Protective and disease-enhancing immune responses to respiratory syncytial virus.

The National Institutes of Health, Centers for Disease Control and Prevention, and World Health Organization jointly sponsored a workshop on protective and disease-enhancing immune responses to respiratory syncytial virus (RSV). The primary purpose of the meeting was to discuss protective and disease-enhancing immune responses to RSV in the context of opportunities and barriers to the development of RSV vaccines. Although both live attenuated and subunit vaccines have been developed, it is not yet clear if any of these vaccines will be safe and effective. The fact that neither the disease-enhancing nor the protective immune response to RSV is well understood or well characterized is an important barrier to development of these vaccines. Studies in animal model systems and newly developed immunologic tools, however, provide hope that these barriers can be overcome and a safe and effective RSV vaccine can be developed.

Poly(A+)RNA levels of growth-, differentiation- and transformation-associated genes in the progressive development of hepatocellular carcinoma in the rat.

The development of chemically induced hepatocellular carcinoma in the rat proceeds through a series of premalignant changes that may ultimately progress to a primary malignant tumor. Using the selection technique based on diminished binding of preneoplastic hepatocytes to tissue culture plates precoated with asialofetuin, we have isolated poly(A+)RNA from early preneoplastic foci as well as preneoplastic persistent nodules and primary hepatocellular carcinoma induced by the Solt-Farber protocol in the Fischer rat. The steady-state poly(A+)RNA levels of genes traditionally associated with growth, differentiation and/or transformation were then determined to address the question of their temporal expression in the multistep nature of cancer development. Ornithine decarboxylase- and P53-specific transcripts did not significantly change in preneoplastic foci but were increased in later-stage preneoplastic nodules and hepatocellular carcinoma. Albumin-specific transcripts were decreased in all hepatocellular carcinoma but there was no consistent coordinated increase in alpha-fetoprotein-specific transcripts. c-myc and raf transcripts increased at the very early preneoplastic foci stage and continued to increase throughout the neoplastic process. No L-myc or N-myc transcripts could be detected in any RNA sample. c-Ha-ras-specific transcripts were essentially unaltered in all RNA samples whereas no c-Ki-ras or N-ras transcripts could be detected throughout the neoplastic process. In addition, no dominant-acting transforming mutations in the ras gene family were detected by DNA transfection experiments using NIH/3T3 cells.

Transformation of Epstein-Barr virus immortalized human B-cells by chemical carcinogens.

Epstein-Barr virus-immortalized human lymphocytes were used to analyze the transition from the benign hyperproliferative to the malignant transformed state. Treatment with N-acetoxy-2-acetylaminofluorene, a potent frameshift mutagen, induced conversion of the Epstein-Barr virus immortalized lymphocytes into high-grade "immunoblastic lymphomas" on injection into athymic mice, whereas injection of the untreated, original cells did not. The tumor cells were all of the B-cell lineage as determined by the presence of surface immunoglobulins and antigens detected by B-cell specific antibodies to B1 and B4, and the absence of the T-cell-specific markers, 3A1 and LEU-1. The N-acetoxy-2-acetylaminofluorene-induced tumor lines displayed abnormal diploid to tetraploid karyotypes. The fewest chromosomal rearrangement, excluding tetraploidy, observed in these chemically induced lymphomas involved a deletion in chromosome 6, and additions on both chromosomes 16 and 4. Neither major rearrangements nor amplifications were found for K-ras, H-ras, N-ras, c-myc, Blym, and c-myb in these tumor lines.

Epstein-Barr virus immortalization of normal cells of B cell lineage with nonproductive, rearranged immunoglobulin genes.

Most continuous cell lines derived by EBV immortalization of peripheral blood cells are composed of phenotypically mature B lymphocytes, and secrete Ig. Occasionally, EBV-immortalized cell lines have failed to secrete Ig. Expansion and characterization of one of these EBV-induced cell lines, VDS-O, showed that in addition to a lack of Ig secretion, surface and intracytoplasmic Ig were absent. Cell surface phenotyping revealed that VDS-O belongs to the B cell lineage, because it expresses the B cell restricted antigens B1 and B4, while it lacks T cell and monocyte-associated determinants. Analysis of the Ig gene organization in VDS-O revealed that the Ig genes are rearranged for both heavy (gamma) and light (kappa) chains. However, the expected gamma-heavy chain and/or kappa-light chain RNA species were not detected. These findings demonstrate the existence in normal peripheral blood of cells of B cell lineage susceptible to EBV immortalization that have Ig genes that are rearranged but are nonproductive.

Studies of gene transcription and translation in regenerating rat liver.

Specific transcriptional and translational products associated with regenerating liver were analyzed by differential hybridization to a cDNA library and by two-dimensional electrophoresis of hepatic proteins, respectively. Comparisons of approximately 800 soluble and 800 particulate liver proteins from normal and 70% partially hepatectomized Fischer rats resulted in the identification of only three apparently unique polypeptides in 70% partially hepatectomized livers, although many quantitative changes were observed. A subset of these quantitative changes were also observed after sham operation. A cDNA library was generated from polyadenylated RNA isolated 18 hr post-70% partial hepatectomy. Comparative analysis of 6,000 transformants with single-stranded cDNA probes prepared from 18 hr post-70% partial hepatectomy and sham-operated animals identified three clones whose sequences were preferentially expressed 4- to 6-fold 18 hr post-70% partial hepatectomy. Southern blot analysis of one clone, REG-A, showed no homology to albumin, alpha-fetoprotein, three different forms of cytochrome P-450, ornithine decarboxylase, globin, or to a putative tumor promotion associated gene called PRO-2. A single, REG-A specific 2.5 kb band was identified by Northern blot analysis of liver samples. REG-A expression was increased 2-fold 18 hr postsham operation; 4-fold 18 hr post-70% partial hepatectomy and following chronic 2,3,7,8-tetrachlorodibenzo-p-dioxin or phenobarbital treatment. REG-A expression returned to control levels 1 week after 70% partial hepatectomy. Furthermore, expression of REG-A was reduced in chemically induced preneoplastic nodules and in primary and transplantable hepatomas. Hybrid selection studies indicated that the REG-A sequence selected a mRNA(s) species, that in an in vitro translation assay, produced two major polypeptides of 21,000 and 25,000 molecular weight with a pI of 6.9. Thus, these data support the hypothesis that liver regeneration is characterized by quantitative changes in genes normally expressed at low levels in the Go hepatocyte and is not the result of major qualitative changes in gene expression.

Tumorigenicity and transcriptional modulation of c-myc and N-ras oncogenes in a human hepatoma cell line.

Tumorigenicity and oncogene expression were examined in HepG2 derived cells, a human hepatoma cell line. HepG2 cells and a single cell clonal HepG2 line, HLD2-6, were equally tumorigenic when injected s.c. into athymic nude mice. Cyclophosphamide pretreatment of both cell lines (500 micrograms cyclophosphamide/ml/two cell cycles) had no effect on tumor incidence or latency (P greater than 0.05). Tumors were nonencapsulated, highly invasive adenocarcinomas and were positive for gamma-glutamyltranspeptidase activity and bile production. Plasma from tumor-bearing mice was positive for human alpha-fetoprotein and negative for hepatitis B virus surface antigen as measured by radioimmunoassay. Two cell lines reestablished into tissue culture from HLD2-6 derived tumors had unaltered cell cycle times. Detailed in vitro translation analysis of RNA isolated from HLD2-6 derived cells and tumors were extremely similar to the translation products of RNA isolated from a normal human liver sample except for a Mr 53,000 polypeptide with an apparent charge shift. c-myc specific transcripts, when compared to a normal human liver sample, were increased in all HLD2-6 cell lines and tumors derived from HLD2-6 cells. This increase in c-myc expression could not be explained by gene amplification or hepatitis B virus integration. N-ras specific transcripts were not elevated in HLD2-6 cells grown in tissue culture but there was a selective increase of the 5.5-kilobase N-ras transcript in HLD2-6 derived tumors grown in nude mice. This increased 5.5-kilobase transcript did not remain elevated if the tumors were reestablished into tissue culture, suggesting some interaction with the host animal. c-Ha-ras expression could not be detected in any HLD2-6 derived tumor or cell line.

NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice.

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.

Abnormally elevated frequency of Epstein-Barr virus-infected B cells in the blood of patients with rheumatoid arthritis.

Patients with rheumatoid arthritis (RA) are known to have in vitro regulatory T cell abnormalities relating to Epstein-Barr virus (EBV). In this report, we asked whether patients with RA have more circulating EBV-infected B cells than normals. To address this question, we determined the frequency of spontaneously transforming B cells in the peripheral blood of 18 normals, 15 patients with RA, and 8 patients with systemic lupus erythematosus (SLE). The mean frequency of spontaneously transforming B cells in RA patients was 10.1/10(6) B cells, which was significantly greater than that of the normal controls, 2.8/10(6) B cells (P less than 0.005). The group of patients with SLE did not differ from the normals (P greater than 0.4). In further studies undertaken to investigate as to whether RA B cells are more easily transformed by EBV than normal B cells, we determined that the frequencies of transforming B cells in the presence of exogenous EBV were similar in RA patients and normals. Lymphocytes obtained from patients with RA demonstrate a profound T cell defect in their EBV-specific suppression, as measured in vitro; there was no direct correlation, however, between this in vitro T cell abnormality and the number of circulating EBV-infected B cells. Thus, patients with RA, as a group, have abnormally elevated numbers of circulating EBV-infected B cells, and this abnormality most likely derives from a complex dysregulation of the defense mechanisms for infection with EBV.

Transcriptional organization of bovine papillomavirus type 1.

Multiple bovine papillomavirus type 1 (BPV-1)-specific polyadenylated RNA species in a BPV-1-infected bovine fibropapilloma were identified and mapped. All of the RNA species were transcribed from the same DNA strand of the BPV-1 genome. Five RNA species previously identified in BPV-1-transformed mouse cells were also present in the bovine fibropapilloma. These five species measured 1,050, 1,150, 1,700, 3,800, and 4,050 bases, mapped within the 69% transforming segment of the BPV-1 genome, and shared a 3' coterminus at 0.53 map units (m.u.). The 5' ends of the bodies of these distinct transcripts were located at ca. 0.03, 0.09, 0.34, 0.39, and 0.41 m.u. Additional polyadenylated RNA species not present in BPV-1-transformed mouse cells were specific for the BPV-1-infected bovine fibropapilloma and measured 1,700, 3,700, 3,800, 6,700, and 8,000 bases. These wart-specific species shared a 3' coterminus at 0.90 m.u. The 5' termini of the bodies of the 1,700- and 3,800-base species mapped at 0.71 and 0.42 m.u., respectively. Exonuclease VII analysis failed to reveal any internal splicing in these two species; however, the presence of small remote 5' leader sequences could not be ruled out. The 3,700-base species hybridized to DNA fragments from the 69% transforming segment as well as from the 31% nontransforming segment of the BPV-1 genome; however, this species was not precisely mapped. The 5' termini of the two largest RNA species (6,700 and 8,000 bases in size) were located at ca. 0.01 and 0.90 m.u., respectively. Since the 5' ends of these mapped adjacent to a TATAAA sequence which could possibly serve as an element of a transcriptional promoter, it is possible that one or both of these species represent nonspliced precursor RNA molecules.

Expression of cellular myc and mos genes in undifferentiated B cell lymphomas of Burkitt and non-Burkitt types.

Burkitt lymphomas contain reciprocal translocations between chromosome 8 and one of the chromosomes containing the immunoglobulin gene loci, prompting speculation that consequent activation of a crucial gene(s) on chromosome 8 might be involved in the generation of these tumors. Recently the human counterparts of the retroviral oncogenes v-myc and v-mos have been mapped to chromosome 8. We have, therefore, analyzed the level of transcription of the cellular myc and mos genes in a variety of undifferentiated B cell lymphomas of Burkitt and non-Burkitt type that possess either an 8;14 or an 8;22 translocation. These lines expressed 2- to 5-fold more myc-specific RNA than do B cell lines without a translocation. Tumor cell lines of American origin with an 8;14 or 8;22 translocation expressed similar amounts of myc-specific RNA. Tumor cell lines of African origin contained slightly higher levels of myc-specific RNA than did those of American origin. However, level of expression does not appear to correlate with the presence or absence of Epstein-Barr virus. Therefore, a major increase in the transcription of this gene secondary to translocation is unlikely to be the cause of Burkitt lymphoma. There was no evidence of mos-related transcripts in any of the cell lines tested.

Cloned human polyomavirus JC DNA can transform human amnion cells.

The genome of the human polyomavirus JC (Mad-1 strain) was molecularly cloned in Escherichia coli by using the plasmid vector pBR322. Recombinant DNA molecules were constructed with the entire JC genome inserted either at its unique EcoRI site at 0.0 map units or at its unique BamHI site at 0.51 map units. Viral DNA from each of these recombinant plasmids was capable of transforming human amnion cells, and cell lines established from transformed foci were positive for JC tumor antigen as assayed by indirect immunofluorescence.

Cloning of human papilloma virus genomic DNAs and analysis of homologous polynucleotide sequences.

The complete DNA genomes of four distinct human papilloma viruses (human papilloma virus subtype 1a [HPV-1a], HPV-1b, HPV-2a, and HPV-4) were molecularly cloned in Escherichia coli, using the certified plasmid vector pBR322. The restriction endonuclease patterns of the cloned HPV-1a and HPV-1b DNAs were similar to those already published for uncloned DNAs. Physical maps were constructed for HPV-2a DNA and HPV-4 DNA, since these viral DNAs had not been previously mapped. By using the cloned DNAs, the genomes of HPV-1a, HPV-2a, and HPV-4 were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm = --28 degrees C), no homology was detectable among the genomes of these papilloma viruses, in agreement with previous reports. However, under less stringent conditions (i.e., Tm = --50 degrees C), stable DNA hybrids could be detected between these viral DNAs, indicating homologous segments in the genomes with approximately 30% base mismatch. By using specific DNA fragments immobilized on nitrocellulose filters, these regions of homology were mapped. Hybridization experiments between radiolabeled bovine papilloma virus type 1 (BPV-1) DNA and the unlabeled HPV-1a, HPV-2a, or HPV-4 DNA restriction fragments under low-stringency conditions indicated that the regions of homology among the HPV DNAs are also conserved in the BPV-1 genome with approximately the same degree of base mismatch.