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Biomedical subjects

C P Muller

Publications and source records attributed to C P Muller.

At least 55 records · Page 3Linked to original sources
PubMed1998

Evaluation of hemagglutinin protein-specific immunoglobulin M for diagnosis of measles by an enzyme-linked immunosorbent assay based on recombinant protein produced in a high-efficiency mammalian expression system.

Recombinant hemagglutinin (H) of the measles virus (MV) expressed in a mammalian high-expression system based on the Semliki Forest virus replicon was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulin M (IgM) and IgG in patients with acute-phase measles. One hundred twelve serum specimens from 70 patients with measles were analyzed. Case definition was based on a commercial IgM ELISA that utilizes MV-infected cells (MV-ELISA) (Enzygnost; Behring Diagnostics); the clinical criteria of the Centers for Disease Control and Prevention (Atlanta, Ga.); and/or the increase in hemagglutinin test titers, neutralization test titers, and levels of MV-specific IgG whenever paired sera were available. The initial time courses of the IgM signal after the onset of rash are similar in the H- and MV-ELISAs. On days 0 to 19, both ELISAs detected IgM in 67 of 68 (98.5%) sera. Average maximal levels of IgM seem to persist, however, about 10 days longer in the MV-ELISA (up to day 25) than in the H-ELISA (day 15). From days 20 to 29 and 30 to 59, the H-ELISA detected only 64.3 (9 of 14) and 19.2% (5 of 26), respectively, of sera that were IgM positive by MV-ELISA. At least up to day 30, the performance of the H-ELISA seemed to be similar to that reported for commercial ELISAs based on whole MV. Our results demonstrate that MV H-specific IgM can be used to diagnose most measles cases from a single serum specimen collected within 19 days after the onset of rash and that the recombinant protein used in this study is suitable for this purpose.

Adolescent↗
PubMed1998

Immunosorbent assay based on recombinant hemagglutinin protein produced in a high-efficiency mammalian expression system for surveillance of measles immunity.

Recombinant hemagglutinin (H) protein of the measles virus (MV) was produced in mammalian cells with a high-yield expression system based on the Semliki Forest virus replicon. Crude membrane preparations of H protein-transfected BHK-21 cells were used to coat microtiter plates to measure specific immunoglobulin G antibodies in 228 serologically defined serum samples mainly from measles late-convalescent adults. The titers by the enzyme-linked immunosorbent assay for the H protein (H-ELISA) closely correlated with neutralization test (NT) titers (R2 = 0.66), hemagglutination inhibition test (HI) titers (R2 = 0.64), with the titers from a certified commercial ELISA based on whole MV-infected cells (MV-ELISA; R2 = 0.45). The correlations described above were better than those of the commercial MV-ELISA titers with the NT (R2 = 0.52) or HI (R2 = 0.48) titers. By using the 2nd International Standard for anti-measles serum, the detection level of the assay corresponds to 215 mIU/ml for undiluted serum, which corresponds to the estimated threshold for protective immunity. The specificity, accuracy, and positive predictive value were, in general, better for the H-ELISA than for a commercial MV-ELISA, independent of whether HI, NT, or HI and NT were used as "gold standards." In contrast, the H-ELISA proved to be slightly less sensitive than the MV-ELISA (sensitivities, 98.6 versus 99.5%, respectively; P was not significant). The assays did not differ significantly in the number of serum samples with positive HI and NT results (n = 212) which measured false negative (H-ELISA, 2 of 212 [0.94%]; MV-ELISA, 1 of 212 [0.47%]), but the H-ELISA detected significantly more measles-susceptible individuals than the MV-ELISA (10 of 11 versus 3 of 11, respectively; P < 0.05) among the individuals whose sera had negative HI and NT results. Our data demonstrate that the H-protein preparation that we describe could be a cost-effective alternative to current whole-virus-based ELISAs for surveillance for immunity to measles and that such an assay could be more efficient in detecting susceptibility to measles. Furthermore, unlike whole MV-based antigens, H-protein would also be suitable for use in the development of a simple field test for the diagnosis of measles.

Adolescent↗
PubMed1997

Characteristics of asymptomatic secondary immune responses to measles virus in late convalescent donors.

Among 44 fully protected, late convalescent adults re-exposed to measles, four developed an asymptomatic secondary immune response (SIR) with a significant increase in measles virus (MV)-specific IgG and low IgM. The boosted antibodies were mainly of the IgG1 subclass and reacted with the nucleoprotein and the haemagglutinin protein. About 30 weeks after re-exposure, antibody levels had decreased by 35-50%, suggesting that the booster effect may only be transient. SIR was only found in individuals with a pre-exposure IgG level below a well defined threshold. Antibody levels above this threshold fully protected against SIR. SIR seems to be an 'all or none response' where the magnitude of increase in specific IgG is independent of pre-exposure antibody levels as long as these are below the above threshold. In combination with pre-exposure neutralizing and haemagglutination inhibiting titres, a threshold was defined below which SIR is likely to occur. This may be useful to predict susceptibility to SIR in a given population, since individuals undergoing clinically inapparent SIR are among seropositive subjects, the most likely candidates to support transmission of virus.

Adult↗
PubMed1997

Antibodies to a new linear site at the topographical or functional interface between the haemagglutinin and fusion proteins protect against measles encephalitis.

The haemagglutinin protein (H) of measles virus (MV) binds to susceptible cells and collaborates with the fusion protein (F) to mediate fusion of the virus with the cell membrane. Binding and fusion activity of the virus can be monitored by haemagglutination and haemolysis, respectively, of monkey erythrocytes. Most monoclonal antibodies (MAbs) with haemolysis inhibiting activity (HLI+) are either MV-F specific and do not inhibit haemagglutination (HI-), or they bind to MV-H and are HI+ by interfering with virus binding. We describe here a small panel of H-specific MAbs (BH47, BH59, BH103, BH129) which bind to a new linear neutralizing epitope, H244-250 (SELSQLS; NE domain), and which prevent virus-cell fusion (HLI+) but not virus binding (HI-). These antibodies also protect against MV encephalitis in an animal model. They do not compete with an HLI+/HI+ antibody (BH216) which binds to the haemagglutinin noose epitope (HNE). The antibodies described here and the HNE-specific antibodies are functionally distinct and define two topographically non-overlapping interfaces, supposedly with a bias towards the host cell MV-receptor and the fusion protein respectively. The proximity of the CD46 downregulating amino acid Arg-243 may suggest a functional link between the domain described here and the CD46 binding domain. This new protective linear site is also of potential interest for the design of a subunit-based vaccine.

Animals↗
PubMed1996

New ligands binding to the human leukocyte antigen class II molecule DRB1*0101 based on the activity pattern of an undecapeptide library.

Major histocompatibility complex (MHC) class II molecules present peptide antigens to CD(4+)-T cells. These heterogeneous peptides are derived from internalized exogenous proteins or from endogenous membrane proteins that are processed by the antigen-presenting cell. Peptides are bound to the MHC class II molecules in an extended conformation and extend out of the binding groove. The aim of this study was to estimate the influence of every amino acid in all the possible undecapeptide amides (2.048 x 10(14) individuals) on the binding to human MHC-DRB1*0101 molecules (HLA-DR1) and to identify new peptide ligands. 220 undecapeptide sublibraries, O/X10, each composed of ten degenerate positions and one defined position, were screened for binding to isolated HLA-DR1. Competition of the sublibraries with a fluorescence-labeled peptide ligand allowed definition of the amino acids favourable or unfavourable for DR1-binding at every sequence position. From the activity pattern of the undecapeptide library, 54 individual peptides were deduced (27 potential hits and 27 potential falls) and prepared by chemical synthesis. As anticipated, 27 positive and 27 negative results were obtained from the competition experiments. The 27 peptides that bind obey the rules for the HLA-DR1-binding motif. The synthetic peptide library approach proved to be valuable for the design of synthetic MHC class II ligands and thus can be considered as a basis for drug design in immunotherapy.

Amides↗
PubMed1996

Hierarchic T-cell help to non-linked B-cell epitopes.

The induction of antibodies against peptides requires the presence of a T helper cell epitope. In the absence of an added T-cell epitope only 10% of the mice, or less depending on the strain, gave an antibody response to a series of peptides of the measles virus (MV) fusion (F) protein. After coimmunization with a non-covalently coupled T-cell epitope more than 60% of the peptides became immunogenic. Considerable differences became apparent when BALB/c mice were immunized with peptides in the presence of different T-cell epitopes. An immunodominant T-cell epitope of the MV-F protein was more efficient than a subdominant or a cryptic T-cell epitope in providing help to a non-linked B-cell epitope. There is both a ranking order of the amount of help which B-cell epitopes require and a ranking order for the help T-cell epitopes are able to provide. The capability of a T-cell epitope to provide help to a B-cell epitope correlated with its own immunogenicity, i.e. the intensity of the antibody response to the peptide representing the T-cell epitope. The data suggest that for each MHC class II allele there is an optimal T-cell epitope which can provide help to a maximal number of B-cell epitopes and that such a peptide can be identified by its ability to induce antibodies against itself. By using this strategy, the authors were able to induce antibodies which cross-reacted with the MV.

Animals↗
PubMed1996

Activation of T cells by the ragged tail of MHC class II-presented peptides of the measles virus fusion protein.

The efficient and sustained immune response of an antigen requires T cell epitopes, capable of inducing a long lasting T cell memory. To detect T cell epitopes of the measles virus fusion protein (MV-F), the proliferation of lymphocytes from late convalescent donors in response to overlapping pentadecapeptides covering the whole protein sequence was studied. Three major immunodominant regions (F51-70, F121-135 and F211-225) containing promiscuous peptides induce proliferation in peripheral blood lymphocytes in approximately 50% of the donors. Potential DR1-restricted epitopes were mapped using an MHC competition binding assay. Both the proliferation and the binding data identified a DR1-restricted T cell epitope (F51-65). Contact sites of the peptide HQSLVIKLMPNITLL with MHC were characterized using substitution analogs. Alanine substitutions at most positions did not interfere with F51-65 binding. These analogs were therefore useful for studying the residues which were recognized by the TCR of MV- and F51-induced T cells lines. In addition to amino acid residues of the core of peptide F51-65 both the C-terminal and the N-terminal amino acids were essential for T cell interaction. Since peptides presented by class II molecules vary in length, these findings suggest that residues of the ragged tail are important for T cell activation. It is speculated that in late convalescent donors the length of the flanking sequence of MHC II-restricted peptides may play a role in controlling the heterogeneity of MV-specific T cell clones recruited as T helper/memory cells.

Adult↗
PubMed1996

Differential activation of T cells by antibody-modulated processing of the flanking sequences of class II-restricted peptides.

Despite poor presentation of measles virus (MV) nucleoprotein (NP) by MHC class II of infected cells, NP-specific antibodies are one of the hallmarks of the early immune response against this virus. To study the influence of antibodies on processing and presentation of NP to three different T cell hybridomas, mAb recognizing distinctive llnear NP epitopes were developed. Two T cell hybridomas TNP408B and TNP408 reacted with the same core epitope of NP (amino acids 383-391), but differed in their sensitivity to the flanking sequences of peptides containing this epitope. TNP408B reacted with minimal concentrations of NP when this was complexed with mAb BNP146. NP alone or saturating concentrations of other mAb did not activate this T cell. Both T cells, TNP408 and TNP408B, were similar in their sensitivity to NP in the presence of saturating concentrations of BNP146 or of appropriate peptide (NP379). TNP408 did not differ from another T cell hybridoma (TNP79) in its sensitivity to different mAb, suggesting a specificity-dependent and a specificity-independent effect of mAb. Antibody-mediated activation was attributed to FcR-mediated uptake independent of the fine specificity of the mAb. In the case of TNP408B, this effect was further enhanced by a specific effect of BNP146. While all NP-specific mAb were sufficient to enhance presentation to TNP408 and TNP79 of their respective peptides derived from processed NP, BNP146 was necessary to generate the peptides with the proper flanking sequences required by TNP408B. Since the binding site of BNP146 coincides with the T cell epitope of TNP408B (and TNP408) it is suggested that binding of this mAb modulates processing of the flanking sequences of the peptides corresponding to this epitope. This study shows that antibodies can influence the T cell response to an antigenic protein quantitatively and qualitatively by taking advantage of the sensitivity of T cells to flanking sequences of class II-restricted peptides.

Amino Acid Sequence↗
PubMed1996

Protection against measles virus encephalitis by monoclonal antibodies binding to a cystine loop domain of the H protein mimicked by peptides which are not recognized by maternal antibodies.

After immunization with measles virus (MV) several monoclonal antibodies (MAbs) were obtained, which reacted with peptides corresponding to the amino acids 361-410 of the haemagglutinin protein (MV-H). Three of these MAbs (BH6, BH21 and BH216) inhibited haemagglutination, neutralized MV in vitro and protected animals from a lethal challenge of rodent-adapted neurotropic MV. These MAbs reacted with the 15-mer peptides H381 and H386 defining their overlapping region 386-395 as a sequential neutralizing and protective epitope, which can be imitated by a short peptide. H381 and H386 share two Cys residues (C(386)KGKIQALC(394)ENPEWA) and for optimal MAb binding of peptide (or MV) disulphide bonds were required in addition to a linear C-terminal extension. Other MAbs bound to peptides C- (BH147, BH195) and N-terminally (BH 168, BH 171) adjacent to the loop but did not neutralize or protect. When sera from measles patients or from women of child-bearing age were tested with the peptides corresponding to this haemagglutinating and neutralizing epitope (HNE), none of the sera recognized the 15-mer peptides of this region, while some reactivity was found to 30-mers homologous to different wild-type mutants. Its lack of recognition by maternal antibodies and its high degree of conservation would make the HNE loop an attractive candidate to include into a subunit vaccine, which could be administered during early childhood, independent of immune status.

Amino Acid Sequence↗
PubMed1995

Intramolecular immunodominance and intermolecular selection of H2d-restricted peptides define the same immunodominant region of the measles virus fusion protein.

The generation of a sustained antibody response requires the participation of MHC class II-restricted T helper cells. We have identified class II-restricted sequences by immunizing BALB/c (H-2d) mice with 108 overlapping synthetic pentadecapeptides covering the whole sequence of the measles virus fusion protein (MV-F). Several strong T cell epitopes were found including a major cluster of H-2d-restricted peptides between amino acids 256 and 305. Some of these peptides including peptide F(421-435) and F(256-270) induced MV-specific T lymphocytes in vivo while other H2d-restricted MV-F sequences did not. Immunization with mixtures of selected peptides indicated a hierarchy among H2d-restricted sequences due to competition between peptides. The dominant peptide F(421-435) impaired the response to other T cell epitopes including F(256-270). The response to F(91-105) was obliterated by F(421-435) and F(256-270) but not by peptides devoid of a T cell epitope. When BALB/c mice were immunized with the MV, the immunodominant sequence F(421-445) was identified which included the synthetic peptide F(421-435). Our data suggest that competition during processing and/or presentation between H2d-restricted peptides defines the immunodominant sequence of the viral protein. Even though only a single immunodominant region was defined after immunization with the MV, peptides from other regions were able to induce MV-specific T cell responses. This finding is of interest for the design of subunit vaccines in general and for studying MV-specific T helper cells in an animal model in particular.

Amino Acid Sequence↗
PubMed1995

Cholera toxin B stimulates systemic neutralizing antibodies after intranasal co-immunization with measles virus.

An efficient mucosal vaccination has a number of obvious advantages over invasive routes of immunization. The immune response to measles virus (MV) was investigated after intranasal and intragastric co-immunization of mice with cholera toxin B (CTB) as an adjuvant. High titres of virus-specific IgG antibodies and a transient IgA response were detected in the sera after intranasal but not after intragastric immunization when CTB was used. In the presence of CTB, higher titres were reached with less antigen and fewer intranasal boosts. Neutralizing antibodies were found in all animals only after co-immunization with MV and CTB. In the nasal wash and the saliva, IgG and IgA titres were significant only in the MV plus CTB groups; IgG levels were comparable to those found after intraperitoneal (i.p.) immunization with complete Freund's adjuvant. Specific IgA was detected in the mucosal fluids only after intranasal immunization with MV plus CTB but not after i.p. or intragastric immunization. The antibody response consisted of 99% IgG1 after MV immunization. In the CTB groups 10% IgG2b and 1% IgG2a were detected in addition to the predominant IgG1 antibodies.

Adjuvants, Immunologic↗
PubMed1994

[Systemic B-cell response in the mouse after mucosal immunization with measles virus].

The respiratory tract represents both the port of entrance of the measles virus (MV) and an important target tissue for measles complications. Therefore, mucosal immunization could be an attractive alternative to the current subcutaneous route. We studied the virus-specific IgA and IgG antibody response in the serum after intranasal and intragastric immunization with MV in the mouse model under non-replicating conditions. Hemagglutination-inhibiting antibodies, mostly without virus-neutralizing activity were found after intranasal immunization. No virus-specific antibodies were detected after intragastric immunization.

Administration, Intranasal↗
PubMed1993

Analysis of antibody response to the measles virus using synthetic peptides of the fusion protein. Evidence of non-random pairing of T and B cell epitopes.

The measles virus induces a life-long immune response associated with antibodies specific for the fusion protein. To map the linear immunodominant recognition sites of the fusion (F) protein of the measles virus, we have reacted a complete set of 108 overlapping pentadecapeptides with purified IgG obtained from donor sera with elevated anti-measles titers. The antibodies recognized about 20% of the peptides and generated a characteristic binding pattern, defining about 6 or 7 distinctive regions (31-75; 111-145; 151-165; 191-215; 271-320; 421-440; 481-530) which include the major hydrophobic segment (111-145) of the intersubunit region and the C-terminal Cys-cluster region. The binding sites were located in close proximity of the few experimentally defined T cell epitopes. This pairing of T and B cell epitopes was corroborated by computer-assisted T cell prediction. The significance of a non-random association of T and B cell epitopes for processing and presentation is discussed. It is speculated that in long-term immunity against measles (F protein), B cells of the same sIg specificity play an important role both as antigen presenting cells and as antibody producing cells. In contrast to human sera from late convalescent donors, mouse and rabbit MV antisera with high neutralizing titers as well as neutralizing MV-F specific monoclonal antibodies did not react with the peptides.

Adult↗
PubMed1993

Analysis of the neutralizing antibody response to the measles virus using synthetic peptides of the haemagglutinin protein.

Infection or immunization with measles virus induces a protective immune reaction including neutralizing antibodies against the haemagglutinin and fusion protein. The reactivity of the polyclonal IgG response of sera obtained from late convalescent donors was studied, using overlapping 15mer peptides covering the complete sequence of the measles virus haemagglutinin. Most sera reacted with a similar set of peptides generating a characteristic binding pattern. The reactive peptides correspond to a region mediating cell hemolysis (aa310-325), to regions which serve as targets to neutralizing antibodies and to a putative transmembrane region (aa35-58). The latter region contains also a human T-cell epitope providing evidence of a non-random association of T- and B-cell epitopes. We also immunized different strains of mice and rabbits with measles virus. In contrast to the human sera, animal sera with strong neutralizing activities did not react with any of the H-protein peptides. The mostly weak reactivities with the linear sequences contrast with the strong neutralizing activities of the human or animal antibodies, suggesting that these primarily recognize the fusion protein or conformational epitopes of the haemagglutinin protein.

Adult↗
PubMed1992

The prognostic significance of total serum cholesterol in patients with Hodgkin's disease.

In the current retrospective study, the authors investigated the prognostic significance of total serum cholesterol values at the time of diagnosis in patients with Hodgkin's disease (n = 179). Cholesterol values were significantly lower in these patients than in age- and sex-matched controls. Subgroups with advanced stages (P less than or equal to 0.01), poor response to therapy (P = 0.04), and relapse after complete response (P = 0.026) (but not with bulky disease) had lower cholesterol values. By univariate analysis (cut-point value, 140 mg/dl), the 5-year survival rate was 2.5 times higher in patients with normal cholesterol values than in hypocholesterolemic patients (P less than 0.00009). Hypocholesterolemia was retained as an adverse, independent prognostic factor by multivariate Cox regression analysis. The authors concluded that total serum cholesterol values at the time of diagnosis may be a parameter with unrecognized significance in Hodgkin's disease.

Adolescent↗
PubMed1992

Mdr1/P-glycoprotein, topoisomerase, and glutathione-S-transferase pi gene expression in primary and relapsed state adult and childhood leukaemias.

In a variety of adult and childhood leukaemia cell samples collected at different states of the disease, we analysed in a series of sequentially performed slot-blot or Northern-blot hybridisation experiments the expression of genes possibly involved in multiple drug resistance (MDR) (mdr1/P-glycoprotein, DNA topoisomerase II, glutathione-S-transferase pi), and the expression of the DNA topoisomerase I and histone 3.1 genes. Occasionally, P-glycoprotein gene expression was additionally examined by indirect immunocytofluorescence using the monoclonal antibody C219. No significant difference in mdr1/P-glycoprotein mRNA levels between primary and relapsed state acute lymphocytic leukaemias (ALL) was seen on average. Second or third relapses, however, showed a distinct tendency to an elevated expression of this multidrug transporter gene (up to 10-fold) in part well beyond the value seen in the moderately cross-resistant T-lymphoblastoid CCRF-CEM subline CCRF VCR 100. Increased mdr1/P-glycoprotein mRNA levels were also found in relapsed state acute myelogenous leukaemias (AML), and in chronic lymphocytic leukaemias (CLL) treated with chlorambucil and/or prednisone for several years. Topoisomerase I and topoisomerase II mRNA levels were found to be very variable. Whereas in all but one case of CLL topoisomerase II mRNA was not detected by slot-blot hybridizations, strong topoisomerase I and topoisomerase II gene expression levels, frequently exceeding the levels monitored in the CCRF-CEM cell line, were seen in many cell samples of acute leukaemia. If topoisomerase II mRNA was undetectable, expression of topoisomerase I was clearly visible throughout. These observations might be valuable considering the possible treatment with specific topoisomerase I or topoisomerase II inhibitors. Significant positive correlations were found (i) for topoisomerase I and histone 3.1 gene expression levels in general (P less than 0.001), and (ii) in the CLL samples additionally for the expression levels of the mdr1 gene, and the histone 3.1, topoisomerase I, and glutathione-S-transferase pi genes, respectively.

ATP Binding Cassette Transporter, Subfamily B, Mem↗