Lars Rymo

Molecular cloning of the complete Epstein-Barr virus genome as a set of overlapping restriction endonuclease fragments

A complete collection of fragments of Epstein-Barr virus DNA, obtained by cleavage with restriction endonuclease Eco RI, has been cloned. Fourteen different internal fragments of the virus genome, derived from linear virion DNA of the B95-8 strain, and sequences corresponding to the terminal regions of virion DNA, derived from intracellular circular EBV DNA isolated from 895-8 cells, were cloned. Sizes of fragments were determined by agarose gel electrophoresis and their sum leads to an estimated molecular weight of 110 x 10(6) for virion DNA. Large Eco RI DNA fragments of special interest were also cloned in cosmids using another source of EBV DNA, that is, to circular viral DNA derived from Raji cells. In order to provide a set of overlapping sequences, all the 29 internal Bam HI fragments of B95-8 virion DNA were cloned in pBR322. The map location within the viral genome of each cloned DNA fragment was identified by hybridizing to blots of virion DNA cleaved with several different restriction endonucleases.

High frequency of mammary adenocarcinomas in metallothionein promoter-human growth hormone transgenic mice created from two different strains of mice

Transgenic mice were developed by injecting a mouse metallothionein promoter-human growth hormone (Mt-hGH) gene fragment into the pronucleus of C57Bl x DBA/2J-f2 or C57Bl x CBA-f2 one cell embryos. Six founder animals with the C57Bl x DBA genetic background grew 1.3-2.2 times larger than littermate controls and had higher levels of hGH in plasma (4.6-279 mU/l). Three of the four female transgenic founders developed malignant papillar adenocarcinomas of mammary origin at 27-43 weeks of age. One male transgenic founder was successfully mated and two of three female transgenic offsprings developed mammary tumors. To examine if the tumor induction was dependent on the strain of mice used the experiments were repeated using animals with different genetic background. Fourteen female hGH transgenic mice from five founder animals were generated using C57Bl x CBA-f2 mice. Thirteen of the animals had elevated levels of hGH in plasma (7-1960 mU/l) and grew larger than control animals. Nine of the animals developed mammary adenocarcinomas. Four of the hGH expressing animals did not demonstrate macroscopic tumor formation but have not yet been analyzed histologically. The present study suggests that markedly elevated endogenous levels of GH cause mammary carcinoma in hGH transgenic mice. The present animal model might prove useful for studying molecular mechanisms involved in the development of hormonally induced mammary tumors.

Disease-related differences in antibody patterns against EBV-encoded nuclear antigens EBNA 1, EBNA 2 and EBNA 6

Antibodies to Epstein-Barr virus (EBV) nuclear antigen family (EBNA) and three of its individual members, EBNA 1, EBNA 2 (A and B) and EBNA 6, were measured by anticomplement immunofluorescence (ACIF) in sera of 75 healthy controls, 13 patients with chronic EBV infection, 38 with non-Hodgkin lymphoma (NHL), 23 with Hodgkins disease (HD), 105 with nasopharyngeal carcinoma (NPC) and 7 patients with infectious mononucleosis (IM). Their anti-EBV lytic antigens were also measured. We observed that: (1) anti-EBNA 2A and E6 rose in parallel 4-6 weeks after IM, followed by anti-EBNA 1 at 3-6 months, (2) all seropositive individuals had anti-EBNA 1; 74% also had anti-EBNA 2A and E6, (3) anti-EBNA 1 accounted for most of the anti-EBNA reactivity in non-IM sera. Striking disease-associated differences were noted on the humoral responses to the lytic and transformation-associated antigens. Compared to the controls, anti-EBNA 1, -EBNA 2A and -EBNA 6 were simultaneously four to 10 times higher in chronic reactivations, whereas only anti-EBNA 1 was elevated (10 times) in NPC. Individual EBNA titres were normal in NHL or HD patients.

Epstein-Barr Virus Shedding in Breast Milk

One hundred healthy women already donating to the Childrens Hospital Breast Milk bank consented to provide a sample of breast milk for this study. Using a DNA-DNA hybridization dot-blot assay Epstein-Barr virus (EBV) genome (Bam HIW region) was detected in cells shed into breast milk of 46 out of 100 women studied and in 60 out of 132 (46%) of samples donated overall. The prevalence of EBV shedding increased postnatally to a peak of 74% (26/35 positive samples) between 3 and 12 weeks postdelivery. Women delivering prematurely had an initially lower prevalence of shedding with only six out of 30 (20%) positive samples in the first week after delivery, compared to 16 out of 35 (46%) for women delivering at term. Of the 18 women donating more than one sample, 13 showed consistently positive (n = 8) or negative (n = 5) results, and the remaining five had intermittent shedding detected. Only seven out of 42 (17%) breast milk samples studied were EBV-IgG antibody positive, and none showed IgM or IgA-EBV antibodies. Further studies and prospective followup of infants are needed to confirm that breast milk is a significant source for early EBV infection of infants, as indicated by serologic studies.

MODULAR ANALYTICS: A New Approach to Automation in the Clinical Laboratory

MODULAR ANALYTICS (Roche Diagnostics) (MODULAR ANALYTICS, Elecsys and Cobas Integra are trademarks of a member of the Roche Group) represents a new approach to automation for the clinical chemistry laboratory. It consists of a control unit, a core unit with a bidirectional multitrack rack transportation system, and three distinct kinds of analytical modules: an ISE module, a P800 module (44 photometric tests, throughput of up to 800 tests/h), and a D2400 module (16 photometric tests, throughput up to 2400 tests/h). MODULAR ANALYTICS allows customised configurations for various laboratory workloads. The performance and practicability of MODULAR ANALYTICS were evaluated in an international multicentre study at 16 sites. Studies included precision, accuracy, analytical range, carry-over, and workflow assessment. More than 700 000 results were obtained during the course of the study. Median between-day CVs were typically less than 3% for clinical chemistries and less than 6% for homogeneous immunoassays. Median recoveries for nearly all standardised reference materials were within 5% of assigned values. Method comparisons versus current existing routine instrumentation were clinically acceptable in all cases. During the workflow studies, the work from three to four single workstations was transferred to MODULAR ANALYTICS, which offered over 100 possible methods, with reduction in sample splitting, handling errors, and turnaround time. Typical sample processing time on MODULAR ANALYTICS was less than 30 minutes, an improvement from the current laboratory systems. By combining multiple analytic units in flexible ways, MODULAR ANALYTICS met diverse laboratory needs and offered improvement in workflow over current laboratory situations. It increased overall efficiency while maintaining (or improving) quality.

Analysis of restriction endonuclease fragments of intracellular epstein-barr virus DNA and isoenzymes indicate a common origin of the Raji, NC-37, and F-265 human lymphoid cell lines

Abstract Circular Epstein-Barr virus DNA molecules isolated from different virus-transformed cell lines usually show similar but distinct restriction enzyme cleavage patterns. It is shown here that EBV DNA from the cell line Raji, which is of African Burkitt lymphoma origin, has the same cleavage pattern on digestion with either Eco RI, Hin dIII, or Bam HI as EBV DNA from two more recently established human lymphoid cell lines, NC-37 and F-265, which are presumed to be of nontumor origin. On the other hand, these three EBV DNA isolates are clearly different from DNA of the standard B95-8 strain of the virus as judged by the same criteria. A restriction enzyme cleavage map of the circular EBV DNA from Raji cells has been made to provide a basis for the comparison. The isoenzyme patterns of 14 different marker enzymes were also investigated for the Raji, NC-37, and F-265 lines and found to be closely similar. These data indicate that the NC-37 and F-265 lines are not independent isolates of human lymphoid cell lines.

Regional assignment of the human 4-hydroxyphenylpyruvate dioxygenase gene (HPD) to 12q24-->qter by fluorescence in situ hybridization.

Using a panel of human-rodent somatic cell hybrids, we have previously mapped the gene (HPD, previously called PPD) encoding 4-hydroxyphenylpyruvate dioxygenase to the distal half of the long arm of human chromosome 12, region q14-->qter. To obtain a genomic probe useful for fluorescence in situ hybridization (FISH) analysis we screened a human leukocyte genomic library and isolated a 13.4-kb phage clone, which by restriction fragment and sequence analyses was shown to contain exons 1-10 of HPD and approximately 2-kb upstream sequences. We now report the subregional localization of HPD to 12q24-->qter based on two color FISH analysis employing this clone.

Epstein-Barr virus (EBV) antigen-specific leukocyte migration inhibition in infectious mononucleosis: II. Kinetics of sensitization against five EBV-encoded nuclear proteins and the latent membrane protein

The T cell-mediated immune response of infectious mononucleosis (IM) patients to five Epstein-Barr virus (EBV)-determined nuclear antigens, EBNAs, and to the membrane antigen associated with growth-transformed cells (latent membrane protein, LMP) was measured by the leukocyte migration inhibition (LMI) assay. Two different antigen sources were used: extracts from cells that only expressed EBNA-1, EBNA-2, or LMP after transfection with the corresponding EBV-DNA fragment, and synthetic peptides deduced from the corresponding genes. Patients in the acute phase of the disease failed to respond to EBNA-1, -5, -6, and LMP, but became responsive during convalescence. The majority of the patients responded to EBNA-2 and/or EBNA-3 in the acute phase (9/15 and 12/15, respectively). The response to EBNA-2 and/or EBNA-3 in the acute phase (9/15 and 12/15, respectively). The response to EBNA-3 disappeared more often in convalescence than the response to EBNA-2: 6 of 15 patients were negative to EBNA-2 and 12 of 15 to EBNA-3 during recovery. In addition to its value in the assessment of host sensitization to virus EBV antigens, these studies and the derived hypotheses also provide certain predictions about the predominant antigen expression in the EBV-infected host under normal and pathological conditions that can be subjected to direct experimental tests.

An Epstein-Barr Virus-Determined Nuclear Antigen Encoded by a Region within the EcoRI a Fragment of the Viral Genome

Large Epstein-Barr virus (EBV) DNA restriction fragments corresponding to regions transcribed in transformed, proliferating cells were cloned into a cosmid derivative of the dominant-acting selection vector pSV2-gpt. Recombinant vectors carrying the EcoRI A fragment of EBV DNA were modified in the region corresponding to the deletion of the virion DNA in the non-transforming viral substrain P3HR-1, to create a series of recombinants lacking parts of this region. The recombinant vectors were introduced into 3T3 mouse fibroblasts under selective conditions, and resistant clones shown to contain EBV DNA sequences were analysed for the expression of EBV-related antigens detectable by direct, indirect, and anticomplement immunofluorescence techniques. Cells that contained the BamHI K fragment expressed a nuclear antigen as expected. It is demonstrated here that cells transfected with recombinant vectors containing the major part of the EcoRI A fragment also express a nuclear antigen detectable with certain anti-EBNA-positive human sera in anticomplement immunofluorescence tests. This antigen is not detected in cells transfected with EcoRI A derived vectors where the BamHI H fragment has been deleted, nor in cells transformed with vectors carrying the BamHI H fragment alone. Direct and indirect immunofluorescence did not reveal the presence of antigens associated with productive infection in any of the EBV DNA transfected fibroblast clones.