Two distinct mechanisms of interleukin-2 gene expression in human T lymphocytes

Interleukin-2 (IL-2) gene regulation was investigated in primary cultures of highly purified human peripheral blood CD28+T cells. Two discrete mechanisms for induction of T-cell proliferation could be distinguished by examining cell cycle progression and the expression of the IL-2 gene. Stimulation of cells by CD3 MoAb induced only transiently expressed, small amounts of IL-2 mRNA that was completely suppressed by cyclosporine. Costimulation of T cells with CD3 MoAb and either CD28 MoAb or PMA, but not calcium ionophore, induced a 50-100-fold increased in IL-2 gene expression and secretion. High levels of IL-2 gene expression could also be achieved by stimulation with calcium ionophore and PMA or CD28 MoAb and PMA, but not by CD28 MoAb plus calcium ionophore. IL-2 gene expression and T-cell proliferation induced by CD3 MoAb plus PMA or calcium ionophore plus PMA were completely suppressible by cyclosporine. In contrast, IL-2 gene expression and T-cell proliferation induced by CD28 MoAb plus PMA were unaffected by cyclosporine. The CD28 signal was dependent on new protein synthesis. Nuclear run-on transcription assays showed that anti-CD28 did not affect lymphokine transcription. A major effect of CD28 stimulation on mRNA stability was shown by studies using actinomycin D; CD28 stimulation substantially increased the half-life of IL-2 and TNF-alpha mRNA. The effects of anti-CD28 stimulation were specific for growth factors, and thus differ from previously described effects of cycloheximide on mRNA stability. These studies suggest the existence of two biochemical pathways for the induction of IL-2 production, one that occurs at the transcriptional level and is mediated by intracellular calcium release and protein kinase C and is cyclosporine-sensitive, and one that acts post-transcriptionally, is mediated by CD28 stimulation, and is cyclosporine-resistant.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27894/1/0000314.pd

Toward the Elucidation of Cytoplasmic Diversity in North American Grape Breeding Programs

Plants have an intriguing tripartite genetic system: Nuclear genome 9 Mitochondria 9 Plastids and their interactions may impact germplasm breeding. In grapevine, the study of cytoplasmic genomes has been limited, and their role with respect to grapevine germplasm diversity has yet to be elucidated. In the present study, the results of an analysis of the cytoplasmic diversity among 6073 individuals (comprising cultivars, interspecific hybrids and segregating progenies) are presented. Genotyping by sequencing (GBS) was used to elucidate plastid and mitochondrial DNA sequences, and results were analyzed using multivariate techniques. Single nucleotide polymorphism (SNP) effects were annotated in reference to plastid and mitochondrial genome sequences. The cytoplasmic diversity identified was structured according to synthetic domestication groups (wine and raisin/table gr.ape types) and interspecific-hybridization-driven groups with introgression from North American Vitis species, identifying five cytoplasmic groups and four major clusters. Fifty-two SNP markers were used to describe the diversity of the germplasm. Ten organelle genes showed distinct SNP annotations and effect predictions, of which six were chloroplast-derived and three were mitochondrial genes, in addition to one mitochondrial SNP affecting a nonannotated open reading frame. The results suggest that the application of GBS will aid in the study of cytoplasmic genomes in grapevine, which will enable further studies on the role of cytoplasmic genomes in grapevine germplasm, and then allow the exploitation of these sources of diversity in breeding

Benefit of early commencement of growth hormone therapy in children with Prader-Willi syndrome

Prader-Willi syndrome (PWS) is a chromosomal disorder and growth failure is a common presentation. Growth hormone (GH) treatment is beneficial in PWS although the optimal age for starting GH is unknown. We investigated whether GH response in PWS was associated with the age of GH commencement by comparing 16 children who commenced GH before 3 years of age (early group) with 40 children who commenced GH after 3 years of age (late group) from the Ozgrow database. Height SDS, body mass index (BMI) SDS, bone age (BA)-chronological age (CA) ratio, change in height (Delta Ht) SDS and change in BMI during 4 years of GH treatment were compared between the groups. The early group had better height SDS and Delta Ht SIDS. BA delay was more pronounced in the early group but BA did not mature beyond CA with GH therapy in either group. Although the initial GH dose for the early group was lower than that of the late group, the former had better height outcome. The starting GH dose seen in the database is lower than the dose used by international centres

Winter curing of Prunus dulcis cv ‘Butte,’ P. webbii and their interspecific hybrid in response to Xylella fastidiosa infections

Clonal replicates of Prunus dulcis cv ‘Butte,’ P. webbii and their interspecific hybrid P 63-61 were inoculated with Xylella fastidiosa strain M23 and evaluated for almond leaf scorch disease and subsequent winter curing of infections during three growing seasons. Initial inoculations established greater than 90% infection in each of the accessions, based on PCR diagnoses from petiole tissues sampled near the inoculation site. Classic leaf scorch symptoms were evident in each population during the first growing season in a controlled greenhouse environment. Trees were removed from the greenhouse during the winters to accumulate chill hours and to provide the possibility of winter curing X. fastidiosa infections. Both PCR diagnostics and in vitro cultivation were used during the second and third growing seasons to determine the persistence of X. fastidiosa in clones among the three populations. Tree survival and the degree of winter cured infections differed among the three populations, with P. webbii and P 63-61 demonstrating enhanced levels of survivorship over ‘Butte.’ After two cycles of ambient winter temperatures and subsequent growth, ‘Butte’ averaged 21.2% winter cured trees with 73.1% mean survival. Tree survival and winter cured infections were nearly 100% for both P. webbii and P 63-61, demonstrating the utility of P. webbii in almond breeding efforts aimed at reducing tree vulnerability to X. fastidiosa infections

Winter curing of Prunus dulcis cv ‘Butte,’ P. webbii and their interspecific hybrid in response to Xylella fastidiosa infections

Clonal replicates of Prunus dulcis cv ‘Butte,’ P. webbii and their interspecific hybrid P 63-61 were inoculated with Xylella fastidiosa strain M23 and evaluated for almond leaf scorch disease and subsequent winter curing of infections during three growing seasons. Initial inoculations established greater than 90% infection in each of the accessions, based on PCR diagnoses from petiole tissues sampled near the inoculation site. Classic leaf scorch symptoms were evident in each population during the first growing season in a controlled greenhouse environment. Trees were removed from the greenhouse during the winters to accumulate chill hours and to provide the possibility of winter curing X. fastidiosa infections. Both PCR diagnostics and in vitro cultivation were used during the second and third growing seasons to determine the persistence of X. fastidiosa in clones among the three populations. Tree survival and the degree of winter cured infections differed among the three populations, with P. webbii and P 63-61 demonstrating enhanced levels of survivorship over ‘Butte.’ After two cycles of ambient winter temperatures and subsequent growth, ‘Butte’ averaged 21.2% winter cured trees with 73.1% mean survival. Tree survival and winter cured infections were nearly 100% for both P. webbii and P 63-61, demonstrating the utility of P. webbii in almond breeding efforts aimed at reducing tree vulnerability to X. fastidiosa infections

Role of the CD28 receptor in T-cell activation

Antigen-specific T-cell activation is initiated through the T-cell receptor. Recent evidence has shown that a number of additional T-cell surface receptors serve to regulate the responses of antigen-activated T cells. One such molecule, CD28, is a member of a heterophilic cell adhesion complex, and is the receptor for the B-cell-restricted B7/BB-1 antigen. As Carl June, Jeffrey Ledbetter, Peter Linsley and Craig Thompson review here, CD28 serves as the surface component of a novel signal transduction pathway that modulates T-cell lymphokine production and increases the resistance of cell responses to various immunosuppressive agents.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28838/1/0000673.pd

CD45 modulates T cell receptor/CD3-induced activation of human thymocytes via regulation of tyrosine phosphorylation

Stimulation of thymocytes or mature T cells via the T cell receptor (TcR)/CD3 complex activates a cascade of processes inducing cells to enter the cell cycle. A key step is the activation of phosphatidylinositol-specific phospholipase C (PI-PLC) within seconds following TcR/CD3 stimulation, an event which is strongly enhanced by co-ligation of the CD4 (or CD8) accessory molecule with TcR/CD3. In contrast, co-ligation of CD45 inhibits the same TcR/CD3 responses. The machinery which couples the TcR/CD3 complex, CD4, and CD45 to PI-PLC appears to involve regulation of tyrosine phosphorylation, as the TcR/CD3 and CD4 receptors are associated with the tyrosine kinases p59 fyn and p56 lck , respectively, and CD45 has intrinsic tyrosine phosphatase activity. Here, we have examined the ability of CD45 to regulate signal transduction via TcR/CD3 in human thymocytes. Co-cross-linking CD45 to the TcR/CD3 complex strongly suppressed the tyrosine phosphorylation of several intracellular substrates normally seen following TcR/CD3 stimulation. This effect of CD45 was associated with inhibition of a rise in intracellular calcium following TcR/CD3 ligation. Since TcR/CD3 stimulation of mature T cells induces tyrosine phosphorylation of PLC Γ 1, we investigated this phenomenon in thymocytes, and asked whether ligation of CD45 might regulate this process. By immunoprecipitation we found that TcR/CD3 stimulation induced tyrosine phosphorylation of PLC Γ 1, an effect which was enhanced by co-cross-linking CD4 to TcR/CD3. In contrast, co-ligation of CD45 strongly blocked PLC Γ 1 phosphorylation induced by either stimulus. Consistent with previous findings in mature T cells, CD45 cross-linking was able to partially inhibit TcR/CD3-induced thymocyte proliferation when interleukin 2 was used as a second signal, but almost completely (80%–90%) blocked proliferation when anti-CD28 mAb was used as the second signal, suggesting that CD45 cross-linking may be able to block interleukin 2 production via the CD28 pathway. These effects of CD45 on TcR/CD3 signaling and proliferation in thymocytes point towards a potential role for this pathway in thymic selection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37960/1/1830220238_ftp.pd