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M Eggers

Publications and source records attributed to M Eggers.

At least 19 recordsLinked to original sources
PubMed2001

Study of GH sensitivity in chilean patients with idiopathic short stature.

We hypothesized that some children with idiopathic short stature in Chile might bear heterozygous mutations of the GH receptor. We selected 26 patients (3 females, 23 males) from 112 patients who consulted for idiopathic short stature at the University of Chile. Their chronological age was 8.3 +/- 1.9, and bone age was 6.1 +/- 1.0 yr. Their height was -3.0 +/- 0.7 SDS; IGF-I, -1.2 +/- 1.1 SD; IGF binding protein 3, -0.7 +/- 2.0 SDS; and GH binding protein, 0.4 +/- 0.8 SDS. Patients were admitted, and blood samples were obtained every 20 min to determine GH concentrations overnight. Coding sequences and intron-exon boundaries of exons 2-10 of GH receptor gene were amplified by PCR and subsequently analyzed through single-strand conformational analysis. Mean serum GH concentration, over 12-h, was 0.20 +/- 0.08 nM; pulse amplitude, 0.40 +/- 0.15 nM; number of peaks, 5.8 +/-1.5 peaks/12 h; peak value of GH during the 12-h sampling, 1.03 +/- 0.53 nM; and area under the curve, 151.4 +/- 56.1 nM/12 h. There were positive correlations between mean GH vs. area under the curve (P < 0.001) and GH peak (P < 0.01). The single-strand conformational analysis of the GH receptor gene showed abnormal migration for exon 6 in 9 patients and for exon 10 in 9 patients, which (by sequence analysis) corresponded to 2 polymorphisms of the GH receptor gene: an A-to-G transition in third position of codon 168 in exon 6 and a C-to-A transversion in the first position of codon 526 in exon 10. We further sequenced all coding exons and intron-exon boundaries in the most affected patients (nos. 6, 9, 11, 14, 15, 16, and 23). This analysis revealed a C-to-T transition in codon 161 of exon 6 in patient 23, which results in an amino acid change (Arg to Cys) in an heterozygous form in the patient and his father. In conclusion, the results of our study suggest that, in Chilean patients with idiopathic short stature, GH receptor gene mutations are uncommon, although we cannot exclude mutations that were missed by single-strand conformational analysis or mutations within introns or in the promoter regions of the GH receptor gene.

Autoradiography↗
PubMed2001

Use of recombinant glycoprotein antigens gB and gH for diagnosis of primary human cytomegalovirus infection during pregnancy.

The major risk factor for intrauterine transmission of human cytomegalovirus (HCMV) is a primary infection during pregnancy. The neutralizing antibody response appeared after an average of 13 weeks after seroconversion and therefore the absence of neutralizing titers in HCMV IgG positive pregnant women is a reliable marker for primary infection. Determination of neutralizing antibody, however, is time-consuming and labor-intensive. For this reason an immunoblot assay for detection of neutralizing antibodies was developed based on the use of recombinant antigens representing neutralizing epitopes of glycoproteins (gp) gB (gpUL55) and gH (gpUL75) of HCMV. In this study, 93.6% of sera of pregnant women with prior infection recognized the gp-specific epitopes corresponding to a nonresponder rate of 6.4% relative to the neutralizing antibody. In primary infection the gp-response in general coincided with the appearance of neutralizing antibody. Intriguingly, lack of HCMV gB-specific antibodies was correlated with a lower risk of intrauterine fetal infection (P < 0.05).

Antibodies, Viral↗
PubMed2001

Kaposi's sarcoma-associated herpesvirus serology in Europe and Uganda: multicentre study with multiple and novel assays.

A multicentre study was undertaken to define novel assays with increased inter-assay concordance, sensitivity, specificity and predictive value for serological diagnosis of human herpesvirus type 8 (HHV-8) infection. A total of 562 sera from European and Ugandan human immunodeficiency virus (HIV)-infected or uninfected individuals with or without Kaposi's sarcoma (KS) and blood donors were examined under code by 18 different assays in seven European laboratories. Sera from KS patients and all non-KS sera found positive by at least 70%, 80%, or 90% of the assays were considered "true positive." The validity of the assays was then evaluated by univariate logistic regression analysis. Two immunofluorescence assays (IFA) for detection of antibodies against HHV-8 lytic (Rlyt) or latent (LLANA) antigens and two enzyme-linked-immunosorbent assays (ELISA) (M2, EK8.1) for detection of antibodies against HHV-8 structural proteins were found to be highly concordant, specific, and sensitive, with odds ratios that indicated a high predictive value. When used together, the two IFA (Rlyt-LLANA) showed the best combination of sensitivity (89.1%) and specificity (94.9%). The performance of these assays indicate that they may be used for the clinical management of individuals at risk of developing HHV-8 associated tumours such as allograft recipients.

AIDS-Related Opportunistic Infections↗
PubMed2000

Combination of microneutralization and avidity assays: improved diagnosis of recent primary human cytomegalovirus infection in single serum sample of second trimester pregnancy.

Estimation of IgG avidity index is a classical serological method. Antibodies with low avidity are detectable at a very early stage of infection whereas high avidity antibodies indicate past infection. Recently, it was shown that the neutralization assay can be routinely used as a reliable method for differentiating between acute primary and non-primary infection in a single serum sample because the first neutralizing titers (NT) appeared after an average of 13 weeks (range, 10-17 weeks). A low positive NT titer in the presence of specific IgM antibodies, however, still represents a diagnostic problem especially if blood sampling occurred after the 12th week of gestation. To overcome this problem the combination of NT and IgG avidity tests was evaluated. Human cytomegalovirus (HCMV) IgG avidity indices of 350 serum samples from 227 pregnant women were investigated using 6M urea in the washing buffer. HCMV specific IgG antibodies reached full maturation approximately 20-22 weeks after seroconversion and low IgG avidity is therefore a marker of primary infection. The combined application of the microneutralization and avidity assays was shown to serve as a helpful tool in diagnosis of a recent primary HCMV infection of second trimester pregnancy particularly when previous serological data were not available.

Antibodies, Viral↗
PubMed2000

The proteasome regulator PA28alpha/beta can enhance antigen presentation without affecting 20S proteasome subunit composition.

PA28alpha/beta is a regulatory complex of the 20S proteasome which consists of two IFN-gamma inducible subunits. Both subunits, alpha and beta, contribute equally to the formation of hexa- or heptameric rings which can associate with the 20S proteasome. Previously, we have shown that overexpression of the PA28alpha subunit enhanced the MHC class I-restricted presentation of two viral epitopes and that purified PA28alpha/beta accelerated T cell epitope generation by the 20S proteasome in vitro, indicating a role for PA28alpha/beta in antigen presentation. This conclusion was recently confirmed in PA28beta gene targeted mice which were severely deficient in MHC class I-restricted antigen presentation. These mice displayed a defect in the assembly of immunoproteasomes, suggesting that a lack of the proteasome subunits LMP2, LMP7, and MECL-1 may account for the deficiency in antigen presentation. In this study we investigated whether the effect of PA28alpha/beta on antigen presentation is dependent on a change of proteasome subunit composition. We have analyzed the assembly and subunit composition of proteasomes in fibroblast transfectants overexpressing both, alpha and beta subunits of PA28. In these transfectants we found a marked enhancement in the presentation of the immunodominant H-2Ld-restricted pp89 epitope of murine cytomegalovirus, although the 20S proteasome composition was the same as in recipient cells. We, therefore, conclude that PA28alpha/beta can enhance antigen processing independently of changes in 20S proteasome subunit composition or assembly.

Animals↗
PubMed2000

Analysis of human cytomegalovirus DNA in urines of newborns and infants by means of a new ultrarapid real-time PCR-system.

BACKGROUND: Amplification techniques such as PCR are becoming increasingly popular in the field of diagnosis of human cytomegalovirus (HCMV) also, thus substituting conventional techniques like the time consuming HCMV antigen or cell culture assays. Current PCR protocols however, are labor intensive, and moreover, the need for extensive postamplification manipulations increases the risk of false positive results due to contamination with amplified products. OBJECTIVES: to overcome these shortcomings, the new ultrarapid and semi-automated real-time LightCycler PCR-system (LC-PCR), which combines amplification and detection in a closed capillary system, was tested for its suitability in diagnosis of HCMV in urines. STUDY DESIGN: 73 urine samples from 64 newborns and infants suspected of having congenitally or postnatally acquired HCMV were tested with the LC-PCR and results were compared with those obtained in parallel with a conventional PCR-ELISA and the rapid shell vial assay for detection of HCMV early antigen (EA-assay). RESULTS: with these methods, 31 newborns/infants were found to be infected with HCMV. HCMV DNA was detected in 39 urines while the EA-assay was positive in 33 urines. All the EA positive samples were also positive for HCMV DNA. In the urines of the remaining 33 newborns (34 urine samples) neither HCMV DNA nor EA were detectable. The overall agreement of the two PCR tests was 100% while a 92% agreement was obtained between the PCR and the EA-assays. As the sensitivity of the three tests turned out to be quite similiar, the discrepancy observed in the positive rate between PCR and EA-assay is due to other factors which will be discussed in detail. However, while LC-PCR takes only about 2 h from sample preparation to result generation, the EA-assay, such as the conventional PCR-ELISA, needs 24-48 h. Furthermore, due to its capability to perform cycle-by-cycle monitoring, the LC instrument enables semi-quantitative analysis of HCMV viral-load. CONCLUSIONS: LC-PCR is a suitable new tool for routine analysis of HCMV in the urines of newborns and infants. Compared to the conventional PCR-ELISA a considerable increase in test rapidity and reliability is achieved without the need to sacrifice sensitivity.

Antigens, Viral↗
PubMed2000

Near final height in pubertal growth hormone (GH)-deficient patients treated with GH alone or in combination with luteinizing hormone-releasing hormone analog: results of a prospective, randomized trial.

To study the effects of delaying puberty in GH-deficient (GHD) children, we studied 21 GHD (9 boys, 14 girls), treatment-naive, pubertal patients in a prospective, randomized trial. Their chronological age was 14.3 +/- 1.6 yr, and their bone age was 11.3 +/- 1.1 yr (mean +/- SD) at the beginning of the study. Four patients who developed hypogonadotropic hypogonadism were subsequently excluded from the study. Patients were randomly assigned to receive GH + LH-releasing hormone analog (LHRH-A) (n = 7), or GH alone (n = 10). GH and LHRH-A treatment started simultaneously in each patient. GH (Nutropin) was administered at a dose of 0.1 U/kg x day sc, until patients reached a bone age (BA) of 14 yr in girls and 16 yr in boys, and LHRH-A (Lupron depot) was administered at a dose of 300 microg/ kg every 28 days in during 3 yr. We defined GH deficiency as patients with a growth velocity less than 4 cm/yr, BA delay more than 1 yr in relationship to chronological age, GH response to two stimulation tests less than 7 microg/L, associated with low serum insulin-like growth factor I and insulin-like growth factor binding protein 3 levels. Statistical analysis was performed by ANOVA or Kruskall Wallis when variances were not homogeneous. We observed a significant decrease in the rate of BA maturation in the group treated with GH+LHRH-A (1.5 +/- 0.2 yr) compared with the group treated with GH alone (4.2 +/-0.5 yr) during the 3 years of LHRH-A therapy (P < 0.05). This delay in BA maturation produced a significant gain in final height in the group treated with GH+LHRH-A, which reached - 1.3 +/- 0.5 SD score compared with -2.7 +/- 0.3 SD score (P < 0.05) in the group treated with GH alone. These results indicate that delaying puberty with LHRH-A in GHD children during treatment with GH increases final height.

Adolescent↗
PubMed1999

Evaluation of two novel enzyme immunoassays using recombinant antigens to detect cytomegalovirus-specific immunoglobulin M in sera from pregnant women.

BACKGROUND: Two novel enzyme linked immunosorbent assays (ELISA) (Abbott IMx CMV IgM 2.0, and Cobas Core CMV IgM EIA recomb, research version) which use recombinant antigens to detect cytomegalovirus (CMV)-specific IgM antibodies were evaluated. OBJECTIVES: A new ELISA is normally evaluated against a gold standard commercial kit, which in this case does not exist. We therefore evaluated the two novel recombinant ELISA against four conventional ELISAs and a recently developed CMV IgM immunoblot containing four purified viral and four recombinant proteins. STUDY DESIGN: A total of 280 sera from pregnant women and 42 potentially cross-reactive sera were investigated using the six ELISAs, including 101 sera which were also tested using the new IgM immunoblot. RESULTS: Relative sensitivity, relative specificity and overall agreement differed according to the reference assay. The Cobas Core CMV EIA recomb showed much higher agreement with the ELISA consensus, and the IMx CMV IgM 2.0 with the immunoblot. CONCLUSION: The evaluation of these new IgM assays in terms of their agreement with either commercial ELISA kits or the IgM immunoblot demonstrates that the question 'which reference method?' is still open. However the recombinant IgM assays may improve the diagnosis of CMV infection in pregnancy since the recombinant technology offers helpful tools for identifying diagnostically relevant proteins and allows the use of standardized pure preparations of antigens. For serological diagnosis of CMV infection in pregnancy two IgM assays that can be relied upon should be performed. IgM positive sera should be tested with supplementary assays to differentiate primary from non-primary infection.

Antibodies, Viral↗
PubMed1999

High-throughput microarray-based enzyme-linked immunosorbent assay (ELISA).

A new generation biochip is described as capable of supporting high-throughput (HT), multiplexed enzyme-linked immunosorbent assays (ELISAs). These biochips consist of an optically flat, glass plate containing 96 wells formed by an enclosing hydrophobic Teflon mask. The footprint dimensions of each well and the plate precisely match those of a standard microplate. Each well contains four identical 36-element arrays (144 elements per well) comprising 8 different antigens and a marker protein. Arrays are formed by a custom, continuous flow, capillary-based print head attached to a precise, high-speed, X-Y-Z robot. The array printing capacity of a single robot exceeds 20,000 arrays per day. Arrays are quantitatively imaged using a custom, high-resolution, scanning charge-coupled device (CCD) detector with an imaging throughout of 96 arrays every 30 s. Using this new process, arrayed antigens were individually and collectively detected using standard ELISA techniques. Experiments demonstrate that specific multiplex detection of protein antigens arrayed on a glass substrate is feasible. Because of the open microarray architecture, the 96-well microarray format is compatible with automated robotic systems and supports a low-cost, highly parallel assay format. Future applications of this new high-throughput screening (HTS) format include direct cellular protein expression profiling, multiplexed assays for detection of infectious agents and cancer diagnostics.

Adsorption↗
PubMed1998

Inactivation of a defined active site in the mouse 20S proteasome complex enhances major histocompatibility complex class I antigen presentation of a murine cytomegalovirus protein.

Proteasomes generate peptides bound by major histocompatibility complex (MHC) class I molecules. Avoiding proteasome inhibitors, which in most cases do not distinguish between individual active sites within the cell, we used a molecular genetic approach that allowed for the first time the in vivo analysis of defined proteasomal active sites with regard to their significance for antigen processing. Functional elimination of the delta/low molecular weight protein (LMP) 2 sites by substitution with a mutated inactive LMP2 T1A subunit results in reduced cell surface expression of the MHC class I H-2Ld and H-2Dd molecules. Surface levels of H-2Ld and H-2Dd molecules were restored by external loading with peptides. However, as a result of the active site mutation, MHC class I presentation of a 9-mer peptide derived from a protein of murine cytomegalovirus was enhanced about three- to fivefold. Our experiments provide evidence that the delta/LMP2 active site elimination limits the processing and presentation of several peptides, but may be, nonetheless, beneficial for the generation and presentation of others.

Animals↗
PubMed1998

Differentiation between acute primary and recurrent human cytomegalovirus infection in pregnancy, using a microneutralization assay.

Acute primary human cytomegalovirus (HCMV) infection in pregnancy, the major cause of congenital symptomatic infection, is often difficult to differentiate from recurrent infection, which presents a considerably smaller risk to the fetus. Therefore, the diagnosis of primary infection in pregnancy is very important, especially if seroconversion is not documented and follow-up sera with declining IgM-titers are not available. To investigate the value of the neutralizing antibody response against HCMV in differentiating acute primary from recurrent and past infection, well-characterized sera from pregnant women were examined. Employing a microneutralization assay, it was found that neutralizing antibodies first appeared approximately 15 weeks after acute infection. However, serum samples of pregnant women with recurrent or past infection consistently displayed neutralizing activity. In conclusion, the neutralization assay can be used as a reliable method for discriminating acute primary from previous or recurrent infection in a single serum sample.

Antibodies, Viral↗
PubMed1997

Effects of luteinizing hormone-releasing hormone analog-induced pubertal delay in growth hormone (GH)-deficient children treated with GH: preliminary results.

To study the effect of delaying epiphyseal fusion on the growth of GH-deficient children, we studied 14 pubertal, treatment naive, GH-deficient patients (6 girls and 8 boys) in a prospective, randomized, placebo-controlled trial. Chronological age was 14.5 +/- 0.5 yr, and bone age was 11.6 +/- 0.3 yr (mean +/- SEM) at the beginning of the study. Patients were assigned randomly to receive GH and LH-releasing hormone (LHRH) analog (n = 8) or GH and placebo (n = 6) during 3 yr, with planned continuation of GH treatment until epiphyseal fusion. Patients were measured with a stadiometer and had serum LHRH tests, serum testosterone (boys), serum estradiol (girls), and bone age performed every 6 months. Patients treated with GH and LHRH analog showed a clear suppression of their pituitary-gonadal axis and a marked delay in bone age progression. We observed a greater gain in height prediction in these patients than in the patients treated with GH and placebo after 3 yr of treatment (mean +/- SEM, 14.0 +/- 1.6 vs. 8.0 +/- 2.4 cm; P < 0.05). These preliminary findings suggest that delaying epiphyseal fusion with LHRH analog in pubertal GH-deficient children treated with GH increases height prediction and may increase final height compared to treatment with GH alone.

Adolescent↗
PubMed1996

A role for the proteasome regulator PA28alpha in antigen presentation.

Cytotoxic T cells recognize viral proteins as peptide fragments which are produced in the cytosol and transported on major histocompatibility complex (MHC) class I proteins to the cell surface. Viral peptides that meet the stringent binding characteristics of class I proteins are generated by the 20S proteasome. The interferon (IFN)-gamma-inducible activator of the 20S proteasome, PA28, strongly influences the proteasomal cleavage pattern in vitro. This led us to investigate whether changes in cellular levels of PA28 affect the efficiency of viral antigen processing. A mouse fibroblast line expressing the murine cytomegalovirus pp89 protein was transfected with either the human or murine gene encoding the PA28alpha subunit, which is sufficient to activate the peptide-hydrolysing activity of the 20S proteasome in vitro. Here we report that enhanced expression of PA28alpha at a level similar to that obtained after IFN-gamma induction resulted in a marked enhancement of recognition by pp89-specific cytotoxic T cells; the presentation of influenza nucleoprotein was also significantly improved. These results demonstrate a fundamental in vivo function for PA28alpha in antigen processing.

Amino Acid Sequence↗
PubMed1996

A single residue exchange within a viral CTL epitope alters proteasome-mediated degradation resulting in lack of antigen presentation.

CTL epitope (KSPWFTTL) encoded by AKV/MCF type of murine leukemia virus (MuLV) differs from the sequence in Friend/Moloney/Rauscher (FMR) type in one residue (RSPWFTTL). CTL experiments indicated defective processing of the FMR peptide in tumor cells. Proteasome-mediated digestion of AKV/MCF-type 26-mer peptides resulted in the early generation and higher levels of epitope-containing fragments than digestion of FMR-type peptides, explained by prominent cleavage next to R in the FMR sequence. The fragments were identified as 10- and 11-mer peptides and were efficiently translocated by TAP. The naturally presented AKV/MCF peptide is the 8-mer, indicating ER peptide trimming. In conclusion, a single residue exchange can cause CTL epitope destruction by specific proteasomal cleavage.

ATP-Binding Cassette Transporters↗
PubMed1995

The cleavage preference of the proteasome governs the yield of antigenic peptides.

Proteasomes degrade endogenous proteins in the cytosol. The potential contribution of the proteasome to the effect of flanking sequences on the presentation of an antigenic epitope presented by the major histocompatibility complex class I allele Ld was studied. Peptides generated in cells from minigenes coding for peptides of 17- and 19-amino acid length were compared with the in vitro 20S proteasome degradation products of the respective synthetic peptides. The quality of generated peptides was independent of ubiquitination. In vivo and in vitro processing products were indistinguishable with respect to peptide size and abundance. Altering the neighboring sequence substantially improved the yield of the final antigenic nonapeptide by 20S proteasome cleavage. These results suggest that, in addition to the presence of major histocompatibility complex class I allelic motifs, the cleavage preference of the proteasome can define the antigenic potential of a protein.

Aldehydes↗
PubMed1995

A review of microfabricated devices for gene-based diagnostics.

The next technologic revolution may well be the miniaturization of solution phase experimentation through the marriage of microelectronics and molecular biology. Already researchers leading this rapidly emerging technology are developing prototype biochemical "microlaboratories" that offer a millionfold reduction of scale; that is, biochemical analyses are being conducted with picoliter sample volumes rather than conventional microliter formats. Such miniaturization is achieved by exploiting the well-honed tools of microelectronics that accommodate highly parallel automated assays, ultrasensitive detection, high throughput, integrated data acquisition, computation, and distributed data storage/retrieval. The subject of this paper is to survey this evolving bioelectronic miniaturization technology with applications to gene-based diagnostics. Several pioneering microchips are described and compared to traditional biochemical methods. Specifically, miniaturized examples of various diagnostic processes such as sample preparation (PCR), assays (electrophoresis and probe arrays), and detection (integrated CCDs) are presented. Although these microdevices are rather embryonic they represent the first steps toward fabricating a fully integrated diagnostic system on a microchip.

Diagnosis↗
PubMed1994

Identification and expression of a murine cytomegalovirus early gene coding for an Fc receptor.

Several herpesviruses, including cytomegalovirus, induce receptors for the Fc domain of murine immunoglobulin G (IgG) molecules. Viral genes coding for these receptors have been characterized only for alphaherpesviruses. In this report, we describe a new approach that led to the identification of an Fc receptor (FcR) of murine cytomegalovirus (MCMV). The Fc fragment of IgG precipitated glycoproteins (gp) of 86 to 88 and 105 kDa from MCMV-infected cells. Deglycosylation by endoglycosidase F resulted in a protein with a molecular mass of 64 kDa. Injection of complete MCMV DNA or of DNA fragments, and the subsequent testing of cytoplasmic binding of IgG by immunofluorescence microscopy, was used to search for the coding region in the MCMV genome. The gene was located in the HindIII J fragment, map units 0.838 to 0.846, where an open reading frame of 1,707 nucleotides predicts a gp of 569 amino acids with a calculated molecular mass of 65 kDa. The sequence of this gp is related to those of the gE proteins of herpes simplex virus type 1 and varicella-zoster virus. The defined length of the mRNA, 1,838 nucleotides, was in agreement with that of a 1.9-kb RNA expressed throughout the replication cycle, starting at the early stages of infection. Expression of the gene fcr1 by recombinant vaccinia virus resulted in the synthesis of gp86/88 and gp105, each with FcR properties, and the correct identification of the gene encoding the FcR was confirmed by the DNA injection method.

Amino Acid Sequence↗