Nippostrongylus brasiliensis can induce B7-independent antigen-specific development of IL-4-producing T cells from naive CD4 T cells in vivo

Th2 immune responses to a number of infectious pathogens are dependent on B7-1/B7-2 costimulatory molecule interactions. We have now examined the Th2 immune response to Nippostrongylus brasiliensis (Nb) in B7-1/B7-2(-/-) mice and show that Th2 effector cells develop that can mediate worm expulsion and produce substantial Th2 cytokines comparable with wild-type infected mice; however, in marked contrast, B cell Ag-specific Ab production is abrogated after B7 blockade. To examine the mechanism of T cell activation, OVA-specific DO11.10 T cells were transferred to recipient mice, which were then immunized with a combination of Nb plus OVA or either alone. Only the combination of Nb plus OVA triggered T cell differentiation to OVA-specific Th2 cells, suggesting that Nb acts as an adjuvant to stimulate Ag-specific naive T cells to differentiate to effector Th2 cells. Furthermore, using the DO11.10 TCR-transgenic T cell adoptive transfer model, we show that blocking B7-1/B7-2 interactions does not impair nonparasite Ag-specific DO11.10 Th2 cell differentiation; however, DO11.10 T cell cycle progression and migration to the B cell zone are inhibited.     

Synthesis and biological activity of N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid

2-Deamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a potent inhibitor of thymidylate synthase. Its analogue, N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid, containing p-aminophenylacetic acid residue substituting p-aminobenzoic acid residue, was synthesized. The new analogue exhibited a moderately potent thymidylate synthase inhibition, of linear mixed type vs. the cofactor, N(5,10)-methylenetetrahydrofolate. The Ki value of 0.34 microM, determined with a purified recombinant rat hepatoma enzyme, was about 30-fold higher than that reported for inhibition of thymidylate synthase from mouse leukemia L1210 cells by ICI 198583 (Hughes et al., 1990, J. Med. Chem. 33, 3060). Growth of mouse leukemia L5178Y cells was inhibited by the analogue (IC50 = 1.26 mM) 180-fold weaker than by ICI 198583 (IC50 = 6.9 microM).     

Memory Th2 effector cells can develop in the absence of B7-1/B7-2, CD28 interactions,and effector Th cells after priming with an intestinal nematode parasite

B7-1/B7-2 interactions are required for many Th2-cell mediated primary immune responses including the response that follows infection with the intestinal nematode parasite, Heligmosomoides polygyrus. However, few studies have examined the role of B7-1/B7-2/CD28 interactions in the development of a Th2 memory immune response. We examined the development of the memory Th2 response to H. polygyrus in BALB/c mice deficient in both B7-1 and B7-2 (B7-1/B7-2(-/-)) and in BALB/c mice deficient in CD28 (CD28(-/-)). Following primary inoculation with H. polygyrus, adult worms in the gut were cleared with an anti-helminthic drug and mice were subsequently challenge-inoculated with H. polygyrus larvae. The memory Th2 response is readily distinguished by its inhibitory effect on adult worm maturation, resulting in marked reductions in adult worm egg production that are not observed during the primary immune response. Following H. polygyrus challenge inoculation, comparable decreases in egg production and similar increases in mesenteric lymph node cell IL-4 production were observed in B7-1/B7-2(-/-) and B7-1/B7-2(+/+) mice. However, elevations in total serum IgG1 and IgE were reduced, while increases in serum Ag-specific IgG1 and IgE and germinal center formation were blocked in H. polygyrus-challenged B7-1/B7-2(-/-) mice. In contrast, in H. polygyrus-challenged CD28(-/-) mice, marked elevations in Ag-specific IgG1 and IgE and increased germinal center formation were observed. The results of these studies demonstrate that effector Th2 memory cells that produce IL-4 and mediate host defense can develop when B7-1/B7-2 interactions, and associated effector Th2 cell development, are blocked during priming. However, humoral immunity is impaired and differentially affected in B7-1/B7-2(-/-) mice and CD28(-/-) mice following H. polygyrus challenge.     

Characterization of the expression of a wheat cystatin gene during caryopsis development

A cDNA coding for phytocystatin, a protease inhibitor, was isolated from wheat embryos by differential display RT-PCR and the corresponding full-length cDNA (named WC5 for wheat cystatin gene 5) subsequently obtained by RACE. The deduced primary sequence of the protein suggests the presence of a 28 amino acid N-terminal signal sequence and a 100 amino acid mature protein containing the three consensus motifs known to interact with the active site of cysteine peptidases. Northern and western analysis revealed a spatio-temporal pattern of the cystatin gene expression during caryopse development. In the embryo, WC5 was only expressed during early embryogenesis whereas, in seed covering layers, WC5 expression was restricted to the maturation stage of grain development. In addition, immunolocalization experiments showed that cystatin accumulated in the aleurone layer of the maturating seed and in the parenchymal tissues of the embryo scutellum. A recombinant form of the wheat cystatin was shown to be able to inhibit peptidase activities present in whole seed protein extracts. In addition, immunological techniques allowed us to identify two putative target peptidases. The possible roles of the cystatin protein are discussed in relation with tissular localization and putative peptidase targets during seed maturation.     

Nuclear export of mRNA by TAP/NXF1 requires two nucleoporin-binding sites but not p15

Metazoan NXF1/p15 heterodimers promote export of bulk mRNA through nuclear pore complexes (NPC). NXF1 interacts with the NPC via two distinct structural domains, the UBA-like domain and the NTF2-like scaffold, which results from the heterodimerization of the NTF2-like domain of NXF1 with p15. Both domains feature a single nucleoporin-binding site, and they act synergistically to promote NPC translocation. Whether the NTF2-like scaffold (and thereby p15) contributes only to NXF1/NPC association or is also required for other functions, e.g., to impart directionality to the export process by regulating NXF1/NPC or NXF1/cargo interactions, remains unresolved. Here we show that a minimum of two nucleoporin-binding sites is required for NXF1-mediated export of cellular mRNA. These binding sites can be provided by an NTF2-like scaffold followed by a UBA-like domain (as in the wild-type protein) or by two NTF2-like scaffolds or two UBA-like domains in tandem. In the latter case, the export activity of NXF1 is independent of p15. Thus, as for the UBA-like domain, the function of the NTF2-like scaffold is confined to nucleoporin binding. More importantly, two copies of either of these domains are sufficient to promote directional transport of mRNA cargoes across the NPC.     

Nucleotide variation,haplotype structure,and association with end-stage renal disease of the human interleukin-1 gene cluster

A dense gene-based SNP map was constructed across a 360-kb region containing the interleukin-1 gene cluster (IL1A, IL1B, and IL1RN), focusing on IL1RN. In total, 95 polymorphisms were confirmed or identified primarily by direct sequencing. Polymorphisms were precisely mapped to completed BAC and genomic sequences spanning this region. The polymorphisms were typed in 443 case-control subjects from Caucasian and African American groups. Consecutive pair-wise marker linkage disequilibrium was not strictly correlated with distance and ranged from D'=0.0079 to 1.000 and D'=0.0521 to 1.0000 in Caucasians and African Americans, respectively. Single markers and haplotypes in IL1 cluster genes were evaluated for association with end-stage renal disease (ESRD). Eleven SNPs show some evidence of association with ESRD, with the strongest associations in two IL1A variants, one SNP, rs1516792-3, in intron 5 (p=0.0015) and a 4-bp insertion/deletion within the 3'UTR, rs16347-2 (p=0.0024), among African Americans with non-T2DM-associated ESRD.     

An X-encoded alloantigenicity between BALB/c and C57BL/6 strains of mice

To detect minor barriers to histocompatibility that might be encoded on the X chromosome in mice, we grafted reciprocal sets of (C57BL/6xBALB/c)F1, (C57BL/6xDBA/2)F1, and (BALB/cxDBA/2)F1 mice with tail skin from the respective paternal inbred strain. Our histogenic analysis suggests that, compared with the C57BL/6 mouse strain, the BALB/c strain generates X-linked antigen loss. In contrast, we detected no X-linked histogenic differences between strains C57BL/6 and DBA/2, or DBA/2 and BALB/c. To localize this X-linked barrier to histocompatibility, we produced a panel of 25 [(BALB/cxC57BL/6)F1xC57BL/6]N2 males that were grafted with C57BL/6 skin to determine which carried the BALB/c-derived component(s) necessary for graft rejection. DNA marker analysis showed one region of overlapping BALB/c-derived X-chromosomal segments among the graft rejecters, suggesting that this antigen-loss haplotype ( H-hix(c), for histoincompatibility on the X chromosome, c haplotype) may be restricted within the DXMit55 to the Xq telomere interval (which excludes only the centromeric tip of the X). Further backcrossing of H-hix(c) to C57BL/6 resulted in fewer rejecter mice than expected by the N4 generation, suggesting that a second, unlinked locus is also involved in this X-linked alloantigenicity. The vigorous rejection of male (C57BL/6xBALB)F1 and female (B6.C- H2(d)xC57BL/6)F1 skin by (BALB/cxC57BL/6)F1 males, as well as the assessment of markers on Chromosome 17 among N2 and N4 graft-recipient males, suggests that this second locus is H2, and that H-hix(b)-encoded alloantigens require both H2(b) and H2(d)-encoded presentation molecules for efficient graft rejection.     

Accurate quantification of basal plasma levels of 3-nitrotyrosine and 3-nitrotyrosinoalbumin by gas chromatography-tandem mass spectrometry

Measurement of 3-nitro-L-tyrosine (NO(2)Tyr) and protein-related 3-nitro-L-tyrosine in human plasma is associated with numerous methodological problems which result in highly divergent basal plasma levels often ranging within two orders of magnitude. Recently, we have described an interference-free GC-tandem MS-based method for NO(2)Tyr which yielded the lowest basal plasma NO(2)Tyr levels reported thus far. This method was extended to quantify protein-associated 3-nitrotyrosine and in particular 3-nitrotyrosinated albumin (NO(2)TyrALB) in human plasma. NO(2)TyrALB and albumin (ALB) were extracted from plasma by affinity column extraction and digested enzymatically at neutral pH. 3-Nitro- L-[2H(3)]tyrosine was used as internal standard. In plasma of 18 healthy young volunteers the molar ratio of NO(2)TyrALB to albumin-derived tyrosine (TyrALB), i.e. NO(2)TyrALB/TyrALB, was determined to be 1.55+/-0.54x1:10(6) (mean+/-SD). The plasma concentration of NO(2)TyrALB was estimated as 24+/-4 nM. The NO(2)Tyr plasma levels in these volunteers were determined to be 0.73+/-0.53 nM. In the same volunteers, NO(2)TyrALB/TyrALB, NO(2)TyrALB and NO(2)Tyr were measured 15 days later and the corresponding values were determined to be 1.25+/-0.58x1:10(6), 25+/-6 nM and 0.69+/-0.16 nM. For comparison, NO(2)Tyr and NO(2)TyrALB were measured in six plasma samples from healthy volunteers by GC-MS and GC-tandem MS. Different values were found for NO(2)Tyr, i.e. 5.4+/-2.8 versus 2.7+/-1.5 nM, and comparable values for NO(2)TyrALB/TyrALB, i.e. 0.5+/-0.2x1:10(6) versus 0.4+/-0.1x1:10(6), by these methods. The ratio of the values measured by GC-MS to those measured by GC-tandem MS were 2.9+/-3.1 for NO(2)Tyr and 1.2+/-0.2 for NO(2)TyrALB/TyrALB. The present GC-tandem MS method provides accurate values of NO(2)Tyr and NO(2)TyrALB in human plasma.     

Hybrid embryonic stem cell-derived tetraploid mice show apparently normal morphological,physiological, and neurological characteristics

ES cell-tetraploid (ES) mice are completely derived from embryonic stem cells and can be obtained at high efficiency upon injection of hybrid ES cells into tetraploid blastocysts. This method allows the immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps. To provide a baseline for the analysis of ES mouse mutants, we performed a phenotypic characterization of wild-type B6129S6F(1) ES mice in relation to controls of the same age, sex, and genotype raised from normal matings. The comparison of 90 morphological, physiological, and behavioral parameters revealed elevated body weight and hematocrit as the only major difference of ES mice, which exhibited an otherwise normal phenotype. We further demonstrate that ES mouse mutants can be produced from mutant hybrid ES cells and analyzed within a period of only 4 months. Thus, ES mouse technology is a valid research tool for rapidly elucidating gene function in vivo.