Behaviour of butyrylcholinesterase in the intestinal epithelial cells of starved and refed rats.

1. The activity and the molecular characteristics of butyrylcholinesterase were studied in the epithelial cells of the following intestinal segments: duodenum, jejunum, ileum, caecum and colon of starved and refed rats. 2. After starvation, the specific activity of the enzyme is found to increase in the jejunum. The same level of activity was maintained after refeeding. No notable changes were observed in the other intestinal segments after either starvation or refeeding. 3. The behaviour of aminopeptidase, a well-characterized intestinal enzyme, is comparable to that of butyrylcholinesterase, except in the duodenum where the aminopeptidase activity is increased after refeeding. 4. In this cell type, BuChE is found only in its globular forms (G1, G2 and G4). Starvation resulted in a higher value of the sedimentation coefficient of the ileal G2 form, suggesting the existence of a complex between the enzyme and non-cholinesterase components. 5. After refeeding, the sedimentation profile was similar to that of control.     

Longitudinal studies of clonally expanded CD8 T cells reveal a repertoire shrinkage predicting mortality and an increased number of dysfunctional cytomegalovirus-specific T cells in the very elderly

The age-associated decrease in functionality of the human immune system is thought to have a negative impact on the capacity to provide protection against infection, in turn leading to increased incidence of mortality. In a previous longitudinal study of octogenarians, we identified an immune risk phenotype (IRP) in the very elderly defined by an inverted CD4/CD8 ratio, which was associated with increased mortality and persistent CMV infection. In this study, we analyzed the CD8 clonal composition of nonagenarians and middle-aged individuals. An increased number of CD8 T cell clones was observed in the nonagenarians, and was associated with CMV-seropositivity. Surprisingly, CMV-seropositive nonagenarians with the IRP had a significantly lower number of clones compared with non-IRP individuals. The decrease in clone numbers in IRP individuals was associated with shorter survival time. MHC/peptide multimer staining indicated that the frequency of CMV-specific T cells was higher in nonagenarians than in the middle-aged, but the ratio of functionally intact cells was significantly lower. The lowest ratio of functional CMV-specific T cells was found in an IRP individual. A thorough longitudinal analysis of the CMV-specific T cells in nonagenarians showed a stable pattern with respect to frequency, phenotype, and clonal composition. We hypothesize that the number of different CD8 T cell clonal expansions increases as the individual ages, possibly, as a compensatory mechanism to control latent infections, e.g., CMV, but eventually a point is reached where clonal exhaustion leads to shrinkage of the CD8 clonal repertoire, associated with decreased survival.     

MHC-based detection of antigen-specific CD8+ T cell responses

The hallmark of adaptive immunity is its ability to recognise a wide range of antigens and technologies that capture this diversity are therefore of substantial interest. New methods have recently been developed that allow the parallel analysis of T cell reactivity against vast numbers of different epitopes in limited biological material. These technologies are based on the joint binding of differentially labelled MHC multimers on the T cell surface, thereby providing each antigen-specific T cell population with a unique multicolour code. This strategy of ‘combinatorial encoding’ enables detection of many (at least 25) different T cell populations per sample and should be of broad value for both T cell epitope identification and immunomonitoring.     

Variability of grain quality in sorghum: association with polymorphism in Sh2, Bt2, SssI, Ae1, Wx and O2

To ensure food security in Africa and Asia, developing sorghum varieties with grain quality that matches consumer demand is a major breeding objective that requires a better understanding of the genetic control of grain quality traits. The objective of this targeted association study was to assess whether the polymorphism detected in six genes involved in synthesis pathways of starch (Sh2, Bt2, SssI, Ae1, and Wx) or grain storage proteins (O2) could explain the phenotypic variability of six grain quality traits [amylose content (AM), protein content (PR), lipid content (LI), hardness (HD), endosperm texture (ET), peak gelatinization temperature (PGT)], two yield component traits [thousand grain weight (TGW) and number of grains per panicle (NBG)], and yield itself (YLD). We used a core collection of 195 accessions which had been previously phenotyped and for which polymorphic sites had been identified in sequenced segments of the six genes. The associations between gene polymorphism and phenotypic traits were analyzed with Tassel. The percentages of admixture of each accession, estimated using 60 RFLP probes, were used as cofactors in the analyses, decreasing the proportion of false-positive tests (70%) due to population structure. The significant associations observed matched generally well the role of the enzymes encoded by the genes known to determine starch amount or type. Sh2, Bt2, Ae1, and Wx were associated with TGW. SssI and Ae1 were associated with PGT, a trait influenced by amylopectin amount. Sh2 was associated with AM while Wx was not, possibly because of the absence of waxy accessions in our collection. O2 and Wx were associated with HD and ET. No association was found between O2 and PR. These results were consistent with QTL or association data in sorghum and in orthologous zones of maize. This study represents the first targeted association mapping study for grain quality in sorghum and paves the way for marker-aided selection.     

Mutational analysis of the nucleotide binding site of Escherichia coli dCTP deaminase

In Escherichia coli and Salmonella typhimurium about 80% of the dUMP used for dTMP synthesis is derived from deamination of dCTP. The dCTP deaminase produces dUTP that subsequently is hydrolyzed by dUTPase to dUMP and diphosphate. The dCTP deaminase is regulated by dTTP that inhibits the enzyme by binding to the active site and induces an inactive conformation of the trimeric enzyme. We have analyzed the role of residues previously suggested to play a role in catalysis. The mutant enzymes R115Q, S111C, S111T and E138D were all purified and analyzed for activity. Only S111T and E138D displayed detectable activity with a 30- and 140-fold reduction in k_cat, respectively. Furthermore, S111T and E138D both showed altered dTTP inhibition compared to wild-type enzyme. S111T was almost insensitive to the presence of dTTP. With the E138D enzyme the dTTP dependent increase in cooperativity of dCTP saturation was absent, although the dTTP inhibition itself was still cooperative. Modeling of the active site of the S111T enzyme indicated that this enzyme is restricted in forming the inactive dTTP binding conformer due to steric hindrance by the additional methyl group in threonine. The crystal structure of E138D in complex with dUTP showed a hydrogen bonding network in the active site similar to wild-type enzyme. However, changes in the hydrogen bond lengths between the carboxylate and a catalytic water molecule as well as a slightly different orientation of the pyrimidine ring of the bound nucleotide may provide an explanation for the reduced activity.     

Vaccination with anti-idiotype antibody ganglidiomab mediates a GD2-specific anti-neuroblastoma immune response

Purpose

Immunotherapy targeting disialoganglioside GD₂ emerges as an important treatment option for neuroblastoma, a pediatric malignancy characterized by poor outcome. Here, we report the induction of a GD₂-specific immune response with ganglidiomab, a new anti-idiotype antibody to anti-GD₂ antibodies of the 14.18 family.

Experimental design and results

Ganglidiomab was generated following immunization of Balb/c mice with 14G2a, and splenocytes were harvested to generate hybridoma cells. Clones were screened by ELISA for mouse antibody binding to hu14.18. One positive clone was selected to purify and characterize the secreted IgG protein (κ, IgG₁). This antibody bound to anti-GD₂ antibodies 14G2a, ch14.18/CHO, hu14.18, and to immunocytokines ch14.18-IL2 and hu14.18-IL2 as well as to NK-92 cells expressing scFv(ch14.18)-zeta receptor. Binding of these anti-GD₂ antibodies to the nominal antigen GD₂ as well as GD₂-specific lysis of neuroblastoma cells by NK-92-scFv(ch14.18)-zeta cells was competitively inhibited by ganglidiomab, proving GD₂ surrogate function and anti-idiotype characteristics. The dissociation constants of ganglidiomab from anti-GD₂ antibodies ranged from 10.8 ± 5.01 to 53.5 ± 1.92 nM as determined by Biacore analyses. The sequences of framework and complementarity-determining regions of ganglidiomab were identified. Finally, we demonstrated induction of a GD₂-specific humoral immune response after vaccination of mice with ganglidiomab effective in mediating GD₂-specific killing of neuroblastoma cells.

Conclusion

We generated and characterized a novel anti-idiotype antibody ganglidiomab and demonstrated activity against neuroblastoma.