Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo.

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN synthesized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been relatively inactive on primate leukocytes in vitro. Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported. In the present study we tested a panel of CpG ODN for their in vitro and in vivo immune effects in mice and identified in vitro activation of B and NK cells as excellent predictors of in vivo adjuvant activity. Therefore, we tested >250 phosphorothioate ODN for their capacity to stimulate proliferation and CD86 expression of human B cells and to induce lytic activity and CD69 expression of human NK cells. These studies revealed that the sequence, number, and spacing of individual CpG motifs contribute to the immunostimulatory activity of a CpG phosphorothioate ODN. An ODN with a TpC dinucleotide at the 5' end followed by three 6 mer CpG motifs (5'-GTCGTT-3') separated by TpT dinucleotides consistently showed the highest activity for human, chimpanzee, and rhesus monkey leukocytes. Chimpanzees or monkeys vaccinated once against hepatitis B with this CpG ODN adjuvant developed 15 times higher anti-hepatitis B Ab titers than those receiving vaccine alone. In conclusion, we report an optimal human CpG motif for phosphorothioate ODN that is a candidate human vaccine adjuvant.

Ontogeny of thymic NK1.1+ cells.

Thymic NK1.1+ cells are a recently described lymphocyte subset whose biologic function is not well defined. There is some controversy as to whether thymic NK1.1+ cells mature in a thymic or an extrathymic pathway. In this study, we examined the ontogeny of murine thymic NK1.1+ cells utilizing direct examination of freshly obtained fetal thymi as well as fetal thymi established in organ cultures (FTOC). We found a reproducible peak (5-40%) of NK1.1+ cells, demonstrable in day 15 to 16 freshly obtained fetal thymi, which was markedly decreased by day 17 of gestation; this peak preceded the appearance of the CD4+ CD8+ thymocytes by 12 to 24 h. Reverse-transcriptase PCR analysis of NK1.1 demonstrated its presence as early as day 9 of gestation, thus placing it as one of the earliest lymphocytic genes to be transcribed. Utilizing FTOC, we found that: 1) day 12 fetal thymi contained a progenitor that can differentiate into an NK1.1+ CD4+ CD8+ lymphocyte; 2) NK1.1+ cells dwindle to <5% in FTOC established from day 14 thymi; 3) NK1.1+ cells dominate in FTOC supplemented with IL-2; and 4) most of the NK1.1+ cells seen in FTOC did not express CD3epsilon on their surface, except for the FTOC supplemented with IL-12. These findings suggest that NK1.1+ cells may play an important role in thymic maturation. Moreover, these findings suggest that fetal thymi contain several novel lymphocyte subsets that can be induced to overgrow the normal thymocytes upon exposure to certain cytokines.

Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA.

We have recently shown that oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides (CpG motif) can induce B cells to proliferate, differentiate, and secrete cytokines. In this study we demonstrate that CpG motifs contained in ODN as short as 15 bases in length were quite effective at inducing NK cell lytic activity in vitro in both human and murine lymphocytes. Such ODN were also effective at inducing NK lytic activity, in vivo, in mice. Experiments designed to determine the cellular and cytokine requirements for NK cell induction revealed that B and T cells are not necessary, that the ODN do not augment the activity of highly purified NK cells, and that the ODN augment NK cell activity indirectly by inducing the secretion of IL-12, IFN-alpha beta, and TNF-alpha. Various ODN sequences were prepared to determine the optimal ODN length, motif, palindrome, backbone modification, and dose requirements. We found no requirement for a palindromic sequence but a definite requirement for an unmethylated CpG motif. While necessary, however, a CpG motif was not sufficient for NK cell induction. Instead, there appeared to be stringent requirements for the immediate flanking bases at the 5' and 3' ends as well as for flanking sequences outside the immediate 5' and 3' bases. In particular poly(G) ends seemed to exert a complex qualitative and quantitative effect which could be up- or down-modulating depending on whether the ODN backbone was phosphorothioate modified or not.

Murine natural killer cells express the Ly24 (Pgp-1) marker on their surface.

The Ly24 (Pgp-1) marker is expressed on some, but not all, mature T lymphocytes. It has recently become apparent that the development of Ly24- T lymphocytes is dependent on the presence of an intact thymus and that virgin Ly24- T cells rapidly acquire this marker upon antigenic or mitogenic stimulation. Although natural killer (NK) cells can develop and function in the absence of an intact thymus, some NK cell subsets express certain markers normally associated with T lymphocytes. The experiments in this report were undertaken to determine if NK cells express Ly24 and whether such an expression could be used to delineate distinct NK cell subsets. We found that mature functional NK cells expressed the Ly24 marker as defined by the monoclonal antibody 9F3. Double-color fluorescence analysis using C57BL/6 splenocytes (whose NK cells express the NK1.1 marker) showed all the NK1.1+ cells to be Ly24+ as well. For C3H/HeN (an NK1.1- strain), double-color fluorescence analysis utilizing asialo GM1 and Ly24 revealed a distinct subset positive for both markers and containing most of the functional NK cell activity. Whereas the Ly24 marker did not illuminate an NK cell subset, these findings demonstrate that this determinant can be useful for the further characterization and isolation of NK cells.

Forms control and error detection procedures used at the Coordinating Center of the Multiple Risk Factor Intervention Trial (MRFIT).

Although methods used for data collection and quality assurance for large-scale clinical trials are important to critical reading of trial results and have been published, such reporting is the exception rather than the rule. In the MRFIT, systematic methods for processing large volumes of data over a long period of time were developed. The methods were designed to detect and control a variety of errors and to leave a complete audit trial of the processing of forms and corrections to forms. Many of these methods evolved and were refined during the course of the study as a result of trial and error. If one were to start over, the methods described herein would be modified. The field of data processing is evolving, and it is important for statistical and data processing staff of coordinating centers to recognize this and continually evaluate and update their methods. For example, the simultaneous entry and computer editing of forms is becoming more feasible with time. Also, more sophisticated intelligent data entry equipment is available for central use. Near the end of MRFIT, some data received at the Coordinating Center were entered and edited on a minicomputer. The parameter-driven edits described previously were performed at the time of data entry. Additional modifications to the content of the data dictionary for future studies are also being considered. The incorporation into the data dictionary of consistency checks (both deterministic and probabilistic) between fields on different forms would facilitate the specification of complex edit checks and would provide better documentation of the edit checks actually performed. Incorporating definitions of the numeric codes for each field would improve the documentation and facilitate reporting using statistical packages. Dedicated computer hardware should also be a major consideration of coordinating centers in future clinical trials. For MRFIT, a dedicated system was used from 1978 to the end of the trial. With the continued decline in hardware costs, dedicated systems can and should be considered, even for trials much smaller than MRFIT. We believe the system developed for processing data in the MRFIT has several advantages. It satisfies the requirements identified by Karrison or a system of data editing and control, it is largely self-documenting as a result of the data dictionary approach taken, and it is easily adaptable to other clinical studies.

An evaluation of the Social Security Administration master beneficiary record file and the National Death Index in the ascertainment of vital status.

To evaluate the relative utility of the Social Security Administration and National Death Index as sources of mortality data, the vital status of 12,866 participants in the Multiple Risk Factor Intervention Trial was identified from these sources and compared to the known mortality experience. The SSA correctly identified 87.8 per cent of the 409 deaths that occurred between 1974 and 1980. Underreporting of deaths by the SSA occurred for participants with certain demographic characteristics, especially marital status. For the years 1979 and 1980, the period for which the SSA and NDI have comparable data, the SSA correctly identified 93.2 per cent and the NDI correctly identified 98.4 per cent of the 191 known deaths. The NDI matching process resulted in a large number of false positive matches.