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S Modrow

Publications and source records attributed to S Modrow.

At least 19 recordsLinked to original sources
PubMed2000

Seroprevalence of parvovirus B19 NS1-specific IgG in B19-infected and uninfected individuals and in infected pregnant women.

Parvovirus B19 is the causative agent of erythema infectiosum in children, but the virus is associated with an increasing range of different diseases. These include acute and chronic arthritis, hydrops fetalis in pregnant women, aplastic anemia, and thrombocytopenia. The host's immune response is directed against the viral structural proteins VP1 and VP2. This study investigated the presence of IgG against the viral nonstructural protein NS1 using Western blot. Serum panels from healthy individuals, B19-infected pregnant women, and various disease groups were tested. The disease groups included patients with symptoms that may be linked to parvovirus B19 infection. The results showed that IgG against the NS1 protein was present in 22% of healthy individuals with past B19 infection. In cases of persistent or prolonged B19 infections, the prevalence of NS1-specific antibodies was as high as 80%. It is concluded that NS1-specific IgG may be used as an indicator of chronic or more severe courses of parvovirus B19 infections.

Adolescent↗
PubMed1999

Frequency of CD8(+) T lymphocytes specific for lytic and latent antigens of Epstein-Barr virus in healthy virus carriers.

We investigated CD8(+) T cell frequencies of five different Epstein-Barr virus-specific cytotoxic T lymphocyte epitopes located within proteins of the replicative cycle and the latent state in healthy long-term virus carriers with IFN-gamma enzyme-linked immunospot assay. Frequencies of the HLA-A3-restricted epitope RVRAYTYSK (RVR) whose minimal length was mapped in this study to amino acid position 148-156 of the immediate-early protein BRLF1 were compared with those of a further known HLA-A3-restricted epitope within EBNA3A, RLRAEAQVK (RLR). Determination of frequencies of CD8(+) T lymphocytes directed against lytic antigen epitope RVR revealed that only one of eight donors recognized this epitope. Frequency was calculated to be 65 RVR-specific CD8(+) T lymphocytes per 10(6) PBMC. None of the HLA-A3-positive donors exhibited IFN-gamma release after antigenic stimulation with the EBNA3A-specific peptide epitope RLR. Furthermore, we chose three known HLA-B8-restricted epitopes, RAKFKQLL (RAK), FLRGRAYGL (FLR), and QAKWRLQTL (QAK), of the lytic protein BZLF1 and the latent protein EBNA3A. Examination of eight HLA-B8-positive virus carriers revealed that the BZLF1-specific epitope RAK was recognized by all donors with a median frequency of 233 RAK-specific CD8(+) T lymphocytes per 10(6) PBMC. Only 50% of these donors reacted against EBNA3A-specific epitope FLR and a minority (25%) reacted against EBNA3A-specific epitope QAK.

CD8-Positive T-Lymphocytes↗
PubMed1999

BiP-binding sequences in HIV gp160. Implications for the binding specificity of bip.

BiP, a resident endoplasmic reticulum member of the HSP70 family of molecular chaperones, associates transiently with a wide variety of newly synthesized exocytotic proteins. In addition to immunoglobulin heavy and light chains, the first natural substrates identified for BiP, a number of viral polypeptides including the human immunodeficiency virus type 1 envelope glycoprotein gp160 interact with BiP during their passage through the endoplasmic reticulum. We have used a computer algorithm developed to predict BiP-binding sites within protein primary sequences to identify sites within gp160 that might mediate its association with BiP. Analysis of the ability of 22 synthetic heptapeptides corresponding to predicted binding sites to stimulate the ATPase activity of BiP or to compete with an unfolded polypeptide for binding to BiP indicated that about half of them are indeed recognized by the chaperone. All of the confirmed binding sites are localized within conserved regions of gp160, suggesting a conserved role for BiP in the folding of gp160. Information on the characteristics of confirmed BiP-binding peptides gained in this and previous studies has been utilized to improve the predictive power of the BiP Score algorithm and to investigate the differences in peptide binding specificities of HSP70 family members.

Adenosine Triphosphatases↗
PubMed1999

Acute parvovirus B19 infection in connection with a flare of systemic lupus erythematodes in a female patient.

BACKGROUND: Since its discovery parvovirus B19-infections could be linked to a growing variety of diseases. Besides the harmless exanthema erythema infectiosum perferentially observed with B19-infections in childhood a panel of rather serious and also chronic courses that may be associated with anemia, thrombocytopenia, arthritis and others have been described. OBJECTIVE: In a 26-year-old female patient an acute parvovirus B19-infection was followed by a serious episode of systemic lupus erythematosus (SLE). Here we demonstrate the clinical and serological parameters which were observed in the patient during that episode in addition to the nucleotide sequence of the virus isolate. RESULTS AND CONCLUSION: In this patient parvovirus B19 was not the initial causative agent for SLE. However the B19 infection was followed by a severe flare of SLE and therefore may be considered as an enhancer of the autoimmune disease. The amount of nucleotide variability observed in the viral genome was in the range known from other B19 isolates. An elevated degree of mutations in antigenic domains was not detectable. Therefore, we would like to emphasize the possible role of parvovirus B19 in the aetiology or the enhancement of autoimmune diseases like SLE and the necessity of an according differential diagnosis.

Adult↗
PubMed1999

Infection of apheresis cells by parvovirus B19.

Parvovirus B19 is the only member of the Parvoviridae family known to cause disease in humans. Owing to the high level of cell tropism the virus can only replicate in proliferating and differentiating erythroid precursor cells, which are present in human bone marrow and foetal liver. As human bone marrow is very difficult to obtain, an alternative in vitro system for the propagation of B19 virus has been developed, based on the application of mobilized haemapoietic progenitor (apheresis) cells. These cells are routinely harvested from cancer patients after treatment with recombinant human granulocyte/macrophage colony-stimulating factor. Replication of parvovirus B19 in vitro is possible in these cells after stimulation with erythropoietin. Therefore, this system is an easily, accessible alternative to the use of human bone marrow in parvovirus B19 infection assays.

Blood Component Removal↗
PubMed1999

Generation of neutralizing human monoclonal antibodies against parvovirus B19 proteins.

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 microgram/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 micrograms/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.

Amino Acid Sequence↗
PubMed1998

Interaction of HLA-E with peptides and the peptide transporter in vitro: implications for its function in antigen presentation.

The assembly of MHC Ia molecules in the endoplasmic reticulum requires the presence of peptide ligands and beta2m and is facilitated by chaperones in an ordered sequence of molecular interactions. A crucial step in this process is the interaction of the class I alpha-chain/beta2m dimer with TAP, which is believed to ensure effective peptide loading of the empty class I molecule. We have previously demonstrated impaired intracellular transport of the class Ib molecule HLA-E in mouse myeloma cells cotransfected with the genes for HLA-E and human beta2m, which is most likely attributable to inefficient intracellular peptide loading of the HLA-E molecule. We therefore analyzed the ability of HLA-E in the transfectant cell line to bind synthetic peptides by means of their ability to enhance cell surface expression of HLA-E. Peptide binding was confirmed by testing the effect on the thermostability of soluble empty HLA-E/human beta2m dimers. Two viral peptides binding to HLA-E were thus identified, for which the exact positioning of the N terminus appeared critical for binding, whereas the contribution of the length of the C terminus seemed to be minor, allowing peptides as short as seven amino acids and up to 16 amino acids to exhibit considerable binding activity. Furthermore, we demonstrate that HLA-E interacts with TAP and that this interaction can be prolonged by the proteasome inhibitor N-acetyl-L-leucyl-L-leucyl-L-norleucinal, which reduces the intracellular peptide pool. The presented data indicate that HLA-E is capable of presenting peptide ligands similar to the repertoire of HLA class Ia molecules.

Amino Acid Sequence↗
PubMed1998

Persistent parvovirus B19 infections in immunocompromised children.

Immunocompromised patients have been shown to suffer from prolonged viral infections often without detectable immune response. Here chronic infections with low virus levels can be frequently observed. In these patients viral DNA can be detected over long periods by polymerase chain reaction (PCR). In this study parvovirus B19 presence was assessed by PCR, immunoblot and enzyme-linked immunosorbent assay in sera from children with mainly oncological and hematological diseases. In 45% of sera B19 DNA was observed. Of the children 25% had IgG antibodies to viral protein 1 and 2 (VP1/2) and 15% to nonstructural protein 1 (NS1). In 6% of children IgM antibodies to VP1/2 were detected. These results indicate that the number of children with immune response to B19 proteins is distinctly lower than the number of children with B19 DNA. Transfusions of blood products might have been a possible route for B19 infection. Establishment and maintenance of a persistent parvovirus B19 infection with or without immune response are enhanced in the analyzed immunocompromised children in comparison with immunocompetent children. A persistence of B19 DNA was demonstrated up to 10 months in patients sera.

Adolescent↗
PubMed1998

Characterization of cis-acting and NS1 protein-responsive elements in the p6 promoter of parvovirus B19.

Parvovirus B19 infections are associated with diverse clinical manifestations, ranging from no symptoms to severe symptoms. The virus shows an extreme tropism for replication in erythroid progenitor cells, possibly due to the activity of the only functional promoter (p6) of the B19 virus genome in combination with both cell- and cell cycle-specific factors and the trans-activator protein NS1. As presented here, p6 promoter sequences derived from several B19 virus isolates proved to be highly conserved. Furthermore, mutations did not affect any of the potential binding sites for transcription factors. One variation of the base at position 223 was identified only in B19 virus isolates derived from patients with persistent infection or chronic arthritis. To determine promoter activity and to characterize regulatory elements, sequences spanning the total p6 promoter and subfragments of them were introduced into a eukaryotic expression vector upstream of the luciferase gene (from Photinus pyralis). After transfection into HeLa, CEM, BJAB, and K562 cells, the p6 promoter was found to be highly active. When introduced into the erythroid cell line K562, p6-controlled transcription exceeded that of the simian virus 40 promoter-enhancer used as a control by more than 25-fold. Sequence elements relevant for promoter activity mapped to the regions from nucleotides (nt) 100 to 190 and 233 to 298. Also, the segment from nt 343 to 400 downstream of the TATA box was important for transcriptional activity in HeLa and K562 cells. By transfecting the promoter-luciferase constructs into a HeLa cell line stably carrying the viral NS1 gene under the control of an inducible promoter, transcriptional activity mediated by the p6 promoter rose significantly after induction of NS1 expression. The region from nt 100 to 160 proved to be essential for NS1-mediated transcriptional activation. Furthermore, NS1-mediated transactivation was dependent on the presence of two GC-rich elements arranged in tandem upstream of the TATA box. These data indicate that NS1-mediated p6 transactivation is dependent on a multicomponent complex combining NS1 with ATF, NF-kappaB/c-Rel, and GC-box binding cellular factors.

Base Sequence↗
PubMed1998

Immediate-early transactivator Rta of Epstein-Barr virus (EBV) shows multiple epitopes recognized by EBV-specific cytotoxic T lymphocytes.

We analyzed the immediate-early transactivator Rta of Epstein-Barr virus (EBV) for its role as a target for specific cytotoxic T lymphocytes (CTL). Panels of overlapping peptides covering the entire amino acid sequence of Rta were synthesized and used to induce and analyze specific CTL responses in EBV-positive donors. Using peptide-pulsed target cells, we found nine different CTL epitopes that are distributed over the entire protein sequence. One epitope restricted by HLA-A24 could be mapped to the decameric sequence DYCNVLNKEF between amino acid positions 28 and 37 of the Rta protein. A second epitope could be assigned to the same region of Rta (residues 25 to 39) and was shown to be restricted by HLA-B18. Another, minimal epitope could be mapped to the nonameric sequence ATIGTAMYK between amino acid positions 134 and 142; this peptide was restricted by HLA-A11. Another four epitopes were proven to be restricted by HLA-A2, -A3, -B61, and -Cw4 and were located between Rta residues 225 and 239, 145 and 159, 529 and 543, and 393 and 407, respectively. For two other epitopes, only the location within the Rta protein is known so far (residues 121 to 135 and 441 to 455); their exact HLA restriction patterns have not yet been identified. Using target cells infected with recombinant vaccinia virus containing the gene for Rta, we showed that six of eight Rta-specific CTL lines recognized the corresponding peptides also after endogenous processing. These data suggest that Rta comprises an important target for EBV-specific cellular cytotoxicity. Together with recent findings of other immediate-early and early proteins also acting as CTL targets, they reveal the role of proteins of the lytic cycle in the immune recognition of EBV-infected cells.

Amino Acid Sequence↗
PubMed1998

Receptor binding sites and antigenic epitopes on the fiber knob of human adenovirus serotype 3.

The adenovirus fiber knob causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s), the interaction of labeled cell membrane proteins to synthetic peptides covering the adenovirus type 3 (Ad3) fiber knob was studied. Peptide P6 (amino acids [aa] 187 to 200), to a lesser extent P14 (aa 281 to 294), and probably P11 (aa 244 to 256) interacted specifically with cell membrane proteins, indicating that these peptides present cell receptor binding sites. Peptides P6, P11, and P14 span the D, G, and I beta-strands of the R-sheet, respectively. The other reactive peptides, P2 (aa 142 to 156), P3 (aa 153 to 167), and P16 (aa 300 to 319), probably do not present real receptor binding sites. The binding to these six peptides was inhibited by Ad3 virion and was independent of divalent cations. We have also screened the antigenic epitopes on the knob with recombinant Ad3 fiber, recombinant Ad3 fiber knob, and Ad3 virion-specific antisera by enzyme-linked immunosorbent assay. The main antigenic epitopes were presented by P3, P6, P12 (aa 254 to 269), P14, and especially the C-terminal P16. Peptides P14 and P16 of the Ad3 fiber knob were able to inhibit Ad3 infection of cells.

Adenoviruses, Human↗
PubMed1997

Inhibition of human immunodeficiency virus type 1 particle formation by alterations of defined amino acids within the C terminus of the capsid protein.

In previous studies, we demonstrated that the substitution of amino acid triplets for alanines in the carboxy-terminal portion (amino acids 341-352: ATL EEM MTA CQC) of the capsid protein domain (p24) of human immunodeficiency virus type 1 (HIV-1) partly led to an inhibitory effect on the capacity to form virus-like particles (VLPs). In these experiments, the uncleaved Pr55gag precursor protein was expressed by recombinant vaccinia viruses. We have now investigated the effects of these mutations with respect to a replication-competent HI-provirus system. Substitution of amino acids 344-346 (EEM) for alanines, which was previously shown to lead to an inhibition of VLP formation, completely blocked assembly and release of HIV. A substantial reduction of HIV synthesis was also observed in the proviral system after exchange of amino acids 347-348 [MT(A)] which, in contrast, was formerly shown to result in an increased formation of VLPs. Western blot analysis of lysates of cells transfected with these mutated proviral constructs revealed an abnormal intracellular processing pattern of the Pr55gag precursor molecules. Further analyses suggest a structural aberration of these altered polyproteins as the basis for the observed block of virus formation.

Amino Acid Substitution↗
PubMed1996

Inhibition of cell adhesion to the virus by synthetic peptides of fiber knob of human adenovirus serotypes 2 and 3 and virus neutralisation by anti-peptide antibodies.

The fiber knob of adenovirus (Ad) causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s) several synthetic peptides derived from Ad2 and Ad3 fiber head sequences and their antisera were tested for interference with virus attachment to HeLa and FL cells and cell adhesion to viruses. The anti-peptide sera were also evaluated in ELISA and virus neutralisation test. Ad2 (of subgroup C) and Ad3 (of subgroup B) attachment was not significantly inhibited by peptides corresponding to the amino acid residues 535-554, 555-573, 562-582 of Ad2 fiber or 210-225, 267-283, 291-306 and 300-319 of Ad3 fiber. However, microplate pre-adsorbed Ad3 fiber residues 210-225 and 267-283 could bind FL and HeLa cells, and 1 mg/ml of Ad3 fiber residues 267-283 inhibited the cell adhesion to Ad3 virus to approximately 90%. This peptide may participate in the receptor binding site of Ad3 fiber. ELISA reactive anti-peptide antibodies against the homologous peptide and virus did not significantly reduce the cell adhesion to the immobilised virus or the virus attachment to cells, but in the neutralisation assay antibodies raised to Ad2 fiber residues 555-573 and 562-582 and Ad3 fiber residues 210-225 caused neutralisation of the homologous virus at serum dilutions of 1:500 and 1:32, respectively. The corresponding peptides and one further peptide of Ad2 fiber and two of Ad3 fiber seem to contain neutralisation epitopes.

Adenoviruses, Human↗
PubMed1996

Sequence variability among different parvovirus B19 isolates.

Parvovirus B19 is the causative agent of a variety of clinical manifestations, ranging from asymptomatic to severe infection. The basis for this complex pattern of B19-associated diseases is as yet poorly understood. In general there are two different possibilities: firstly, the infected individuals may have a genetic or acquired predisposition, which renders them susceptible for a certain course of infection; secondly, differences in the B19 genome may result in different outcomes of infection. In order to investigate this second possibility we have partially sequenced the genomes of 20 different B19 isolates derived from serum samples from patients with various B19-associated diseases. Four distinct regions, which cover nearly half of the genome and include parts of the coding regions of all three major B19 proteins-NS1, VP1 and VP2, were selected for sequencing. Comparisons between the different extracted virus isolates at the DNA and protein levels revealed that isolates from patients with persistent parvovirus B19 infection show a tendency towards higher genome variability with respect to isolates derived from persons with acute infection.

Base Sequence↗
PubMed1996

Lymphoproliferative responses after infection with human parvovirus B19.

Immunity after infection with the parvovirus B19 is assumed to be conferred by a humoral immune response with development of neutralizing antibody. In contrast, little is known about the nature of T-cell-mediated responses to parvovirus B19 infection in humans. We used recombinant proteins VP1, VP2, and NS1, as well as a recombinant VP1-specific amino-terminal sequence, to test the proliferative responses of peripheral blood mononuclear cells after infection of otherwise healthy individuals with parvovirus B19. These proteins were used as antigens for the stimulation of freshly isolated cells. The results show that a B19 virus-specific cellular immunity develops that is directed against the capsid proteins VP1 and VP2. We also demonstrate that viral determinants are presented to CD4+ T cells by HLA class II molecules.

Antigens, Viral↗
PubMed1996

Defined amino acids in the gag proteins of human immunodeficiency virus type 1 are functionally active during virus assembly.

A structurally highly ordered arrangement of the polyprotein precursor, Pr55gag is a necessary prerequisite for assembly, budding and maturation of the human immunodeficiency virus type 1 (HIV-1). In particular, distinct regions of the matrix protein (p17) and the capsid protein (p24) contained within Pr55gag are functionally active during these processes. In order to determine such regions we exchanged amino acid triplets within p17 (amino acids 46-61) and p24 (amino acids 341-352) for alanine residues and deleted the whole regions. Synthetic peptides derived from these regions had been shown previously to inhibit the production of infectious virus. The effect of the mutations on the release of viral particles was investigated by using recombinant baculoviruses for the expression of mutated Pr55gag as virus-like particles and by use of the respective HI proviruses for monitoring the production of infectious particles.

Amino Acids↗
PubMed1995

BiP binding sequences in antibodies.

During the process of folding and assembly of antibody molecules in the endoplasmic reticulum, immunoglobulin heavy and light chains associate transiently with BiP, a resident endoplasmic reticulum protein that is a member of the Hsp70 family of molecular chaperones. BiP is thought to recognize unfolded or unassembled polypeptides by binding extended sequences of approximately seven amino acids that include bulky hydrophobic residues not normally exposed on the surface of native proteins. We used a computer algorithm developed to predict BiP binding sites within protein primary sequences to identify sites within immunoglobulin chains that might mediate their association with BiP. Very few of the sequential heptapeptides in the heavy or light chain sequences were potential BiP binding sites. Analysis of the ability of synthetic heptapeptides corresponding to 24 potential sites in heavy chains to stimulate the ATPase activity of BiP indicated that at least half of them were authentic BiP binding sequences. These sequences were not confined to a single domain of the heavy chain but were distributed within both the VH and CH domains. Interestingly, when the BiP binding sequences were mapped onto the three-dimensional structure of the Fd antibody fragment, the majority involve residues that participate in contact sites between the heavy and light chains. Therefore, we suggest that in vivo BiP chaperones the folding and assembly of antibody molecules by binding to hydrophobic surface regions on the isolated immunoglobulin chains that subsequently participate in interchain contacts.

Adenosine Triphosphatases↗