A pilot trial targeting the ICOS-ICOS-L pathway in nonhuman primate kidney transplantation.

Costimulation blockade with the B7-CD28 pathway-specific agent belatacept is now used in clinical kidney transplantation, but its efficacy remains imperfect. Numerous alternate costimulatory pathways have been proposed as targets to synergize with belatacept, one of which being the inducible costimulator (ICOS)-ICOS ligand (ICOS-L) pathway. Combined ICOS-ICOS-L and CD28-B7 blockade has been shown to prevent rejection in mice, but has not been studied in primates. We therefore tested a novel ICOS-Ig human Fc-fusion protein in a nonhuman primate (NHP) kidney transplant model alone and in combination with belatacept. ICOS-Ig did not prolong rejection-free survival as a monotherapy or in combination with belatacept. In ICOS-Ig alone treated animals, most graft-infiltrating CD4(+) and CD8(+) T cells expressed ICOS, and ICOS(+) T cells were present in peripheral blood to a lesser degree. Adding belatacept reduced the proportion of graft-infiltrating ICOS(+) T cells and virtually eliminated their presence in peripheral blood. Graft-infiltrating T cells in belatacept-resistant rejection were primarily CD8(+) CD28(-) , but importantly, very few CD8(+) CD28(-) T cells expressed ICOS. We conclude that ICOS-Ig, alone or combined with belatacept, does not prolong renal allograft survival in NHPs. This may relate to selective loss of ICOS with CD28 loss.

Display of epitopes on the surface of tobacco mosaic virus: impact of charge and isoelectric point of the epitope on virus-host interactions.

The biophysical properties of the tobacco mosaic tobamovirus (TMV) coat protein (CP) make it possible to display foreign peptides on the surface of TMV. The immunogenic epitopes G5-24 from the rabies virus (RV) glycoprotein, and 5B19 from murine hepatitis virus (MHV) S-glycoprotein were successfully displayed on the surface of TMV, and viruses accumulated to high levels in infected leaves of Nicotiana tabacum Xanthi-nn. The peptide RB19, which contains an arginine residue plus the 5B19 epitope fused to the CP (TMV-RB19), resulted in the induction of necrotic local lesions on inoculated leaves of N. tabacum Xanthi-nn and cell death of infected BY2 protoplasts. RNA dot blot assays confirmed that expression of the acidic and basic pathogenesis-related PR2 genes were induced in infected Xanthi-nn leaf tissue. TMV that carried epitope 31D from the RV nucleoprotein did not accumulate in inoculated tobacco leaves. Analysis of hybrid CPs predicted that the isoelectric points (pI):charge value was 5.31:-2 for wild-type CP, 5.64:-1 for CP-RB19, and 9.14:+2 for CP-31D. When acidic amino acids were inserted in CP-RB19 and CP-31D to bring their pI:charge to near that of wild-type CP, the resulting viruses TMV-RB19E and TMV-4D:31D infected N. tabacum Xanthi-nn plants and BY2 protoplasts without causing cell death. These data show the importance of the pI of the epitope and its effects on the hybrid CP pI:charge value for successful epitope display as well as the lack of tolerance to positively charged epitopes on the surface of TMV.

Cloning of tobacco genes that elicit the hypersensitive response.

We used a functional screening method to isolate genes whose products elicit the hypersensitive response (HR) pathway of defense against plant pathogens. A cDNA library derived from tobacco leaves undergoing the HR was cloned into a tobacco mosaic virus (TMV)-based expression vector. Infectious transcripts were generated and used to inoculate tobacco plants lacking the N resistance gene (genotype Xanthi nn). Approximately 1/1000 of the infectious transcripts produced local lesions, and may thus elicit the HR. The cDNA inserts from 50 lesion-forming clones were recovered by RT-PCR, and 12 unique clones were sequenced. Comparisons with protein databases revealed homologies to (a) ubiquitin, (b) tobacco tumor-related protein, similar to Kunitz-type trypsin inhibitors and (c) ribosomal protein S14. The remaining nine clones revealed no homology to known proteins and are thus considered novel. Five clones were able to induce the expression of PR2, a gene which is specifically activated in the tobacco HR. Northern and western blot analyses of leaves infected by the clone encoding ubiquitin strongly suggest that the infection produced a co-suppression response; the endogenous level of ubiquitin mRNA and protein in infected leaves are ca. 50% less than those found in healthy leaves. This observation supports a previous report on the involvement of the ubiquitin system in the tobacco HR [2], and validates and utility of the functional cloning method.

Three cis-elements required for rice alpha-amylase Amy3D expression during sugar starvation.

Expression of alpha-amylase genes during seedling development plays a key role in production of sugar from the starch stored in the cereal seed. Rice alpha-amylase Amy3D promoter/GUS constructs in transgenic rice cell lines were studied to identify cis elements in the promoter of this metabolite-regulated gene. Three sequences having the greatest effects on Amy3D gene expression included the amylase element (TATCCAT), the CGACG element, and a G box-related element (CTACGTGGCCA). These promoter cis elements are needed for high-level expression of Amy3D under conditions of sugar starvation. The involvement of G box cis-elements in environmental stress responses suggest a link between the nutrient stress and the environmental stress responses of the plant.

In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries.

A method for isolating and cloning mRNA populations from individual cells in living, intact plant tissues is described. The contents of individual cells were aspirated into micropipette tips filled with RNA extraction buffer. The mRNA from these cells was purified by binding to oligo(dT)-linked magnetic beads and amplified on the beads using reverse transcription and PCR. The cell-specific nature of the isolated mRNA was verified by creating cDNA libraries from individual tomato leaf epidermal and guard cell mRNA preparations. In testing the reproducibility of the method, we discovered an inherent limitation of PCR amplification from small amounts of any complex template. This phenomenon, which we have termed the "Monte Carlo" effect, is created by small and random differences in amplification efficiency between individual templates in an amplifying cDNA population. The Monte Carlo effect is dependent upon template concentration: the lower the abundance of any template, the less likely its true abundance will be reflected in the amplified library. Quantitative assessment of the Monte Carlo effect revealed that only rare mRNAs (< or = 0.04% of polyadenylylated mRNA) exhibited significant variation in amplification at the single-cell level. The cDNA cloning approach we describe should be useful for a broad range of cell-specific biological applications.

Metabolic regulation of rice alpha-amylase and sucrose synthase genes in planta.

Isolated rice embryos were used to investigate the regulatory effects of endosperm extracts and pure sugars on the expression of alpha-amylase gene RAmy3D and a sucrose synthase gene homologous to the maize isozyme Ss2. The high-level expression of RAmy3D in the scutella of isolated embryos could be inhibited by a variety of sugars as well as endosperm extracts from germinated rice grains. Glucose, at a concentration of 250 mM, was most effective in repressing RAmy3D mRNA accumulation. Furthermore, this repression was reversible. Interestingly, RAmy3D repression was always accompanied by the induction of sucrose synthase gene expression. These results support a model in which the expression of alpha-amylase and sucrose synthase genes in the rice scutellum are counter-regulated by the influx of sugars from the endosperm.

Differential expression of alpha-amylase genes in germinating rice and barley seeds.

Steady-state levels of mRNA from individual alpha-amylase genes were measured in the embryo and aleurone tissues of rice (Oryza sativa) and two varieties of barley (Hordeum vulgare L. cv. Himalaya and cv. Klages) during germination. Each member of the alpha-amylase multigene families of rice and barley was differentially expressed in each tissue. In rice, alpha-amylase genes displayed tissue-specific expression in which genes RAmy3B, RAmy3C, and RAmy3E were preferentially expressed in the aleurone layer, genes RAmy1A, RAmy1B and RAmy3D were expressed in both the embryo and aleurone, and genes RAmy3A and RAmy2A were not expressed in either tissue. Whenever two or more genes were expressed in any tissue, the rate of mRNA accumulation from each gene was unique. In contrast to rice, barley alpha-amylase gene expression was not tissue-specific. Messenger RNAs encoding low- and high-pI alpha-amylase isozymes were detectable in both the embryo and aleurone and accumulated at different rates in each tissue. In particular, peak levels of mRNA encoding high-pI alpha-amylases always preceded those encoding low-pI alpha-amylases. Two distinct differences in alpha-amylase gene expression were observed between the two barley varieties. Levels of high-pI alpha-amylase mRNA peaked two days earlier in Klages embryos than in Himalaya embryos. Throughout six days of germination, Klages produced three times as much high-pI alpha-amylase mRNA and nearly four times as much low-pI alpha-amylase mRNA than the slower-germinating Himalaya variety.

The isolation and characterization of a barley 1,3-1,4-beta-glucanase gene.

The barley gene encoding isozyme I of 1,3-1,4-beta-glucanase was isolated and sequenced. The 6260-bp region sequenced included 1885 bp of the 5'-flanking region, the entire coding region, an intron of 2490 bp, and 792 bp of the 3'-flanking region. The 1,3-1,4-beta-glucanase mRNA was found to be regulated at the level of RNA accumulation by both gibberellins (positively) and abscisic acid (negatively) in barley aleurones. The mRNA for isozyme II preferentially accumulated (70%) relative to the mRNA for isozyme I (30%) in poly(A)-rich RNA isolated from material including both the aleurone and the scutellum tissues. The gene family encoding 1,3-1,4-beta-glucanase enzymes in barley was found to be comprised of two closely related genes, isozymes I and II, as well as several related sequences that could be identified by Southern blot analysis. The nucleotide sequence for the 5' untranslated leader and the coding region for the signal peptide of the isozyme II transcript were determined from a cDNA produced by the polymerase chain reaction. The structure of the protein encoded by the isozyme I gene is also discussed.

Polyamines in bacteriophage phi W-14 and in phi W-14-infected Pseudomonas acidovorans.

Bacteriophage phi W-14 is sensitive to osmotic shock. It contain sufficient free putrescine, 2-hydroxyputrescine and spermidine to neutralize about 15% of the DNA phosphates. The alpha-putrescinylthymine residues of the DNA could neutralize a further 25% of the phosphates. Label is transferred from ornithine to the alpha-putrescinyl residues of phi W-14 DNA. The rates of polyamine synthesis in Pseudomonas acidovorans are increased by phi W-14 infection.

Polyamines of Pseudomonas acidovorans.

Pseudomonas acidovorans contains putrescine, 2-hydroxyputrescine, and spermidine.