Combination of immunoglobulins and natural killer cells in the context of CMV and EBV infection.

Cytomegalovirus (CMV)-specific hyperimmunoglobulin (CMV-HIG) is used to treat and prevent CMV infection in immunocompromised patients, and anti-CD20 monoclonal antibody is successfully used in the treatment for post-transplant lymphoproliferative disease caused by Epstein-Barr virus (EBV). Two immunological approaches have been suggested to further improve the control of viral reproduction in patients with active disease: first, the use of monoclonal antibodies with specificity against viral epitopes and second, coadministration of cells with the capacity to promote antibody-dependent cell-mediated cytotoxicity. Here, we have evaluated the effectiveness of these strategies in vitro (alone and in combination) with neutralization and cytotoxicity assays. Our results indicate that monoclonal antibodies (in particular SM5-1) can be as effective as CMV-HIG in neutralizing-cell-free CMV. Moreover, our data indicate that antibody-mediated elimination (either by moAb or by HIG) of EBV-infected cells can be significantly enhanced by NK cells. Using human NK cells that have been purified, cultured and expanded under GMP conditions, we were able to demonstrate that the combination of NK cells and antibodies could represent a feasible and highly effective clinical approach to achieve control of EBV infections. Especially in leukopenic patients with low numbers of ADCC-promoting cells, the combination of adoptively transferred NK cells and antiviral antibodies offers a promising strategy that should be tested in clinical trials.

Topological complexity of different populations of pBR322 as visualized by two-dimensional agarose gel electrophoresis.

Neutral/neutral two-dimensional (2D) agarose gelelectrophoresis was used to investigate populations of the different topological conformations that pBR322 can adopt in vivo in bacterial cells as well as in Xenopus egg extracts. To help in interpretation and identification of all the different signals, undigested as well as DNA samples pretreated with DNase I, topoisomerase I and topoisomerase II were analyzed. The second dimension of the 2D gel system was run with or without ethidium bromide to account for any possible changes in the migration behavior of DNA molecules caused by intercalation of this planar agent. Finally, DNA samples were isolated from a recA-strain of Escherichia coli , as well as after direct labeling of the replication intermediates in extracts of Xenopus laevis eggs. Altogether, the results obtained demonstrated that 2D gels can be readily used to identify most of the complex topological populations that circular molecules can adopt in vivo in both bacteria and eukaryotic cells.

Glucocorticoid hormones upregulate interleukin 2 receptor alpha gene expression.

We present evidence that glucocorticoid hormones increase expression of IL-2Rec alpha chain on T cells by regulating IL-2Rec alpha gene transcription. We have previously reported that glucocorticoids can upregulate IL-2Rec alpha mRNA and protein expression in some T cell hybrids. In the present study we show that the glucocorticoid analogue dexamethasone increases mRNA levels of the endogenous IL-2Rec alpha gene and the expression of plasmids containing 5'-flanking sequences of the IL-2Rec alpha gene linked to CAT reporter genes transiently transfected into different cell lines. We show that the dexamethasone effect depends on cis-acting regulatory elements in a segment (-1835/-802) of the mouse gene that also contains cytokine response elements and a inducible DNase I-hypersensitive site. Dexamethasone responses of IL-2Rec alpha-CAT reporter gene constructs were observed in a CTL line, in an IL-3-dependent bone marrow-derived cell line, and in COS7 monkey kidney cells. In the latter the response depended on cotransfection of a glucocorticoid receptor expression vector. The biological relevance of the glucocorticoid-mediated upregulation of the IL-2Rec alpha gene is discussed.

Conserved features in the mode of replication of eukaryotic ribosomal RNA genes.

It was previously shown that a 1.5 kb fragment located in the non-transcribed spacer (NTS) is the earliest replicating region of pea (Pisum sativum) rDNA in synchronized root cells. In the present report the structure of this region was characterized. It contains a cluster of four 11 bp near matches to the Saccharomyces cerevisiae ARS consensus sequence (ACS). These near matches are embedded in an A+T rich domain located upstream from the transcription initiation site. We identified and mapped an intrinsic DNA bending locus 5' to the cluster of near matches. Several eukaryotic origins including the ARS from the budding yeast show very similar structural features. This observation strengthens the notion that pea rDNA replication initiates at or near this region. Replication of the entire pea rDNA repeat was analysed by two-dimensional (2D) agarose gel electrophoresis. The results obtained indicate that only a small fraction of the potential origins is used in each replication round. Forks moving in the direction opposite to rRNA transcription are stalled at a polar replication fork barrier (RFB), which mapped near the 3' end of the transcription unit. Consequently, most of pea rDNA appears to replicate in a unidirectional manner. These results show that the strategy used to replicate pea and yeast rRNA genes is very similar, suggesting that it has been conserved and might be common to most eukaryotes.

Initiation of DNA replication in ColE1 plasmids containing multiple potential origins of replication.

We have investigated the frequency of replication origin usage in bacterial plasmids containing more than one potential origin. Escherichia coli recA- cells were selectively transformed with pBR322 monomers, dimers, or trimers. Plasmid DNA was isolated and digested with a restriction enzyme that cut the monomer only once, and the replicative intermediates (RIs) were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis. Evidence for initiation outside the linearized plasmid was found only for oligomers. Moreover, in dimers, the intensity of the signal indicative for external initiation was equivalent to that reflecting internal initiation, whereas it was approximately twice as strong in trimers. To determine whether initiation could occur simultaneously at two origins in a single plasmid, we studied the replication of a neodimer in which both units could be unambiguously distinguished. The results showed that although both origins were equally competent to initiate replication, only one was active per plasmid. These observations strongly suggest that in ColE1 plasmids, replication initiates at a single site even when there are several identical potential origins per plasmid. In addition to the conventional two-dimensional gel patterns, novel specific patterns were observed with intensities that varied from one DNA sample to another. These unique patterns were the result of breakage of the RIs at a replication fork. This type of breakage changes both the mass and shape of RIs. When the entire population of RIs is affected, a new population of molecules is formed that may generate a novel pattern in two-dimensional gels.

Unidirectional replication as visualized by two-dimensional agarose gel electrophoresis.

Two-dimensional (2D) agarose gel electrophoresis is progressively replacing electron microscopy as the technique of choice to map the initiation and termination sites for DNA replication. Two different versions were originally developed to analyze the replication of the yeast 2 microns plasmid. Neutral/Neutral (N/N) 2D agarose gel electrophoresis has subsequently been used to study the replication of other eukaryotic plasmids, viruses and chromosomal DNAs. In some cases, however, the results do not conform to the expected 2D gel patterns. In order to better understand this technique, we employed it to study the replication of the colE1-like plasmid, pBR322. This was the first time replicative intermediates from a unidirectionally replicated plasmid have been analyzed by means of N/N 2D agarose gel electrophoresis. The patterns obtained were significantly different from those obtained in the case of bidirectional replication. We showed that identification of a complete are corresponding to molecules containing an internal bubble is not sufficient to distinguish a symmetrically located bidirectional origin from an asymmetrically located unidirectional origin. We also showed that unidirectionally replicated fragments containing a stalled fork can produce a pattern with an inflection point. Finally, replication appeared to initiate at only some of the potential origins in each multimer of pBR322 DNA.

Evidence that replication initiates at only some of the potential origins in each oligomeric form of bovine papillomavirus type 1 DNA.

In a subclone of ID13 mouse fibroblasts latently infected with bovine papillomavirus type 1 (BPV-1) DNA, the viral genome occurred as a mixture of extrachromosomal circular monomers and oligomers. Multiple copies were also associated with the host cell genome, predominantly at a single site in a head-to-tail tandem array. We examined the replicative intermediates of extrachromosomal forms of BPV-1 DNA by using two-dimensional gel electrophoresis. The results obtained indicate that initiation of DNA replication occurred near the center of the EcoRI-BamHI 5.6-kilobase fragment. In some molecules, however, this fragment was replicated from one end to the other by means of a single fork initiated elsewhere. Termination also occurred within this fragment. The EcoRI-BamHI 2.3-kilobase fragment replicated as a DNA molecule containing a termination site for DNA replication and also by means of a single fork traversing the fragment from one end to the other. Thus, replication forks proceeded through these fragments in different manners, apparently depending on whether they were part of a monomer, a dimer, a trimer, or higher oligomers. These observations lead to the conclusion that initiation of DNA replication in BPV-1 DNA takes place at or close to plasmid maintenance sequence 1. From this point, replication proceeds bidirectionally and termination occurs approximately 180 degrees opposite the origin. The results obtained are consistent with one or more replication origins being quiescent in BPV-1 DNA oligomers.