Sensitivity of an epstein-barr virus-positive tumor line, Daudi, to alpha interferon correlates with expression of a GC-rich viral transcript.

The exquisite sensitivity of the Burkitt's lymphoma (BL)-derived cell line Daudi to type I interferons has not previously been explained. Here we show that expression of an Epstein-Barr virus (EBV) transcript, designated D-HIT (Y. Gao et al., J. Virol. 71:84-94, 1997), correlates with the sensitivity of different Daudi cell isolates (or that of other EBV-carrying cells, where known) to alpha interferon (IFN-alpha). D-HIT, transcribed from a GC-rich repetitive region (IR4) of the viral genome, is highly structured, responding to RNase digestion in a manner akin to double-stranded RNA. Comparing EBV-carrying BL cell lines with differing responses to IFN-alpha, we found the protein levels of the dsRNA-activated kinase, PKR, to be similar, whereas the levels of the autophosphorylated active form of PKR varied in a manner that correlated with endogenous levels of D-HIT expression. In a classical in vitro kinase assay, addition of either poly(I)-poly(C) or an in vitro-transcribed D-HIT homolog stimulated the autophosphorylation activity of PKR from IFN-alpha-treated cells in both EBV-positive and EBV-negative B lymphocytes. By transfection experiments, these RNAs were shown to reduce cell proliferation and to sensitize otherwise relatively insensitive Raji cells to IFN-alpha. The data lead to a model wherein the D-HIT viral RNA also serves as a possible transcriptional activator of IFN-alpha or cellular genes regulated by this cytokine.

Epstein-Barr virus in epithelial cell tumors: a breast cancer study.

The human herpes virus Epstein-Barr (EBV) is clearly associated with African Burkitt's lymphoma and the undifferentiated from of nasopharyngeal carcinoma, although its role in oncogenesis is still poorly defined. Recently EBV has been implicated in other types of lymphomas, as well as in some nonlymphomatous neoplastic processes. Its possible association with human breast cancer has been investigated here. DNA from 91 cases of breast carcinoma and blood samples from the same patients were amplified with the PCR over a region in the EBV BamHIW major repeat sequence following a single-step amplification protocol. Nineteen samples (21%) were found to be positive; 10 samples of blood (only 3 of them from patients with EBV-positive tumors) were found by the adopted protocol to contain EBV DNA. Another series of PCR amplifications using primers covering a unique (nonreiterated) fragment in BamHIC encoding the EBERs (two short nonpolyadenylated RNAs generally highly expressed in cells latently infected with EBV) confirmed these findings. A good correlation between the two sets of experiments was observed, and only five differences in results were obtained on samples tested. In situ hybridization was carried out using BamHIW biotinylated DNA probes or EBER-1 digoxigenin-labeled riboprobes with the aim of confirming as well as localizing the signal to the epithelial cell. Twelve sections (63%) among the PCR-positive samples were found positive by in situ hybridization with the DNA probe, and six (31.5%) sections were found with the RNA probe. Twenty-one samples from benign breast tumors or normal breast tissue were used as controls for PCR amplification in this study, none of which was found positive. This is the first known report showing positive results for EBV in breast cancer. No statistical association was found in these studies between the presence of EBV and the histological type of the tumor, however. Its role therefore remains for the moment unknown, as well as does the significance of the association of EBV with only a subset of the cases.

Cell fractionation in non-ionic detergent distinguishes sub-populations of polyoma virus middle T antigen and reveals a novel form.

The transforming function of polyoma virus, middle T antigen (MT), interacts with several cellular enzymes, essential to its oncogenic activity. We have used cell fractionation to study the various MT/cellular protein complexes. We demonstrate that MT can be separated into three sub-species, dependent upon extraction in two buffers that we designate A and B: Antigen extracted from whole cells by both buffers (called MT1) is associated with most of the phosphorylated phosphatidyl-inositol kinase 85 kD subunit, pp85, and protein phosphatase 2A. Antigen (MT2), associated with the greater portion of pp60c-src, is extracted by buffer B, but not buffer A. A third population (MT3), resistant to extraction by either buffer, is not detectably associated with protein phosphatase 2A or pp85. It is, however, associated with a low level kinase activity. The interaction between pp60c-src and MT appears to influence the formation of both MT2 and MT3. MT2 fractionates with the cellular microtubule network, but does not appear to be directly associated with it. MT3, a previously undescribed population, comprises about one third of MT in wild type antigen-containing cells. It is missing in mutants incapable of interacting with pp60c-src, but exists in the absence of an interaction with pp85. We suggest that MT3 may be an intermediate in, or product of, one of the MT/pp60c-src signalling pathways, distinct from that involving pp85.

Regulation of cytoskeletal association by a basic amino acid motif in polyoma virus middle T antigen.

The subcellular localization of many oncogenic proteins is thought to be important for their function. In the case of the middle T antigen of the DNA tumour virus, polyoma, localization to membranes in a specific manner is essential for its cellular transforming activity. To investigate factors that influence this localization, heterologous membrane targetting sequences were substituted for the middle T antigen transmembrane domain and the properties of the resulting proteins studied. Whereas C-terminal lipid modification derived from the H-ras CaaX box restored oncogenic activity to non-transforming truncated middle T antigen species, N-terminal myristylation from pp60c-src did not. Furthermore, a region, rich in basic amino acids and adjacent to the middle T transmembrane domain, was found to mediate association with detergent-insoluble cytoskeleton. Co-operation between the basic motif and neighbouring membrane binding domains resulted in specific localization of proteins to particular membrane sites, characterized by the association with subcellular structures, likely to be cytoskeletal in nature. These results demonstrate that the cellular localization of MT is regulated by at least two determinants, a transmembrane sequence which confers membrane binding and a basic motif which specifies a particular site within the membrane.

Immortalization and transformation of human fibroblasts by regulated expression of polyoma virus T antigens.

We have established conditions for the immortalization of human fibroblasts by the large T antigen of the rodent virus polyoma. This allows the mechanism of immortalization to be studied, without interference by transformation events, in cells with relatively stable chromosomes. Large T antigen could immortalize human fibroblasts if expression was driven by a heterologous promoter like the immediate early promoter/enhancer of cytomegalovirus or the inducible mouse mammary tumour virus (MMTV) promoter. Using the latter promoter and dexamethasone, three clones were obtained, the immortalized phenotype of which was strictly dependent on the induction of T-antigen expression. At least one of these clones became mortal after removal of the inducing agent. The expression of large T antigen was paralleled by PCNA gene expression, as shown by nuclear run-off transcription, whereas none of a number of other known proto-oncogenes was influenced in its activity. Immortalized fibroblasts were readily transformed by polyoma virus middle T antigen expressed from the MMTV promoter or by the activated c-Ha-ras oncogene. The reversibility of immortalization and transformation is considered.

Stimulation of cell division and fibroblast focus formation by antisense repression of retinoblastoma protein synthesis.

Circumstantial evidence supports a role for the retinoblastoma susceptibility gene, Rb-1, in the maintenance of normal cell growth, in that loss of its function results in abnormal growth and malignancy. Here we report that a high rate of mitosis and efficient dense focus formation in human embryonic lung fibroblasts (HEL cells) is induced by antisense oligonucleotide-directed inhibition of synthesis of p105-Rb, the product of the Rb-1 gene. mRNA specific for p105-Rb is truncated at the site of base pairing with the antisense oligonucleotide, and no synthesis of p105-Rb is observed. The rate of mitosis is considerably increased and the frequency of dense focus formation is extremely high in treated cells. However, although phosphothioate oligodeoxyribonucleotides taken up by the cells remain stable for at least 4 weeks, the recipient cells do not become immortal; nor are they able to induce tumor formation in nude mice. Thus, loss of Rb-1 function is not sufficient per se to allow malignant transformation.

Alterations in the structure of the EBV nuclear antigen, EBNA1, in epithelial cell tumours.

The EBV nuclear antigen, EBNA1, is the only viral protein consistently expressed in all virus-infected cells. It is required in trans for viral replication, maintenance of EBV extrachromosomal episomes, and transcriptional transactivation in latently-infected B-cells. It binds RNA suggestive of a regulatory role in post-transcriptional events and in transgenic mice, it is tumorigenic. In RNase protection studies relating to the EBV-associated tumour, nasopharyngeal carcinoma (NPC), we show that a C-terminal EBNA1 RNA probe from the prototype B95-8 marmoset strain can protect its own mRNA from enzymatic digestion, but does not fully protect EBNA1 mRNA from NPC cells. This finding is consistent with changes in the coding region for the antigen. We thus determined the sequences of EBNA1 genes derived from an NPC xenograft and numerous patient biopsies and identified a number of mutations in the gene in these human cells, relative to B95-8. Many of the nucleotide changes would lead to non-conservative amino acid alterations in apparently functionally significant regions of the protein. We show that although some of the mutations lie in regions designated as critical to DNA binding, they have negligible effect on this property of EBNA1. The basic regions in EBNA1 that may bind to RNA, at least in vitro, are exempt from mutation. Thus, unless the alterations are 'silent', which for such a critical viral function seems unlikely, they may relate to as yet unmapped viral activities, such as a role in tumorigenesis and the ability of EBNA1 to evade the cellular immune system, or be associated with the ability of the antigen to regulate gene transcription.

A Preliminary Genetic Investigation of VOLVOX CARTERI.

A preliminary genetic analysis of a number of genetic variants of Volvox carteri f. nagariensis is presented. Techniques are outlined for mutagenesis of Volvox, isolation of mutants and routine genetic analysis. All of the mutants show simple Mendelian segregation patterns and have been tentatively placed in 14 linkage groups.

Polyoma virus pseudocapsids as efficient carriers of heterologous DNA into mammalian cells.

Polyoma virus VP1 pseudocapsids, generated from a recombinant baculovirus, have been successfully used to transfer exogenous DNA stably into rodent (rat-2) cells. To evaluate the efficiency and biological usefulness of this route for introducing heterologous DNA into cells, the gene for a transforming deletion mutant of the middle T antigen of polyoma virus, dl8 MT, was used initially. Whereas the amount of DNA packaged together with pseudocapsids was found to be variable (2-30%), even at low efficiency its transfer as biologically functional information was high. The dl8 MT gene was stably transferred and integrated in low copy numbers into the host chromosome. Transformed cell lines (derived from single foci) were shown to produce high levels of the corresponding mutant protein, which was active in an in vitro protein kinase assay. In comparisons with the calcium phosphate DNA coprecipitation procedure (or lipofectin route), the VP1 pseudocapsid approach was shown to have many advantages in terms of maintenance of DNA fidelity and increased efficiency of gene expression. This system was also assessed for its ability to transfer into and express the chloramphenicol acetyl transferase (CAT) gene in a human liver cell line. Here again, the assay for functional CAT expression showed the pseudocapsid transfer procedure to compare favorably with lipofectin transfer. In another transient assay, a low-level endogenously expressed gene, p43, was complexed with pseudocapsids and transferred into human embryo lung fibroblasts, thereby increasing the expression levels. The ease of production of VP1 pseudocapsids, coupled with their efficient transfer of biologically useful information, should make this route of gene delivery an attractive proposition for further exploration with regard to gene therapy.

Efficient oligodeoxyribonucleotide-directed deletion mutagenesis using pEMBL vectors: removal of early region introns from polyoma virus mutants.

We have used oligodeoxyribonucleotide-directed deletion mutagenesis to remove early region introns from polyoma virus mutants. To this end we compared single priming, double priming, and gapped duplex approaches using either priming at 37 degrees C or at the critical temperature. The gapped duplex approach, coupled with priming at the critical temperature, resulted in up to 70% yield of the desired product. In conjunction with the use of the pEMBL vector system this method was simplified to yield specific deletions from cloned large DNA fragments with high efficiency. The resulting mutant plasmids could be used directly for biological assays without retransformation or recloning. RNA and protein analyses showed that removal of the large T- or middle T-antigen introns from polyoma early region mutants dl23 and dl8 was specific and resulted in DNA competent for the synthesis of only one T antigen.

Interactions of heterologous DNA with polyomavirus major structural protein, VP1.

'Empty' polyomavirus pseudocapsids, self-assembled from the major structural protein VP1, bind DNA non-specifically and can deliver it into the nuclei of mammalian cells for expression [Forstová et al. (1995) Hum. Gene Ther. 6, 297-3061. Formation of suitable VP1-DNA complexes appears to be the limiting step in this route of gene delivery. Here, the character of VP1-DNA interactions has been studied in detail. Electron microscopy revealed that VP1 pseudocapsids can create in vitro at least two types of interactions with double-stranded DNA: (i) highly stable complexes, requiring free DNA ends, where the DNA is partially encapsidated; and, (ii) weaker interactions of pseudocapsids with internal parts of the DNA chain.

Meningitis, ventriculitis, and hydrocephalus: a complication of fetal monitoring.

A case is presented of meningitis, ventriculitis, and hydrocephalus secondary to the use of fetal scalp monitoring. The most commonly reported fetal complication related to the application of the scalp electrode is the development of a scalp abscess; the incidence varies between 0.45 and 4.5%, but several other complications have also been reported and the overall incidence is not well established. Infants in whom a scalp electrode has been used should be carefully examined to detect potential serious complications.

African Burkitt's lymphoma: a new perspective.

High titres of antibody to Epstein-Barr virus (EBV) late genes identify individuals at risk of developing endemic Burkitt's lymphoma (eBL). Viral lytic cycle early and intermediate-early gene expression in BL is associated with a favourable tumour response to chemotherapy. Our study investigated whether serological data identifying antibody expression to zta, a viral function that activates lytic-cycle gene expression, correlate with expression of its gene in tumours, and could have prognostic value. Studies on 10 Malawian patients, with presumed BL on clinical grounds, showed good correlations, suggesting that serum antibody responses might predict treatment responsiveness. The results with 1 patient were particularly striking. When admitted in January 1998, prognosis was poor as he was unable to walk, and had tumour cells, characteristic of stage IV disease, in his bone marrow. Laboratory investigations showed particularly high levels both of serum zta antibodies and of gene expression in his tumour. Follow-up confirmed him alive 6 months after hospital discharge. Among the EBV-positive cases, 2 were ultimately diagnosed as rhabdomyosarcoma, a tumour not previously associated with this virus. The findings from this small study, if confirmed, should have value for future BL management in resource-poor parts of the world.

Pulse oximetry: what's normal in the newborn nursery?

The objective of this study was to establish normal values for pulse oximetry saturation (POS) in healthy newborn infants in the nursery. POS values were obtained from the right (R) hand and R foot at admission, 24 hr, and at discharge. The following information was recorded: postnatal age, activity state, gender, gestational age (GA), birth weight (BW), mode of delivery (MOD), and Apgar scores. Charts were reviewed and follow-up information was obtained for newborns with measurements < or =92%. The study group consisted of a convenience sample of newborn infants, excluding those on supplemental oxygen. Seven hundred eighteen patients were studied: 51% males, 28% cesarean sections, gestational age 39.3+/-1.6 weeks (mean +/- SD), birth weight 3370+/-550 g, and median Apgar scores 8 and 9. The mean POS was 97.2 +/-1.6%, and the median value was 97%. Only postnatal age and activity state affected POS significantly. POS increased 0.17% per 24 hr in the nursery (P = 0. 0001). POS values obtained while the infants were fussy and crying were lower compared to measurements obtained while sleeping [mean decreases: 0.44% while fussy (P = 0.001), 0.98% while crying (P = 0.0001)]. We conclude that newborns in the nursery have an overall mean POS of 97.2% (+/-2 SD: 94-100%). Mean POS values increase to a small degree with increasing postnatal age. Fussy and crying newborns have lower POS values compared to quiet and sleeping newborns. These reference data can be used in the evaluation of POS measurements in symptomatic newborn infants.

Epstein-Barr virus (EBV) and human disease: facts, opinions and problems.

The human herpesvirus, Epstein-Barr virus (EBV), has classically been associated with two pathologies with frequencies approaching 100%. One of these, Burkitt's lymphoma (BL), is of B-cell origin and the other, nasopharyngeal carcinoma (NPC), is a tumour of poorly differentiated epithelial cells. More recently, EBV had been identified with frequencies from a few percent to 100% (in one case) with a variety of other malignancies. These include Hodgkin's disease (HD; where in the west, the frequency of association is about 50%), sino-nasal T-cell lymphomas, lymphoepitheliomas, some sarcomas and breast cancers, other cancers from the head and neck, and lymphomas arising in patients with immune dysfunctions. Since EBV is ubiquitous, with the vast majority of the world's population having met and seroconverted to the virus, the diversity of tumours with which it has now been associated represents a substantial health burden. In a recent IARC monograph, EBV was classified as a group 1 carcinogen. Here, the data on BL and NPC, as they relate to geographical restrictions, viral strain variation, co-factors in disease, and genetic components are reexamined. We raise the question whether in their origins, these tumours genuinely reflect distinct and independent events, as deemed at present, or may represent a response by different cell types to common extracellular factors. For example, a situation in Kenya apparently existed in the past, where both BL and NPC were observed in ethnic Africans with roughly equal frequencies; more recently, in Kenya, EBV has been identified in nearly 100% of the tumours in children with HD. We also consider tumours where the viral association is reportedly of low frequency, and offer explanations for these data, including the possibility of loss of the viral genome once malignancy has been initiated. If this phenomenon occurs as a frequent secondary event, EBV could be an even greater health risk than presently believed.

A cluster of atypical skin lesions in well-baby nurseries and a neonatal intensive care unit.

We describe an epidemiologic investigation that elucidated the cause of vesicular and bullous skin lesions of the hands and feet that occurred in three otherwise well neonates during a 24-hour period. The investigation encompassed two well-baby nurseries of 28 and 17 beds and one level III neonatal intensive care unit (NICU) of 31 beds located in a 440-bed university-affiliated community hospital. Work-up for infectious causes of the skin lesions in the initial three cases had negative results. Expanded case surveillance disclosed seven additional cases that had occurred within the previous 2 weeks in the NICU. Analysis of risk factors focused attention on the insertion technique for peripheral intravenous catheters. This led to the discovery of a defective transillumination device, the tip of which reached a temperature of 88 degrees C within 20 seconds, causing thermal burns. The cause of the malfunction was the failure to install an infrared filter during the manufacture of the device. No additional cases were observed after the defective unit was removed from service. In summary, a defective transilluminating device caused a cluster of thermal burns in a newborn nursery and NICU. Epidemiologic investigation of the cluster allowed the investigators to focus on techniques of intravenous catheter insertion, which thus led to the identification of the cause of the injuries. With the increasing emphasis on health outcomes measurement, hospital epidemiologists will likely have an expanding role in investigating clusters of noninfectious adverse events.

Serological diagnosis of nasopharyngeal carcinoma by enzyme linked immunosorbent assay: optimization, standardization and diagnostic criteria.

OBJECTIVES: To produce an enzyme linked immunosorbent assay (ELISA) to detect antibodies against Epstein Barr virus (EBV) specified nuclear antigen 1 (EBNA 1) and the 18 kD EBV matrix protein, and to determine and optimize its sensitivity and specificity for the diagnosis of nasopharyngeal carcinoma (NPC). METHODS: We used a combination of highly purified glutathione transferase fusion proteins of the 40 kD carboxy domain of EBNA1 and the 18 kD EBV matrix protein for coating ELISA plates. In three separate studies, we tested for IgA antibodies in serum specimens from 28 EBV seronegative donors, 284 EBV seropositive donors and 160 newly diagnosed NPC patients. By comparing the sensitivity and specificity of diagnosis obtained for different cutoff values, we derived several quantitative parameters to evaluate assay performance, establish objective diagnostic criteria which optimize the intrinsic diagnostic capability of the assay and assess the significance of individual test results, respectively. Optimum cutoff optical density (OD) is defined as the cutoff OD where sensitivity of the assay equals its specificity, and resolution of the assay is indicated by the value of sensitivity (or specificity) determined at the optimum cutoff OD. Diagnosis of NPC was achieved by setting a cutoff zone at +/- 20% of this value. RESULTS: All the EBV seronegative donors tested were not reactive, and most of the EBV seropositive donors were weakly reactive, while the majority of NPC patients were moderately or strongly reactive. While the assay was thus shown to be specific for EBV, there was an overlap in the level of these serum antibodies between few individuals of the two latter groups. It was shown that the assay performed equally well in two separate studies conducted under different testing conditions and using different collections of sera in that assay resolution determined on these occasions were 86% and 87% respectively. Diagnosis of NPC can be achieved at the same expected sensitivity of 89% and 83% determined at the lower and upper limits of the cutoff zones, with the corresponding values of specificity being 78% and 91%. It was further shown in the third study that resolution of the assay can be increased to 90% using an assay produced with a higher concentration of the same antigens, and that diagnosis of NPC can be achieved at a higher sensitivity ranging between 86% and 95% at a corresponding specificity of 93% and 86%. CONCLUSIONS: After optimization and standardization, the ELISA can achieve a sensitivity ranging from 86% to 95%, with corresponding specificities of 93% and 86% respectively for the diagnosis of NPC.