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G Bein

Publications and source records attributed to G Bein.

At least 55 records · Page 3Linked to original sources
PubMed1996

The intracellular localization of human cytomegalovirus DNA in peripheral blood leukocytes during active infections by high-resolution fluorescence in situ hybridization.

Although viremia is an integral part of the pathogenesis of human cytomegalovirus (HCMV) disease, the interaction between HCMV and circulating leukocytes of actively infected patients remains an area of uncertainty. It is still a matter of dispute, whether leukocytes support viral replication with subsequent production of infectious virus. In a new approach we developed and applied a sensitive fluorescence in situ hybridization assay for the precise intracellular localization of HCMV genomes in leukocytes. It was shown that in vivo HCMV genomes were exclusively localized in the cytoplasm of leukocytes, indicating that the majority of these cells are virus carriers or abortively infected. Though this method easily detects single copy genes in metaphase chromosomes, the number of HCMV DNA positive leukocytes was significantly lower than the number of HCMV pp65 antigen positive cells. In relation to the pp65 antigen positive cells, only 1-4% of these cells were DNA positive. In addition, the much lower frequency of HCMV immediate early antigen positive leukocytes in comparison to the pp65 antigen positive cells and the impossibility of detecting other viral antigens support the hypothesis that the origin of pp65 found in leukocytes results mainly from protein uptake.

Antigens, Viral↗
PubMed1996

Fluorescence in situ hybridization with cosmid clones for the detection of human cytomegalovirus DNA in peripheral blood leukocytes.

Radioactive in situ hybridization techniques or enzymatic detection procedures of hapten-modified human cytomegalovirus (HCMV) probes have been widely used for studying the infection of peripheral blood leukocytes with HCMV. This report describes significant improvements in terms of signal resolution which can be obtained by applying a highly sensitive fluorescence in situ hybridization (FISH) technique in conjunction with a large subgenomic HCMV DNA probe. Three cosmid clones spanning 119.1 kb of the HCMV genome (230 kb) were used to construct the digoxigenin-11-dUTP-labeled probe which was found to be superior to a total HCMV probe representing the entire genome. Crucial hybridization parameters were analyzed systematically in order to ensure optimal resolution power and sensitivity. The protocol was successfully applied to HCMV-infected fibroblasts and peripheral blood leukocytes of 12 transplant patients and unambiguously facilitated the precise intracellular localization of HCMV genomes in infected cells. Because of its excellent resolution properties, accompanied by the virtual absence by any background staining, we recommend the use of this protocol as a sensitive approach for further virological analyses of the interactions between HCMV and peripheral blood leukocytes at the single-cell level.

Cloning, Molecular↗
PubMed1996

IgM-specific serodiagnosis of acute human cytomegalovirus infection using recombinant autologous fusion proteins.

Portions of three human cytomegalovirus (HCMV) polypeptides, which were shown previously to be highly reactive with patient sera, were expressed in Escherichia coli as autologous fusion proteins. Purified recombinant polypeptides were used as antigens in enzyme linked immunosorbent assay (ELISA) and compared against assays which use natural viral antigen from cell culture for their ability to improve IgM-specific serology of acute HCMV-infection. A fusion protein (CM2) which contained two copies of the C-terminal portion of pUL44 (p52, aa 297-433) and one copy of a highly reactive fragment of the major DNA-binding protein pUL57 (aa 545-601) proved to be superior in sensitivity and specificity compared to assays which use culture derived antigen. A construct expressing one copy of the fragments from pUL44 and pUL57 in fusion with the 54 amino terminal residues of pUL32 (pp150, aa 994-1048) did not lead to an improved sensitivity compared to CM2. Adversely, this polypeptide reacted with a number of sera from asymptomatic blood donors infected latently with HCMV indicating low specificity of this antigen for the detection of acute infection. Concordant results were obtained with an antigen that combined only the C-terminal portions of pUL44 and pUL32 (CM3). ELISA experiments with sequential sera from renal transplant recipients demonstrated that detection of IgM-antibodies using CM2 as antigen correlated closely with acute infection, whereas high levels of IgM-antibodies against CM1 and CM3 persisted for a month following acute HCMV-infection. These results indicate that the application of a single autologous fusion protein like CM2 as antigen for recombinant ELISAs can improve significantly IgM-serodiagnosis of acute HCMV infection.

Antibodies, Viral↗
PubMed1996

Morphometric techniques in the evaluation of pulmonary vascular changes due to congenital heart disease.

Open lung biopsy is performed in patients with congenital heart disease to determine the suitability for surgical correction. Controversy exists as to whether arterial density can be graded with certainty. We studied the influence on the grading (Heath-Edwards) of two morphometric techniques. Lung tissue from 14 controls and biopsy specimens from 80 patients with atrial septal defect (n=10) ventricular septal defect (n=27), complete atrioventricular canal (n=27), miscellaneous lesions (n=8) and tetralogy of Fallot (n=8) were analyzed with a planimetric method. Additionally, wall thickness was determined in 52 patients by distance measurements. The ratio of alveoli/arteries per area of lung tissue was measured. Medial thickness was "increased" on distance measurements in 15% of the cases where planimetric data showed normal wall thickness. The ratio of alveoli/arteries varied up to 43% from the mean. Hemodynamic data obtained at a median (range) time of 2 months (1 day to 18 months) before the operation did not correlate with morphologic findings. In 27 patients hemodynamic data were recorded at a median time of 1 year (3 days to 44 months) after the operation. Intimal fibrosis occupying more than 10% of the vessel lumen was associated with persistent high pulmonary vascular resistance. We conclude that morphometric techniques are useful to determine the degree of fibrosis in advanced vascular lesions. Arterial density cannot be determined in biopsy specimens with adequate certainty.

Adolescent↗
PubMed1996

Rapid typing for human platelet antigen systems-1, -2, -3 and -5 by PCR amplification with sequence-specific primers.

Typing for human platelet antigens (HPA) is useful in a variety of clinical situations. We developed a method for genotyping for HPA-1, -2, -3 and -5 by means of the PCR amplification with sequence-specific primers (PCR-SSP) technique. Primer sets were designed to allow PCR amplification for all systems using the same assay conditions. Specificity and sensitivity of the method were assessed in a blind quality control study (n = 112). In 111 cases, results obtained by PCR-SSP were identical as compared with PCR-restriction fragment length polymorphism technique. One discrepancy was found to be due to a typing error in the data sheet. The results of the PCR-SSP technique were available within 3 h. We conclude that genotyping based on PCR-SSP enables rapid typing for HPA systems, which makes this technique feasible in most clinical settings where urgent HPA typing is required.

Antigens, Human Platelet↗
PubMed1996

Immunoglobulin A-specific serodiagnosis of acute human cytomegalovirus infection by using recombinant viral antigens.

The immunoglobulin A-specific reactivities of recombinant viral proteins from nine different reading frames of human cytomegalovirus were evaluated in enzyme-linked immunosorbent assay experiments. Antigen fragments of reading frames pUL32, pUL44, and pUL57 were identified as preferable antigens for immunoglobulin A serodiagnosis. Application of autologous fusion proteins which combine these polypeptides may be useful especially for the early detection of acute secondary human cytomegalovirus infection.

Acute Disease↗
PubMed1996

[Blood leukocytes are not a primary latent site of cytomegalovirus].

Conflicting results do exist regarding blood leukocytes as a site of CMV latency. In an attempt to detect latently infected blood cells we studied CMV major immediate early (MIE) antigen expression in monocytes or mononuclear leukocytes from 80 blood donors (71 seropositive) after in vitro stimulation and subsequent antigen detection by immunocytochemistry (detection limit 1 positive cell/10(6) cells). None of the more than 800 cytospin preparations investigated turned out to be positive. Additionally the granulocyte macrophage colony stimulating factor (GM-CSF)/hydrocortisone (HC) stimulated monocytes were analysed by reverse transcriptase PCR for the presence of MIE mRNA and were also completely negative. In contrast to the negative findings in blood samples, we detected CMV DNA by PCR in 6 out of 20 suddenly deceased individuals in tissue samples of the lung, but not in those of other organs. We conclude that we have no evidence that blood leukocytes are a site of latent CMV. The transmission of CMV by blood transfusions may be mediated by rare phagocytes that transiently bear CMV after uptake from infected endothelial cells.

Blood Donors↗
PubMed1995

Coincidence of Epstein-Barr virus reactivation, cytomegalovirus infection, and rejection episodes in renal transplant recipients.

Reactivation of the Epstein-Barr virus was reported to occur frequently under immunosuppressive therapy following organ transplantation. However, little is known about the clinical significance of these EBV reactivations. Therefore, we searched for correlations among the treatment with various immunosuppressive drugs, the incidence of CMV infections, rejection crises, and serological signs of EBV reactivation. EBV-specific antibodies were measured with novel ELISAs, utilizing the recombinant antigens p72 (for anti-EBV nuclear antigen [EBNA]1-IgG), p54, and p138 (anti-early antigen [EA]-IgM, -IgG, -IgA) in a follow-up study of 79 renal transplant recipients. Patients receiving antithymocyte globulin or antilymphocyte globulin therapy showed increasing anti-EA-IgG and -IgA more often than did patients not receiving antithymocyte globulin or antilymphocyte globulin therapy (P < 0.05). In patients receiving OKT3 antirejection therapy, anti-EA-IgM seroconversion was found more frequently (P < 0.01). A significant correlation was also found between groups of patients who had had at least one rejection episode versus patients without any sign of organ rejection, and the incidence of increasing anti-EA-IgG (P < 0.05). Since in most of these patients signs of EBV reactivation followed the appearance of the rejection episode, this may not be due to viral-induced rejection but may be caused by the reinforced immunosuppression during antirejection therapy. As opposed to patients with no signs of CMV infection and with nonsymptomatic CMV infection, patients undergoing symptomatic CMV infection showed anti-EA-IgM seroconversion (P < 0.01), increasing anti-EA-IgA (P < 0.01), and decreasing anti-EBNA-IgG (P < 0.01) more frequently. Our results confirm the role of immunosuppressive therapy in the pathogenesis of EBV reactivation. We further demonstrate a striking coincidence of EBV reactivation and symptomatic CMV infection.

Antibodies, Viral↗
PubMed1995

Plasma endothelin-1 in patients with left-to-right shunt.

Endothelin-1 (ET-1) has been implicated in the pathogenesis of pulmonary vascular disease in patients with congenital heart disease. We studied the effect of pulmonary blood flow on plasma ET-1 concentrations. Systemic venous, pulmonary arterial, and pulmonary venous blood samples were obtained from 40 patients with atrial septal defect II (n = 21), ventricular septal defect (n = 10), persistent ductus arteriosus (n = 6), and complete (n = 2) and partial (n = 1) atrioventricular canal at cardiac catheterization and analyzed by radioimmunoassay. Median age (range) was 5.2 years (8 months to 66 years), and the resistance ratio (Rp/Rs) was 0.08 (0.03 to 0.67). Pulmonary vein to pulmonary artery ET-1 ratio in patients with "high flow" (n = 26, Qp/Qs > or = 1.5; ET-1 ratio = 0.8) did not differ from those with "low flow" (n = 14, Qp/Qs < 1.5; ET-1 ratio = 1.0). We conclude that an increase in pulmonary blood flow alone does not result in an increase in plasma ET-1.

Adolescent↗
PubMed1995

Failure to detect type 1 interferon production in human umbilical cord vein endothelial cells after viral exposure.

Cytokine production by endothelial cells is known to occur after the reception of signals such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). In the present study, we demonstrate cytokine release and upregulation of adhesion molecule expression of human endothelial cells derived from umbilical cord veins in response to stimulants. The cells were exposed to the plant lectin phytohemagglutinin A (PHA), the viral inducers Newcastle disease virus (NDV) and Sendai virus, and the nucleic acid analog polyinosinic/polycytidylic acid (polyI:C). All stimulants induced expression of IL-1 beta and IL-6. The titers of these cytokines were comparable to those obtained in human leukocytes, whereas no TNF-alpha release was detectable. A further feature of activation was upregulation of intercellular adhesion molecule 1 (ICAM-1), which was analyzed after contact with these stimulants. These experiments revealed an increased expression of ICAM-1 in response to all stimulants tested. A major part of our studies was devoted to the ability of endothelial cells to produce interferons as an important function of a number of cell types in response to viral infections. To ensure stimulation of the cells, the inducers NDV, Sendai virus, and polyI:C were analyzed. Expression of interferon (IFN) was not detected, although viral inducers mediated the release of IL-1 beta and IL-6, suggesting that endothelial cells are quite responsive to these stimulants. In conclusion, these findings show that the ability of endothelial cells to secrete cytokines is not restricted to mediators of the immune system, such as IL-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1995

Low resolution DNA typing of the HLA-B5 cross-reactive group by nested PCR-SSP.

We have established a DNA typing system for the HLA-B5 serologically cross-reactive group (CREG) by means of a two-step PCR amplification with nested sequence-specific primers (nPCR-SSP). The present study provides a low resolution definition of the HLA-B5 CREG, i.e. identifying polymorphism equivalent to serology. Two different primer combinations allow group-specific amplification of all HLA-B5 CREG alleles and other related HLA class I alleles from genomic DNA. The amplified DNA is subjected to a second amplification step using eleven nested primer pairs. This assay permits the detection of the HLA-B5 CREG specificities B35, B51, B52, B53, and B7801 in all homozygous and heterozygous combinations. Sensitivity and specificity as judged by a blind quality control study investigating a reference panel (n = 50) is 100%. Extension of this approach should allow rapid DNA typing of all serologically defined HLA-B specificities by nPCR-SSP.

Alleles↗
PubMed1995

The DNA-binding protein pUL57 of human cytomegalovirus is a major target antigen for the immunoglobulin M antibody response during acute infection.

A small polypeptide from pUL57 of human cytomegalovirus was identified as a major target for the immunoglobulin M antibody response. This antigen seems to be superior to antigenic fragments from pp150 and p52 in the identification of sera from acutely infected patients. It may therefore represent an essential antigen for recombinant immunoglobulin M antibody tests for human cytomegalovirus.

Acute Disease↗
PubMed1994

Semiautomated HLA-DQB1 typing by fluorescent dye photometry of amplified DNA on microtiter plates.

We report a simple semiautomated HLA class II typing method that suits the demands of a 24-hour-duty transplantation service for preorgan retrieval donor typing. The procedure consists of sequence-specific amplification of HLA alleles by the polymerase chain reaction (nested PCR-SSP) followed by fluorescent dye photometry of the resulting PCR products on microtiter plates. The entire typing procedure is semiautomated and completed in less than 90 minutes after DNA isolation. The test was evaluated for the definition of the specificities DQ1-DQ9 (generic HLA-DQB1 typing). Sensitivity and specificity as judged by DNA reference typing in a different laboratory was 99.5% (n = 202 alleles).

Alleles↗
PubMed1994

[HLA diagnosis and DNA technology].

The invention of the polymerase chain reaction, for which Mullis was awarded the Nobel prize for chemistry in 1993, is one of the most significant advances to occur in the field of molecular biology during recent years. Today, precise DNA typing of the HLA class II alleles can be established as early as 90 min after DNA extraction. The classical methods of serological HLA class II typing have an error rate of up to 25%. Retrospective analyses show that the success rate of transplantations could be improved considerably by the introduction of DNA typing into donor-recipient matching in clinical kidney transplantation. The present survey attempts to provide an evaluation of the various DNA techniques in clinical practice.

Alleles↗
PubMed1993

A longitudinal prospective study of cytomegalovirus pp65 antigenemia in renal transplant recipients.

Cytomegalovirus (CMV)-encoded pp65 antigen in peripheral blood leukocytes (CMV antigenemia) was investigated in 1017 serial samples from 64 patients for 16 weeks after renal transplantation in a prospective study. In 110 samples from 24 patients, at least one antigen-positive leukocyte was identified. The median number of stained cells was 4 (range 1-1000) per 4 x 10(5) leukocytes. Twenty-one of 24 patients with serological signs of an active CMV infection were antigen-positive (sensitivity 87.5%), whereas 3 patients with antigenemia did not show serological signs of infection during the observation period (specificity 92.5%). Positive results were obtained 19 days (median) before serological response and 9 days (median) before the onset of CMV syndrome. The sensitivity in defining a CMV syndrome was 100% (n = 8). In all patients who presented with CMV syndrome, antigenemia was present prior to the onset of symptoms or on the same day. In contrast, serological monitoring rendered the diagnosis of CMV infection possible at the onset of clinical symptoms in only two of eight patients. We conclude that (1) insufficient results obtained with the CMV antigenemia assay by other investigators are mainly due to technical problems that can easily be overcome by the protocol presented and (2) the detection of CMV pp65 antigen in peripheral blood leukocytes is an excellent tool for rapid and early diagnosis of CMV infection.

Antibodies, Viral↗