A study of 386 white clover (Trifolium repens L.) mapping population F1 progeny was conducted to quantify the type and magnitude of genotypic variation for a range of root morphology traits. Clones of each of the 386 white clover progeny were grown in sand. There were significant (P < 0.05) genotypic variance components and repeatability estimates for all the root traits examined. Progeny genotypes with high expression of key traits, including number of root tips and number of root forks were identified. These types may improve phosphate uptake as their highly branched roots will explore a large volume of soil per unit root weight. A strong positive phenotypic and genotypic correlation between several root traits was identified. This suggests an opportunity for indirect selection. For example, selection for high root fork number, a trait that is relatively less complicated to measure, should result in the concurrent increase in expression of the following root traits: surface area, number of tips, volume, and dry weight. Comparison of results from the sand‐based trial with an earlier trial using hydroponic conditions, with clones of the same 386 progeny, showed similar correlations exist among the root traits in both systems. The progeny genotype‐by‐trait Best Linear Unbiased Predictor matrix generated from the sand study is currently being used for the identification of root trait quantitative trait loci.

Background. Cellular rejection of xenografts is predominantly mediated by CD4(+) T cells. Little is known of the effectiveness of CD4(+)CD25(+) T regulatory (Treg) cells at suppressing this strong T-cell mediated immune response. In this study, we evaluated the activity of fresh Treg cells and expanded Treg cells to suppress the xeno immune response in vitro.Methods. Human Treg cells were preferentially expanded by CD3/CD28 expand beads, interleukin (IL)-2, and rapamycin. Human CD4(+)CD25(-) T cells were stimulated with irradiated porcine peripheral blood mononuclear cells in the presence or absence of fresh or expanded human Treg cells for 5 days before proliferation assay. In a separate experiment, the porcine xenoantigen-stimulated CD4(+)CD25(-) T cells were separated from Treg cells by transwells and assessed for cytotoxicity of porcine peripheral blood mononuclear cells target cells. Cytokine-producing cells and cytokine release in the cocultures were examined by enzyme-linked immunosorbent spot and enzyme-linked immonosorbent assay, respectively.Results. Human Treg were expanded up to 3500-fold after 14 days in culture. The addition of fresh Treg suppressed the T-cell mediated xenoimmune response. Compared with fresh Treg cells, expanded Treg cells were more Potent at suppressing CD4(+)CD25(-) T-cell-mediated antiporcine xenogeneic responses. This suppression required cell contact. However, the enhanced suppression by expanded Treg cells was associated with increased secretion of IL-4 and IL-10 when compared with their nonexpanded Treg counterparts.Conclusion. This study shows that expanded human Treg cells were capable of suppressing antiporcine xenogeneic responses in vitro and involve both contact dependent and cytokine mediated mechanisms.

OBJECTIVE:Our previous study has shown that porcine antigen-primed and CD4+ T cells activated macrophages are capable of the Recognition and rejection of porcine xenografts but not mouse allografts, and therefore suggested the involvement of signaling between the graft and macrophages in this specific graft recognition and destruction.METHODS:NOD-SCID mice were transplanted with fetal pig pancreatic fragment (FPP) before adoptive transfer with exogenous macrophages isolated from rejecting FPP xenografts of BALB/c recipient mice. The exogenous macrophages were tracked by Ly5.1 surface antigen or via CSFE staining. Gene expression of CCR2 and CCR5 and their chemokines in transplanted FPP xenografts was evaluated by real-time PCR.RESULTS:After the adoptive transfer, recently transplanted but not established FPP xenografts were rejected by exogenous activated macrophages. In the meantime, greater level of chemokine gene expression was detected in recently-transplanted compared with the established xenografts. Furthermore, expression of both CCR2 and CCR5 genes was enhanced significantly in activated macrophages when compared with non-activated macrophages.CONCLUSION:Upregulated chemokines were associated with macrophage recruitment and destruction of islet xenografts.

Background. Porcine antigen primed and CD4+ T-cell activated macrophages are able to migrate to and destroy porcine xenografts. However, the specific signaling mechanisms involved remain to be identified. Methods. In this study macrophages which lack the universal toll-like receptor (TLR) adaptor MyD88 were used to investigate the role of TLR in the recognition and activation of macrophages in islet xenograft rejection. Macrophages were isolated from rejecting MyD88−/− and wild-type C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts, and were transferred to NPCC recipient NOD-SCID mice. Results. Both wild-type C57BL/6 and MyD88−/− mice rejected NPCC xenografts 8 and 10 days, respectively after transplantation, and the grafts were heavily infiltrated with CD4+ T cells and macrophages. However, graft infiltrating macrophages from rejecting MyD88−/− recipients demonstrated impaired up-regulation of TLR expression and impaired activation phenotype, when compared to those from rejecting C57BL/6 recipients. Transfer of NOD-SCID recipients with macrophages from rejecting C57BL/6 mice resulted in NPCC xenograft rejection along with massively infiltrated macrophages 8 days after transfer, whereas NPCC xenografts in NOD-SCID mice transferred with macrophages from rejecting MyD88−/− mice remained intact until the end of this study, 90 days after transfer, with insulin-positive islets and no infiltration by macrophages. Conclusion. This study demonstrates that deletion of MyD88 causes impaired macrophage activation after pig islet xenotransplantation. However, graft survival is not prolonged and xenografts are rejected rapidly by alternate mechanisms.

Introduction: Porcine antigen primed and CD4+ T cell activated macrophages are able to migrate to and destroy porcine xenografts. However, the specific signaling mechanisms involved remain to be identified. In this study macrophages which lack the universal TLR adaptor MyD88 were used to investigate the role of LTR in the recognition and activation of macrophages in islet xenograft rejection. Methods: Macrophages were isolated from rejecting MyD88 mice and wildtype C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts, and were transferred to NPCC recipient NOD-SCID mice. Graft rejection was determined by immunohistochemistry. Macrophage activation phenotype and gene expression profile in macrophages were analyzed by FACS and real-time PCR, respectively. Results: Both wildtype C57BL/6 and MyD88 mice rejected NPCC xenografts 8 and 10 days, respectively after transplantation. The grafts were heavily infiltrated with CD4+ T cells and macrophages in both rejecting MyD88 and C57BL/6 mice. Graft infiltrating macrophages from rejecting MyD88 recipients demonstrated impaired upregulation of TLR1, 4, 5, 6, 7, 8, 9, reduced expression of both cell surface and intracellular activation markers and down-regulated CCR5 and activating receptor NKG2D, when compared to those from rejecting C57BL/6 recipients. Recipient NODSCID mice that were transferred with macrophages from rejecting C57BL/ 6 mice rejected NPCC xenografts 8 days after transfer. Whereas NODSCID mice infused with macrophages from rejecting MyD88 recipients did not reject their xenografs at day 60 after transfer. The grafts at this time point remained intact with insulin producing function and no infiltration by macrophages while grafts from rejected C57BL/6 mice showed a large macrophage infiltrate. Conclusions: This study provides direct evidence that the MyD88 signaling plays a critical role not only in macrophage activation but also their recruitment in response to pig islet xenotransplantation.

Background. Porcine antigen primed and CD4+ T-cell-activated macrophages are capable of both recognition and rejection of porcine xenografts. However, the specific signaling mechanisms involved remains to be addressed. The aim of this study was to examine the role of chemokine receptor and CD40 signaling in macrophage recruitment and graft destruction. Methods. Macrophages were isolated from rejecting CCR2−/−, CCR5−/−, CD40−/− and control C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts and were transferred to NPCC recipient NOD-SCID mice. Results. Macrophages isolated from rejecting NPCC xenografts in CD40−/− and wildtype C57BL/6 mice demonstrated upregulated expression of macrophage activation markers as well as CCR5 and CCR2 genes, and caused pig islet xenograft destruction 8 days after transfer to NOD-SCID recipients. Graft infiltrating macrophages from rejecting CCR2−/− mice showed a similar activation phenotype and destroyed NPCC xenografts 10 days after transfer to NOD-SCID mice. Blockade of MCP-1 by anti-MCP-1 mAb did not prolong graft survival in CD4+ T cell reconstituted NPCC recipient NOD-SCID mice. By contrast, the graft infiltrating macrophages from rejecting CCR5−/− recipients showed impaired macrophage activation when compared to control C57BL/6 recipients, and transfer of these macrophages did not result in xenograft destruction in NOD-SCID recipients until day 16 after transfer. Analysis of graft infiltrating macrophages from these rejecting NOD-SCID mice showed an impaired activation phenotype. Conclusion. These results demonstrate that CCR5 is involved in both the activation and recruitment of macrophages to rejecting islet xenografts but other pathways are involved.

The 1:1 adduct ion formation between a series of permethylated monosaccharides (M; I(a)-I(h)) and an organic or a metallic cation (A+) has been examined in quantitative FAB mass spectrometry. In a careful comparison under the same FABMS conditions, the relative (M + A)+ peak intensities increase in the following order of the monosaccharides, and further increase at nearly the same extents in spite of using three different cations such as octylammonium, (methoxycarbonyl)methylammonium, and potassium ions: beta-Glc < alpha-Glc < alpha-Gal < beta-Gal less-than-or-equal-to alpha-Man < beta-Man < alpha-Tal < beta-Tal. The order and cation-independency clearly indicate the OCH3 configurational effects of M on (M + A)+ adduct ion formation. The findings can be interpreted in terms of multisite electrostatic interaction of oxygens with the cation. Coupled with the results of gas-phase behavior by FABMS/MS(CAD), solution behavior by H-1 NMR, and model calculations by MNDO, the characteristic structure of the 1:1 adduct ion is deduced as host-guest type association between permethylated monosaccharides and cationic species in particular cases, at least beta-Tal and alpha-Tal cases.

Relative FABMS peak-intensities of the cluster ions between a set of permethylated aldopyranoses and potassium ion provide a lose relation with relative FABMS/MS area-intensities of the corresponding cluster ions between a set of methyl aldopyranosides and potassium ion.

The relative FABMS intensities of the cluster ions between permethylated monosaccharides and glycine methyl ester hydrochloride strongly suggest the configuration effect of the OCH3 groups, reflecting the relative stabilities of the cluster ions in gas phase.