Isolation of a cDNA from the virus responsible for enterically transmitted non-A, non-B hepatitis.

Major epidemic outbreaks of viral hepatitis in underdeveloped countries result from a type of non-A, non-B hepatitis distinct from the parenterally transmitted form. The viral agent responsible for this form of epidemic, or enterically transmitted non-A, non-B hepatitis (ET-NANBH), has been serially transmitted in cynomolgus macaques (cynos) and has resulted in typical elevation in liver enzymes and the detection of characteristic virus-like particles (VLPs) in both feces and bile. Infectious bile was used for the construction of recombinant complementary DNA libraries. One clone, ET1.1, was exogenous to uninfected human and cyno genomic liver DNA, as well as to genomic DNA from infected cyno liver. ET1.1 did however, hybridize to an approximately 7.6-kilobase RNA species present only in infected cyno liver. The translated nucleic acid sequence of a portion of ET1.1 had a consensus amino acid motif consistent with an RNA-directed RNA polymerase; this enzyme is present in all positive strand RNA viruses. Furthermore, ET1.1 specifically identified similar sequences in complementary DNA prepared from infected human fecal samples collected from five geographically distinct ET-NANBH outbreaks. Therefore, ET1.1 represents a portion of the genome of the principal viral agent, to be named hepatitis E virus, which is responsible for epidemic outbreaks of ET-NANBH.

Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent.

An RNA virus, designated hepatitis G virus (HGV), was identified from the plasma of a patient with chronic hepatitis. Extension from an immunoreactive complementary DNA clone yielded the entire genome (9392 nucleotides) encoding a polyprotein of 2873 amino acids. The virus is closely related to GB virus C (GBV-C) and distantly related to hepatitis C virus, GBV-A, and GBV-B. HGV was associated with acute and chronic hepatitis. Persistent viremia was detected for up to 9 years in patients with hepatitis. The virus is transfusion-transmissible. It has a global distribution and is present within the volunteer blood donor population in the United States.

Establishment and characterization of a cloned giant cell line from a patient with Hodgkin's disease.

The phenotypic characteristics of a cloned giant cell line, SU/RH-HD-1, established from the spleen of a patient with Hodgkin's disease were studied. The cells grew slowly, adhered to the culture vessel surface, and had an elongated, irregular shape. After trypsinization, they became spherical and measured 30-100 micron in diameter. Although most cells were mononuclear, binucleated and multinucleated cells could be identified in expanded cultures. The cells phagocytized latex and ink particles and were nonspecific esterase-positive, but they did not secrete lysozyme. They were Epstein-Barr nuclear antigen-negative, and their culture fluid supernatants were devoid of reverse transcriptase activity. Electron microscopy revealed cells with a pronounced smooth endoplasmic reticulum, free ribosomes, some filaments, and mitochondria. Many 0.5- to 1.0-micron invaginations (pits) were seen along the cell membrane. Nucleoli were enlarged and prominent in the very heterochromatic nuclei. The SU/RH-HD-1 cells had 10- to 100-micron-long pseudopodia that were sometimes forked or branching, as well as multiple stress fibers. Electron microscopic appearance was suggestive of that of macrophages. This interpretation of the results was substantiated by monoclonal antibody studies, which revealed that the cells express antigenic determinants distinctive for cells of the monocyte-macrophage lineage and by functional studies demonstrating that the cells are capable of specific antigen presentation to immune T-cells. The SU/RH-HD-1 cells were aneuploid and could be cloned, first in liquid culture by limiting dilution and later in semisolid medium. It was likely that the SU/RH-HD-1 cells were derived from the neoplastic giant cell population in Hodgkin's disease and that they originated from cells of the mononuclear phagocyte-reticulum cell lineage.

Electron microscopic studies of purified eukaryote messenger RNA.

Rat liver mRNA, hen and rabbit globin messenger RNA (mRNA) were investigated by electron microscopy. No secondary structures were visible in the molecules of these mRNAs under conditions where short secondary structures of other types of RNA, i.e. MS2 phage RNA and 28 S rRNA, were clearly demonstrated. The contour lengths of these mRNAs were also determined by electron microscopy and compared with the sizes estimated by other techniques. The contour lengths of rabbit short and long globin mRNA are 0.1498 +/- 0.0019 and 0.1908 +/- 0.0021 micrometer, respectively. The former is assumed to be globin mRNA for alpha chain and the latter for beta chain. Similarly, hen "alpha" and "beta" globin mRNA have mean lengths of 0.1449 +/- 0.0011 and 0.1891 +/- 0.0017 micrometer, respectively. Hen and rabbit reticulocytes contain 1.8-2.0 times as much mRNA for alpha globin chains as for beta.

Patterns of thymocyte differentiation markers on virus and radiation induced lymphomas of C57BL/Ka mice.

To better understand the biology of tumorigenesis in virus and radiation lymphomas of C57Bl/Ka mice, we have examined the cell surface phenotypes of a large series of primary tumors induced by both agents. Data derived using flow cytometry and recently available monoclonal antibodies to thymocyte differentiation antigens supports three major conclusions. First, tumor cell populations are unimodal for staining with most antibodies and are probably of clonal origin. Second, many, but not all, tumor cells show surface phenotypes similar to those of previously defined subpopulations of normal thymocytes. Third, at the cell surface level, no major differences between virus- and radiation-induced lymphomas can be discerned. Our data thus further define the relationship between thymomas induced by these two agents.

Message amplification phenotyping (MAPPing): a technique to simultaneously measure multiple mRNAs from small numbers of cells.

A rapid and highly sensitive technique (MAPPing: message amplification phenotyping) has been developed to simultaneously analyze the array of messenger RNAs made by small numbers of cells. The technique incorporates a micro-procedure for isolating RNA, reverse transcription of total cellular RNA to produce cDNA, and enzymatic amplification of cytokine-specific DNA fragments using the polymerase chain reaction. In this study, the technique has been applied to the analysis of cytokines produced by lymphoid cells ranging in number from a single cell to 10(6) cells. The technique should be applicable to virtually any tissue or cell type.

Variation in antibody reactivity to the hepatitis C virus by comparative immunoscreening and enzyme immunoassay.

The detection of antibody to the hepatitis C virus C100-3 antigen from the nonstructural region (NS3/NS4) of the viral genome was the first useful marker developed to detect past or potentially active infection with the hepatitis C virus. A systematic epitope survey of the nonstructural region has uncovered other immunogenic antigens. In order to assess the possible diagnostic utility of these antigens, their reactivity against a limited panel of sera from patients with chronic liver disease due to hepatitis C virus and other etiologies was tested. Antibody assays were performed using an immunoblot plaque assay and an enzyme-linked immunosorbent assay (ELISA). In a study of 16 C100-3-reactive individuals, all 16 patients were reactive using the plaque assay for the NS3 3' (409-1-1) and NS3 5' (C33u). In this same group of patients, antibodies by ELISA were reactive to NS3 3' in 12 of 16 patients (75%), NS3 5' in 15 of 16 patients (93%), and a capsid antigen (NC450) in 14 of 16 patients. In a group of five patients who were diagnosed with cryptogenic liver disease (C100-3 negative), 4 of 5 patients were reactive for antibody to all of the above epitopes. In a survey of 23 patients with other forms of chronic liver disease (nonviral liver disease, hepatitis B, alcoholic liver disease, cholestatic liver disease, and autoimmune hepatitis), only 1 of 23 patients was reactive for antibody to the C100-3 and 4 of 23 patients were reactive for antibodies to structural and nonstructural regions of the virus.(ABSTRACT TRUNCATED AT 250 WORDS)

VH gene use by HIV type 1-associated lymphoproliferations.

The pathogenesis of polyclonal HIV-associated lymphomas lacking traditional B cell cofactors (i.e., Epstein-Barr virus [EBV] infection, c-myc translocations) is poorly understood. A multistep pathogenesis model has been proposed in which polyclonal lymphomas represent an earlier stage in HIV-associated lymphomagenesis before the emergence of a dominant malignant clone. Chronically present antigens have been proposed as a likely stimulus for polyclonal B cell proliferation; if so, polyclonal lymphoma-associated immunoglobulins (Igs) should have molecular evidence of somatic hypermutation, a process by which antibody affinity maturation in response to chronic antigenic stimulation occurs. Molecular analyses of Ig heavy chain variable (V(H)) gene use by B cells in a polyclonal HIV-associated large cell lymphoma lacking EBV and c-myc rearrangement was undertaken. Eighteen randomly selected clones generated from RT-PCR yielded 15 unique V(H) sequences, all of which were most homologous to only three previously identified germline V(H)1 genes. Two sets of clones (consisting of three and two clones, respectively) had identical V(H) gene sequences, and one pair of clones had identical third complementarity determining regions (CDR3s) but different V(H) gene sequences; eight clones were <95% homologous to their most related germline V(H)1 genes. We compared these results with Ig V(H)1 gene use by B cells present in a reactive hyperplastic lymph node obtained from an HIV-1-infected individual. Fifteen clones randomly selected from RT-PCRs yielded 15 unique V(H)1 sequences, all of which were most homologous to 5 previously identified germline V(H)1 genes; 10 clones were <95% homologous to their most related germline gene. Binomial probability analysis revealed that only 1 of the 15 unique V(H)1 sequences derived from the polyclonal lymphoma (i.e., 7%), as compared with 5 of 15 unique V(H)1 sequences derived from the reactive lymph node (i.e., 33%), had a low probability of occurrence by random chance (p < 0.05). These data provide molecular evidence of polyclonality in an HIV-associated polyclonal lymphoma, demonstrate a qualitative difference in somatic hypermutations of Ig V(H) genes associated with malignant versus reactive B cell lymphoproliferations, and support an antigen-mediated multistep pathogenesis model of HIV-1-associated lymphomagenesis.

Specific amplification of rearranged immunoglobulin variable region genes from mouse hybridoma cells.

In this article we show how the polymerase chain reaction (PCR) and primers designed for conserved sequences of leader (L), framework one (FR1) and constant (CONST) regions of immunoglobulin light and heavy chain genes can be used for the cloning and sequencing of rearranged antibody variable regions from mouse hybridoma cells. RNA was extracted from the mouse hybridoma cells secreting MAbs: IOR-T3a (anti-CD3), C6 (anti-P1 of N. meningitidis B385), IOR-T1 (anti-CD6), CB-CEA.1 (anti-carcinoembryonic antigen), and CB-Fib.1 (anti-human fibrin). First strand cDNA was synthesized and amplified using PCR. The newly designed primers are superior to others reported recently in the literature. Isolated PCR DNA fragments of C6 and IOR-T3a were sequenced after asymmetric amplification, or M13 cloning. The FR1/CONST primer combinations selectively amplified mouse lights chain of groups kappa II, V, and VI, and heavy chains of groups IIa and IIc. The L/CONST primers for light chains amplified light chains from all four hybridomas. These methods greatly facilitate structural and functional studies of antibodies by reducing the efforts required to clone and sequence their variable regions.

Some factors affecting the Hill reaction activity in cotton chloroplasts.

A method of plant culture was developed for growing large leaves of glandless cotton on single stems. Chloroplasts isolated from these leaves actively reduced ferricyanide when assayed for the Hill reaction. Hill reaction activity increased 133% when the 0.5 m sucrose isolation medium was replaced with 10% (w/v) polyethylene glycol, both buffered at pH 7.6. The presence of 2 or 5% (w/v) bovine serum albumin in the sucrose buffer did not increase Hill activity. Ferricyanide reduction in the dark occurred in all assays, and the possibility of gossypol as the reductant is discussed. Half-life of the chloroplasts stored in 10% glycerol at -23 C was 23 days. The ammonium ion at 0.01 m enhanced Hill reaction activity up to 171%. Leaves containing chloroplasts with the highest Hill reaction activity were found near the 8th node below the apex. Leaf water potentials less than -28 bars reduced the activity about 50%. Daylight conditions during the winter months in the greenhouse reduced the activity about 30%.

Recombinant antibodies.

Many fundamental advances in our understanding of the structure and function of eukaryotic genes were derived from the study of antibody genes. Examples include mRNA splicing and rearrangement to generate antibody diversity. The capacity to immortalize an individual B cell using cell fusion permitted the generation of monoclonal antibodies. Monoclonal antibodies have had wide application in many fields of the life sciences and beyond. Recent advances permitting manipulation of antibody genes using recombinant DNA techniques offer many advantages over conventional somatic cell hybridization techniques. Rodent monoclonals can be "humanized" and antibody isotype readily changed. Grafting of the complementarity determining regions from rodent to human framework regions demonstrated the importance of these hypervariable portions of the immunoglobulin to the integrity of the antibody combining site. Recombinant monoclonal antibodies (rMAb) or fragments thereof have been successfully produced in both prokaryotic and eukaryotic hosts at levels equal to those produced by hybridomas. Successful efforts to express rMAbs in plants and other large capacity systems suggest that rMAbs can be produced inexpensively. Use of antibody catalysis and antibodies mimicking various receptors or ligands have numerous applications. Technology developed to immortalize the heavy and light chain repertoire permits the generation in vitro of recombinatorial libraries of antibodies. The capacity to artificially generate high-affinity antibodies in vitro using the methods of recombinant DNA technology has enormous pharmaceutical and industrial potential.

Molecular characterization of the hepatitis G virus.

A novel virus associated with acute and chronic hepatitis has been cloned and characterized. The single-stranded RNA genome is approximately 9000 nucleotides long and has the structure of a virus in the Flaviviridae family. The genes of the virus have been identified and characterized by a number of molecular techniques and a genetic map has been determined.

Variations in genetic recombination due to amber mutations in T4D bacteriophage.

Recombination experiments were performed to assess the affect of amber mutations in 12 genes of T4D bacteriophage on genetic recombination. Crosses were performed in various suppressor-containing bacterial hosts to permit the production of progeny phage. Amber mutations in genes 32, 46, and 47 caused decreased recombination, amber mutations in genes 30, 41, 42, 43, 56, 61, and 62 caused increased recombination, whereas mutations in genes 63 and 37 showed no demonstrable effect on recombination.

Characterization of an infective molecular clone of the B-tropic, ecotropic BL/Ka(B) murine retrovirus genome.

Using molecular cloning techniques, we amplified the unintegrated, linear proviral DNA of the BL/Ka(B) virus, a non-leukemogenic retrovirus of mouse strain C57BL/Ka. Two independent clones in lambda phage vector 607 and one subclone in pBR322 were infective when transfected into mouse fibroblasts. Analysis of the progeny virus revealed biological properties and a restriction map identical to those of the parental viral shock. Comparison of the restriction map with the maps of other ecotropic murine viruses reveals many similarities. Particularly interesting is the comparison of the N-tropic Akv virus and the B-tropic BL/Ka(B) virus. The long terminal repeats of the two viruses are virtually identical, as are 22 of 23 restriction sites located outside of the region which spans from 1.8 to 3.8 kilobases from the left end of the genome. Within this region, however, only three of nine sites examined are shared. This suggests that the BL/Ka(B) virus was derived from an endogenous N-tropic virus closely related to Akv by recombinational events which altered the sequence in the last half of the gag gene and the first third of the pol gene. This change is probably responsible for the observed difference in the Fv-1 tropism of the two viruses.

Identification of potential diagnostic and vaccine candidates of Helicobacter pylori by "proteome" technologies.

BACKGROUND: There is great interest in characterizing the proteins of the gastric pathogen, Helicobacter pylori, especially those proteins to which humans respond immunologically. Such proteins have potential importance in diagnosis and vaccine development. METHODS: Two-dimensional gel electrophoresis in combination with Western blotting was used to separate and identify potential antigens of Helicobacter pylori strain Z-170. Proteins found to be reactive with pooled sera from 14 infected patients were individually digested in situ with endoproteinase Lys-C, and the resulting fragments were analyzed by matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Over 20 proteins were reactive in Western blots with pooled sera from 14 infected patients. The mass spectral data was compared with predictions from the H. pylori genome DNA sequence. Each of the 20 proteins was readily identified. CONCLUSIONS: We propose that this "proteome" approach for identification of previously unknown proteins will be useful in examining regulation of H. pylori gene expression and protein localization in the development of improved serologic tests to detect and monitor H. pylori infection. This approach will also be useful for identifying potential targets for antimicrobial or vaccine development for H. pylori and other pathogens whose genomes have been sequenced.

Molecular cloning and partial characterization of unintegrated linear DNA from gibbon ape leukemia virus.

We have cloned the complete genome of an oncogenic primate retrovirus, the San Francisco isolate of gibbon ape leukemia virus, in a lambda phage vector. DNA sequence analysis and restriction endonuclease mapping of the inserted linear provirus demonstrated 9-base pair inverted repeats at its ends, flanking direct terminal repeats 470 base pairs in length. The (-) strong stop region of this DNA showed surprisingly low sequence homology to that of another gibbon ape leukemia virus isolate from an animal with similar disease. Analysis of the clone also revealed the terminal phosphate configuration of the linear provirus. The recombinant phage is suitable for direct use as a hybridization probe to detect homologous retroviral sequences in human cell lines.

Altered regulation of c-myc expression in adenovirus-transformed cells.

Expression of the oncogenes c-myc, c-raski, and p53 is studied in normal primary mouse cultures and in two adenovirus-transformed mouse cell lines. In all cases oncogene expression is measured in cells arrested in G1 (or G0 for primary cells) by serum starvation and at different times after cell cycle traverse is stimulated by addition of high serum. For primary mouse cells, c-myc mRNA levels are found to increase four- to six-fold within 1 h of serum addition and then decline by 4 h to nearly the level observed in serum-starved cells. This level is maintained throughout the remainder of the cell cycle. The early induction of c-myc is dependent on serum concentration and is independent of cell density. These results confirm and extend previous observations for primary cells. By contrast, expression of c-raski does not vary at all through the cell cycle and p53 increases with time after mitogenic stimulation. In the adenovirus-transformed cell lines, the regulation of expression of c-myc with respect to the cell cycle is altered. There is an increase in c-myc in S phase cells which is dependent on cell density and the early induction in response to serum addition as seen in primary cells is absent. Expressions of c-raski and p53 are found to show similar profiles to those observed for primary cells.

Therapeutic human antibodies derived from PCR amplification of B-cell variable regions.

Despite advances in the in vitro immunization of human B cells (Borrebaeck et al. 1988) and the development of immunodeficient mice (McCune et al. 1988) for the reconstitution of the human immune system ex vivo, immortalization of antigen-specific human B cells remains the limiting step in the generation of human monoclonal antibodies. Typically this is performed with the aid of Epstein-Barr virus transformation followed by subcloning, confirmation of antigen binding and hybridization of the B lymphoblasts to a suitable fusion partner such as GLI-H7. This general approach is effective and widely used; however, it is time-consuming with erratic results. These were the immediate reasons we and others devised methods to directly obtain the variable regions from small numbers of human B cells (Larrick et al. 1987). The success of the PCR-based approach is illustrated above. In the present studies we successfully captured and stably produced antibodies from the V regions of two potent human anti-tetanus antibodies secreted by heteromyelomas that were too unstable for scale-up production. Although further preclinical evaluation of these antibodies is in progress, results to date indicate that the recombinant antibodies produced in myeloma-based cell lines or CHO cells are equivalent in binding specificity and activity to the native heteromyeloma-derived antibodies. Recent studies from this laboratory indicate that effective anti-tetanus protection will require a cocktail of anti-tetanus antibodies. Details of this work will be the subject of a future communication (Lang et al., in preparation).

Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction.

A general method to directly obtain the DNA sequence of the variable regions of any immunoglobulin chain using a mixture of oligomer primers and the polymerase chain reaction (PCR) is described. Mixed oligonucleotide primers corresponding to the 5' signal peptide and a conserved 3' constant region primer were used for enzymatic amplification of each of the heavy and light chain variable regions of a human hybridoma producing a monoclonal antibody recognizing an epitope of gp120 of the human immunodeficiency virus 1. The amplified DNA segments were cloned and the sequence was determined for the heavy chain variable region. This method will greatly facilitate structural and functional studies of immunoglobulins by reducing the effort to clone and sequence the members of the immunoglobulin as well as other multigene families.