Autoimmune ovarian disease in day 3-thymectomized mice: the neonatal time window, antigen specificity of disease suppression, and genetic control.

Discovery of the CD4+CD25+ T cells has stemmed from investigation of the AOD in the d3tx mice. Besides CD4+CD25+ T cell depletion, d3tx disease induction requires effector T cell activation prompted by lymphopenia. This is supported by other neonatal AOD models in which T cell-mediated injury has been found to be triggered by immune complex or Ag immunization. In addition, there is growing evidence that support a state of neonatal propensity to autoimmunity, which depends on concomitant endogenous antigenic stimulation, concomitant nematode infection, resistance to CD4+CD25+ T cell regulation, and participation of the neonatal innate system. The suppression of d3tx disease by polyclonal CD4+CD25+ T cells appears to be dependent on endogenous Ag and the persistence of regulatory T cells. Thus, suppression of AOD occurs in the ovarian LN, and AOD emerges upon ablation of the input regulatory T cells; and in AIP, the hormone-induced expression of prostate Ag in the CD4+CD25+ T cell donors rapidly enhances the capacity to suppress disease over Ag negative donors. Finally, genetic analysis of AOD and its component phenotypes has uncovered seven Aod loci. As the general themes that emerged, significant epistatic interactions among the loci play a role in controlling disease susceptibility, the majority of the Aod loci are linked to susceptibility loci of other autoimmune diseases, and the genetic intervals encompass candidate genes that are differentially expressed between CD4+CD25+ T cells and other T cells. The candidate genes include Pdcd1, TNFR superfamily genes, H2, Il2, Tgfb, Nalp5 or Mater, an oocyte autoAg that reacts with autoantibody in sera of d3tx mice.

Expression library immunization protects mice against a challenge with virulent rodent malaria.

Although several candidate vaccine antigens have been developed for malaria, there is as yet no effective single vaccine available. There is a growing consensus that the ultimate malaria vaccine will be multivalent, requiring the identification of a suitable cocktail of antigens. However, evaluation of the multitude of potential malaria vaccine antigens in suitable combinations is a daunting task. Here we describe the validation of expression library immunization (ELI) as a tool for the discovery of sequences protective against malaria infection. A genomic Plasmodium chabaudi expression library was constructed comprising ten separate pools of 3000 plasmids. Over three vaccine trials using biolistic delivery of pools composed of 616 to 30,000 plasmids we report up to 63% protection of mice from a challenge with P. chabaudi adami DS, a highly virulent strain. Overall, ELI protected 36% of vaccinated mice against virulent challenge compared with only 3.2% survival of control mice. These results demonstrate that ELI is a suitable approach for screening the malaria genome to identify the components of multivalent vaccines.

Expression pattern of tobacco cyclin genes.

We have previously isolated three cDNA clones (Ntcyc25, Ntcyc27, Ntcyc29) encoding mitotic cyclins from tobacco (Nicotiana tabacum), and in this report we describe the expression patterns of these genes. RNA gelblot analysis showed that the mitotic cyclin genes were expressed predominantly in actively dividing meristematic tissues such as the shoot apex and young developing leaves. In situ hybridization analysis indicated that while theNtcyc29 gene was expressed consistently at low levels in shoot apex, but its transcripts were found at relatively high levels in the flower bud, indicating the possibility that theNtcyc29 product plays a role in flower development. TheNtcyc25 andNtcyc29 genes were expressed differentially in the root apex during the cell cycle, confirming the result obtained using synchronized-cultured tobacco cells. These results suggest that the transcriptionally regulated cyclin genes may be important in controlling cell division in tobacco.

Molecular cloning and characterization of a cDNA clone that encodes a Cdc2 homolog from Nicotiana tabacum.

We have isolated a cDNA clone (cdc2Nt1) that encodes a homolog of p34(cdc2/CDC28) kinase from tobacco (Nicotiana tabacum). The cdc2Nt1 protein showed extensive similarity to other homologs of Cdc2 from plants. Complementation studies showed that the cdc2Nt1 gene was able to overcome cell cycle arrest at both the G1/S and the G2/M transitions of cdc28ts mutants of budding yeast, demonstrating that the cdc2Nt1 protein was able to replace the Cdc28 kinase at both the G1/S and the G2/M transitions. Analysis of gene expression demonstrated that the cdc2Nt1 gene was transcribed constitutively throughout the cell cycle but that it was preferentially expressed in activity dividing tobacco BY-2 cells.

Tobacco mitotic cyclins: cloning, characterization, gene expression and functional assay.

Three cyclin cDNA clones (Ntcyc25, Ntcyc27, Ntcyc29) have been isolated from tobacco (Nicotiana tabacum) using the PCR cloning method. The encoded Ntcyc cyclins were highly homologous to mitotic cyclins. In synchronized tobacco suspension cultured cells, the mRNA levels of Ntcyc25 and Ntcyc27 were detectable through S, G2 and M phases, while the Ntcyc29 mRNA was detected from G2 to M phase. These expression patterns classified the Ntcyc25 and Ntcyc27 into A-type cyclin and Ntcyc29 into B-type cyclin. The three genes were expressed in growing tobacco cultured cells but ceased to be expressed when cells entered the stationary phase, indicating that the expression of these cyclin genes was well correlated with cell growth. The N-terminal truncated Ntcyc25 and Ntcyc29 cDNAs were able to rescue G1 cyclin mutant of Saccharomyces cerevisiae, while the full-length of Ntcyc27 protein could also partially perform G1 cyclin functions.