Lars Schaade

Application of Virus-Specific Immunoglobulin M (IgM), IgG, and IgA Antibody Detection with a Polyantigenic Enzyme-Linked Immunosorbent Assay for Diagnosis of Epstein-Barr Virus Infections in Childhood

ABSTRACT The Enzygnost anti-Epstein-Barr virus enzyme-linked immunosorbent assay (ELISA) system, which is based on a defined antigen mixture and on detection of antibodies of the immunoglobulin G (IgG), IgM, and IgA classes, was evaluated for its reliability in diagnosing Epstein-Barr virus infections in childhood. With samples from 66 children, the Epstein-Barr virus status and the infection phase were defined by indirect immunofluorescence and anticomplement fluorescence assays: 11 children were seronegative, 8 had a primary infection, 20 had a recent primary or past infection, and in 27 a reactivated Epstein-Barr virus infection was diagnosed. When applying the Enzygnost ELISAs, 15 serum samples (22.7%) were not interpretable due to indeterminate results in at least one of the assays used and were therefore excluded from further evaluation. The respective sensitivities and specificities for the diagnosis of seronegativity were 100 and 100%, those for the diagnosis of primary infection were 100 and 97%, those for the diagnosis of recent primary or past infection were 100 and 52%, and those for the diagnosis of reactivated infection were 10 and 100%. This poor performance of the Enzygnost system with reactivated infections is due to the prerequisite of an IgG antibody value of >650 IU/ml for the diagnosis of viral activity, which was fulfilled in only two of the children. Despite the high rate of indeterminate results, the Enzygnost system is useful in diagnosing acute and past Epstein-Barr virus infection in childhood. For serological diagnosis of viral activity in childhood, a supplementary assay is necessary.

Detection of Cytomegalovirus DNA in Human Specimens by LightCycler PCR: Melting Point Analysis Is Mandatory To Detect Virus Strains with Point Mutations in the Target Sequence of the Hybridization Probes

In our recently published paper we described the application of the LightCycler technology for the detection of cytomegalovirus (CMV) DNA in human plasma and urine (1). We are now able to give further information gained since September 2000, when this PCR procedure was introduced as the standard assay for the detection of CMV DNA in our diagnostic laboratory. As of June 2001, we had tested 912 specimens using this assay. According to the described test protocol, 185 specimens (20.3%) were positive for CMV DNA. A melting point (Tm )o f 59.2°C was observed for the hybridization probes with PCR amplicons of all positive specimens. Another 12 specimens (1.3%) were negative in quantitative analysis but generated a significant peak, with a Tm of 53.1°C, in the melting point analysis. Gel electrophoresis revealed a distinct PCR product of about 250 bp with these specimens that corresponds to the targeted 254-bp amplicon. DNA sequencing of the PCR products with the decreased melting point confirmed the specific amplification of CMV DNA using these specimens as a template. The decline of the melting point is caused by a point mutation in position 630 of the CMV glycoprotein B gene (GenBank accession no. A13758), resulting in a shift from cytosine to thymidine. This strain variant is not included in the databases and causes a mismatch with position 5 of the LCRed 640-labeled acceptor fluorophore probe. However, the existence of further strain variations in the amplified region cannot be excluded. The melting point analysis allows the reliable detection of CMV strain variants harboring this point mutation. Specificity of the signal should be further confirmed by gel electrophoresis. Melting point analysis is mandatory to detect virus strain variants in the target sequence of the hybridization probes with the described LightCycler CMV PCR assay.

MRI follow-up of herpes simplex virus (type 1) radiculomyelitis

Most genital infections by herpes simplex virus (HSV) are associated with HSV-2, whereas HSV-1 has rarely been implicated in these conditions.1 Following primary genital infection, the virus persists in a latent stage within neurons of dorsal root ganglia (DRG). Occasionally, HSV reactivation may be followed by radiculomyelitis, which variably affects the cauda equina, conus, and thoracic spinal cord.1 We report a 48-year-old immunocompetent man with acute HSV-1 radiculomyelitis that occurred in the absence of any preceding or concomitant herpetic skin lesions. The patient was admitted to the hospital owing to progressive numbness, which started in both feet the day before and ascended proximally to the legs. He also complained of gait disturbance and urinary incontinence. On neurologic examination, signs of meningeal irritation were absent and cranial nerves were normal. Deep-tendon reflexes were ++ in both arms and absent in the legs; plantar responses were flexor. Muscle strength was normal in the arms and slightly reduced in the legs (4/5). There was a severe sensory loss for all modalities in both legs and in the perianal region, as well as marked sensory ataxia of the lower limbs with corresponding gait disturbance. Anal sphincter muscle tone was reduced, resulting in stool incontinence; there was also overflow urinary incontinence. Motor nerve …

Cloning and expression of the human single copy homologue of the mouse zinc finger protein zfr

A human homologue of the murine zinc finger protein zfr is transcriptionally induced in the Epstein-Barr virus-positive Burkitt lymphoma cell line Raji upon treatment with the granulocyte/macrophage lineage ganglioside IV(3)NeuAc-nLcOse(4)Cer. The gene was cloned by a rapid amplification of cDNA ends approach based on a cDNA clone. The resulting hzfr sequence is 3393 base pairs in length coding for a protein of 1057 amino acids. Sequence alignments between hzfr and zfr reveal an identity of 92% on the nucleotide level and an identity of 96.4% on the amino acid level, respectively. Based on Southern blot data hzfr can be addressed as a single copy gene. Tissue-specific expression was determined by semi-quantitative PCR of normalized cDNA populations from various human tissues with glyceraldehyde-3-phosphate dehydrogenase as an internal control. Highest levels of transcripts were found in brain. hzfr transcripts could not be detected in skeletal muscle.

Enhanced transcription of the s-adenosylhomocysteine hydrolase gene precedes Epstein-Barr virus lytic gene activation in ganglioside-stimulated lymphoma cells

Abstract Stimulation of Epstein-Barr virus (EBV) genome-positive Burkitt lymphoma cells with the ganglioside IV3NeuAc-nLcOse4Cer leads to the induction of cell differentiation processes and activates the EBV lytic viral cycle. In cells of the Burkitt lymphoma line Raji differential expression of host cell genes was analysed in the early phase (150 min) post stimulation with the ganglioside to display the cell activities that precede the activation of the EBV lytic cycle using the differential display reverse transcription-polymerase chain reaction technique. Multiple fragment cDNAs derived from control cells and ganglioside-stimulated cells were amplified using random primers and displayed via polyacrylamide gel electrophoresis. The expression pattern of 8,400 bands was analysed. Eleven differentially expressed fragment cDNAs were reamplified and identified by nucleotide sequencing. Six of these could be identified as coding for proteins that may take part in virus reactivation and differentiation. The most striking finding was the induction of s-adenosylhomocysteine hydrolase (AHCY) expression. The cellular enzyme AHCY plays an important role in transmethylation reactions controlling the replication of several viruses. Thus, an involvement in EBV replication can be suggested.

Early steps in termination of the immortalization state in Burkitt lymphoma: induction of genes involved in signal transduction, transcription, and trafficking by the ganglioside IV3NeuAc-nLcOse4Cer

Stimulation by the ganglioside IV(3)NeuAc-nLcOse(4)Cer leads to growth arrest in the Burkitt lymphoma cell line Raji. In order to analyze the primary response of Raji cells to that stimulus, a cDNA array screen and a suppression subtractive hybridization-PCR approach were performed. Twenty-four genes with assigned functions were confirmed to be induced by the ganglioside in reverse Northern blot experiments covering e.g. protein kinase B, phospholipase C, the MAP-kinase ERK3, the transcription factors YY1, DR1 and NSEP, the membrane traffic protein TAP, and the nuclear export protein CRM1. Most of the genes identified are involved in signal transduction, transcription, and cell trafficking. For selected genes, the induction of expression was quantified by semiquantitative RT-PCR.

A membrane-located glycosphingolipid of monocyte/granulocyte lineage cells induces growth arrest and triggers the lytic viral cycle in Epstein-Barr virus genome-positive Burkitt lymphoma lines.

Abstract Gangliosides are known to influence cell growth and differentiation. The neolacto series ganglioside IV3NeuAc-nLc4 (2→3-sialosylparagloboside) is present in members of the monocyte/granulocyte lineage, but is not found in cells that belong to the lymphocyte lineage. In this study we demonstrated that IV3NeuAc-nLc4 inhibits the proliferation of Epstein-Barr virus (EBV) genome-positive Burkitt lymphoma cells of the lines Raji and P3HR-1K. IV3NeuAc-nLc4-induced growth inhibition is associated with an increase in G0/G1 phase cells and a reduced expression of CD21 and HLA-DR antigens on Raji cells. These data suggest that IV3NeuAc-nLc4 may affect differentiation of lymphoma cells. Additionally, the increased expression of viral mRNA species which are characteristic for the lytic viral cycle in the non-producer line Raji and the enhanced release of virions from the producer line P3HR-1K demonstrate that IV3NeuAc-nLc4 activates the replication of EBV. Growth inhibition and termination of the viral latency suggest that IV3NeuAc-nLc4 present in monocyte/granulocyte lineage cells may be an effector of the natural defense against EBV persistency and transformation.

Induction of growth rate and transcriptional modulation of growth promoters and growth inhibitors in epithelial cells by EBV-LMP1

LMP1 is a genuine oncogene encoded by the Epstein-Barr virus (EBV). The cellular response to expression of the EBV-encoded gene LMP1 in the epithelial cell line Wish was studied. Cells were stably transfected with pCEP-LMP, an expression vector for LMP1. On transcript level a transient expression of the LMP1-gene with a maximum 2 days post transfection was observed. Six days post transfection the rate of DNA synthesis of LMP1-transfected Wish cells was increased by 80% compared to control cells, after 2 further days the number of cells was increased by 32%. A human cDNA-array was screened with probes from LMP1-transfected and control cells showing induction of transcription for proliferation associated genes and repression for growth suppressor genes.

Differentiation in Murine Mastocytoma Induced by Macrophage Gangliosides

In the membrane of mouse macrophages two gangliosides were detected which inhibit the division of murine mastocytoma P815 tumor cells. The two gangliosides were incorporated into the cytoplasmatic membrane of mastocytoma cells. The concentration necessary to achieve a complete inhibition of P815 tum or cell division is about 1 [_im for both effective gangliosides. Macrophage ganglioside-induced inhibition of cell division is accompanied by morphological changes of the mastocytoma cells. While the cells are rounding, their diameter increases and serotonin and granules appear in the cytoplasm of the enlarged cells. Our findings suggest that macrophage gangliosides may differentiate mastocytoma cells into mast cells.

Characterization of Cytostatically Active Glycosphingolipids Isolated from Thioglycollate‐Elicited Murine Macrophages

Two acidic glycosphingolipids (gangliosides) derived from mouse macrophage membranes and separated by thin‐layer chromatography have a strong cytostatic effect on human and mouse tumor cells. The structure of the two gangliosides, named MphiG1 and MphiG2, was elucidated by application of physicochemical and immunochemical methods. Gas chromatography and mass spectrometry of MphiG1 and MphiG2 classified them as isomeric monosialogangliosides with ceramide moieties composed of sphingosine as the long‐chain base, C16 and C18 fatty acids, respectively, and a lacto‐tetraose backbone. For MphiG1, additional immunochemical findings led to the proposed structure IV3NeuAc‐nLcOse4Cer. The immunochemical reactions of MphiG2 suggest a branched structure for the oligosaccharide moiety.