment 2, increasing the delay between the reactivation treatment and exposure to the novel context also failed to facilitate retention in the novel test context. In Experiment 3, the reactivated memory was updated when the contingency was briefly experienced in the novel context immediately after the reactivation treatment. Under these circumstances, previously trained infants exhibited retention in the novel context, but infants who had not been trained 3 weeks earlier or whose original memory had not been reactivated exhibited none. The resistance of a reactivated memory to contextual updating unless the new context is predictive apparently buffers infants' memories against revision after long delays by contexts that could be inappropriate.

Attention↗
PubMed1993

Identification of human endogenous retroviruses with complex mRNA expression and particle formation.

Retroviruses comprise strains with considerable disease potential in animals and humans. In addition to exogenous strains transmitted horizontally, endogenous proviruses are transmitted through the germ line. Some of these endogenous retroviruses can be pathogenic in mice and possibly in other animal species. They may also be considered as mobile genetic elements with the potential to produce mutations. In humans, genomic DNA contains numerous endogenous retroviral sequences detected by their partial relatedness to animal retroviruses. However, all proviruses sequenced so far have been found to be defective. In this communication, we describe the expression of a family of human endogenous retrovirus sequences (HERV-K) in GH cells, a teratocarcinoma cell line producing the human teratocarcinoma-derived retrovirus (HTDV) particles previously described by us. Four viral mRNA species could be identified, including a full-length mRNA. The other three subgenomic mRNAs are generated by single or double splicing events. This expression pattern is reminiscent of the more complex control of virus gene regulation observed, for example, with lenti- or spumavirus strains, although HERV-K shows no sequence homology to human T-lymphotropic virus or human immunodeficiency virus. Sequence analysis of expressed HERV-K genomes revealed non-defective gag genes, a prerequisite for particle formation. Open reading frames were also observed in pol and env. Antisera raised against recombinant gag proteins of HERV-K stained HTDV particles in immunoelectron microscopy, linking them to the HERV-K family.

Base Sequence↗
PubMed1993

Evidence that HERV-K is the endogenous retrovirus sequence that codes for the human teratocarcinoma-derived retrovirus HTDV.

Human teratocarcinoma-derived viruses (HTDV) are retrovirus-like particles that are regularly observed by electron microscopy at low frequency in cell lines established from human teratocarcinomas. Over the last years, one of our teratocarcinoma cell lines spontaneously began to produce high amounts of HTDV. This cell line is stained in immunofluorescence tests by an antiserum raised against recombinant gag protein of HERV-K, an expressed human endogenous retrovirus sequence. In immunoelectron microscopy of ultrathin frozen sections, this anti HERV-Kgag-specific antiserum reacts specifically with HTDV particles. In Western blots, the antiserum recognizes predominantly a protein with an apparent molecular weight of 30 kDa, presumably the major core protein of HTDV particles. Taken together, these results provide evidence that HERV-K codes for HTDV.

Fluorescent Antibody Technique↗
PubMed1993

Infants' eyewitness testimony: effects of postevent information on a prior memory representation.

In eyewitness testimony research, postevent information impairs retention of the original event and increases the probability that interpolated information will be identified as part of the original event. The present experiments studied these effects with 3-month-olds. Infants learned to kick to move a particular crib mobile and then were briefly exposed to information about a novel mobile. The novel postevent information impaired recognition of the original mobile when it immediately followed training but not when it was delayed by 1 day. Like adults, infants treated the postevent information as part of the original training event, continuing to do so for at least 2 weeks. We propose that postevent information displaces conflicting information coactive with it in primary memory and creates a new, updated memory token of the event. Once the new token leaves primary memory, however, it is protected; only a copy can be retrieved and modified in the future.

Association Learning↗