ALBERT

Description: The Nares Strait is a waterway separating NW-Greenland and North America. The nature of the Nares Strait has been subject of discussion for decades, especially if it represents a transform fault that compensated the opening of the Baffin Bay in the Paleogene as Alfred Wegener supposed in 1912. The Kane Basin in the central part of Nares Strait provides an opportunity to cross the proposed fault. Geophysical data were acquired in 2001 and 2010, including amongst others multichannel and wide-angle seismic data. The eastern part of the Kane Basin is characterized by a solid platform most likely representing a continuation of the Paleoproterozoic Inglefield-Mobile-Belt (Greenland). In the western part, a sedimentary basin with northwestward tilted and eroded layers of Cretaceous age can be resolved. The transition between those two units shows the plate boundary between Greenland and North America and can be considered as a relic of the Wegener Fault.

Description: The ability to perturb individual genes in genome-wide experiments has been instrumental in unraveling cellular and disease properties. Here we introduce, describe the assembly, and demonstrate the use of comprehensive and versatile transcription activator-like effector (TALE) libraries. As a proof of principle, we built an 11-mer library that covers all possible combinations of the nucleotides that determine the TALE-DNA binding specificity. We demonstrate the versatility of the methodology by constructing a constraint library, customized to bind to a known p53 motif. To verify the functionality in assays, we applied the 11-mer library in yeast-one-hybrid screens to discover TALEs that activate human SCN9A and miR-34b respectively. Additionally, we performed a genome-wide screen using the complete 11-mer library to confirm known genes that confer cycloheximide resistance in yeast. Considering the highly modular nature of TALEs and the versatility and ease of constructing these libraries we envision broad implications for high-throughput genomic assays. Scientific Reports 4 doi: 10.1038/srep04857

Description: Multiply-transfused individuals are at higher risk for BM rejection. We show that whereas allosensitization resulted in the priming of both cellular and humoral immunity, preformed antibody was the major barrier to engraftment. The generation of cross-reactive alloantibody led to rejection of BM of a different MHC-disparate strain. Imaging studies indicated that antibody-mediated rejection was very rapid (〈 3 hours) in primed recipients, while T-cell–mediated rejection in nonprimed mice took more than 6 days. Antibody-mediated BM rejection was not due to a defect in BM homing as rejection occurred despite direct intra-BM infusion of donor BM. Rejection was dependent upon host FcR+ cells. BM cells incubated with serum from primed mice were eliminated in nonprimed recipients, indicating that persistent exposure to high-titer antibody was not essential for rejection. High donor engraftment was achieved in a proportion of primed mice by mega-BM cell dose, in vivo T-cell depletion, and high-dose immunoglobulin infusion. The addition of splenectomy to this protocol only modestly added to the efficacy of this combination strategy. These data demonstrate both rapid alloantibody-mediated elimination of BM by host FcR+ cells and priming of host antidonor T cells and suggest a practical strategy to overcome engraftment barriers in primed individuals.

Description: Abstract 3530 Poster Board III-467 FTY720 (FTY), a sphingosine-1-phosphate receptor agonist, inhibits lymphocyte egress from lymphoid tissues although the complete mechanism of its immunomodulatory effects is not fully understood. We previously published that FTY inhibited but did not prevent graft-versus-host disease by multiple mechanisms. Using the same dose and schedule (3 mg/kg orally d0-28) we evaluated FTY for its effect on allogeneic bone marrow (BM) engraftment in sublethally-irradiated mice. C57BL/6 mice were irradiated with 5.0 Gy total body irradiation (TBI) on day -1, and received 107 T-cell depleted BALB/c BM cells on day 0. At 5 wks, FTY-treated mice had a mean 84% ± 4% (mean ± SEM, n=47) donor chimerism in peripheral blood leukocytes (PBL) versus 5% ± 2% in water-treated controls (n=38, p90% donor at 5 wks, only 6 were 〉50% donor at 3 months indicating that even high level donor chimeras were subject to delayed graft rejection. We found that although FTY promoted robust donor engraftment in the NK, myeloid and B cell lineages in BM, spleen, and lymph nodes by the first week after transplantation, thymopoiesis was severely impaired at 1 month resulting in near absent donor (and also host) thymic T cell production. FTY-treated mice had very low thymocyte cellularity (

Description: KGF (FGF-7), an epithelial cell growth factor, can lessen the injurious effects of post-BMT lung injury (IPS) and GVHD when given prior to conditioning of the recipient. We reported that mice treated with KGF pre-conditioning had elevated systemic levels of Th2-type cytokines at the time of BM infusion which was associated with less GVHD. In non-conditioned SCID mice that developed lethal GVHD from allogeneic T cells, KGF pretreatment also increased Th2 cytokines implying that KGF alone was responsible for the systemic Th2 milieu and not a response that was dependent on conditioning. In the post-BMT lung, KGF pretreatment did not alter the cellular composition of the inflammatory infiltrate but resulted in decreased levels of immune activation markers compared to non-treated control IPS mice. The mechanism of this immunomodulatory effect is unknown. Since Th2 responses are dependent on the STAT-6 transcription factor, we studied the effects of KGF in our established murine IPS model using lethally-conditioned STAT-6−/− mice (B6 background) as recipients of allogeneic B10.BR BM and T cells and compared to WT hosts. STAT-6−/− mice pretreated with KGF (5 mg/kg s.c. for 3 days) had an accelerated mortality rate and poorer survival compared to WT recipients. Examination of GVHD target organs revealed that KGF pretreatment ameliorated liver GVHD in WT and STAT-6−/− recipients. In contrast, the benefit of KGF for GVHD of the colon was only seen in the WT but not in STAT-6−/− recipients, consistent with post-BMT body weights and survival. Whole body plethysmograph was done to evaluate lung function at 30 cm water pressure on day 7 post-BMT, a time when acute IPS is manifested in this model. WT but not STAT-6−/− mice pretreated with KGF had improved lung compliance compared to their non-treated counterparts (p

Description: Although allosensitization results in the priming of both cellular and humoral immunity, our data indicate that preformed antibody is the major barrier to successful bone marrow transplantation (BMT) in the sensitized recipient. A single priming event led to rapid, high, long-lived antibody levels that practically precluded waiting for sufficient waning of antibody titer for successful BMT. C57BL/6 (B6) mice, whether primed against BALB/c alloantigen 7 days or 4 months before BMT, died of graft failure by 2 wks after transplantation. Antibody-mediated BM rejection was not entirely antigen specific as B6 mice primed against BALB/c also rejected B10.BR BM albeit more slowly than BALB/c BM. However, B6 mice primed against B10.BR rejected B10.BR BM but not BALB/c BM. Examination of serum from primed B6 mice indicated that BALB/c priming resulted in a more vigorous and broadly cross-reactive antibody response than did B10.BR priming perhaps due to the multiple minor antigen differences (as well as MHC disparity) that exist between BALB/c and B6 mice but not between B10.BR and B6 mice. Moreover, BM incubated with serum from primed mice was rejected in non-primed recipients indicating that ex vivo antibody-coating of donor BM was sufficient for its elimination and that continual in vivo exposure to high titer antibody was not essential for rejection. Antibody-mediated rejection was formally shown to be dependent upon host Fc receptor+ cells. We hypothesized that if donor BM cells could be delivered directly to the bone marrow cavity they might bypass destruction by the RES system or a BM homing defect, but direct intra-BM infusion of donor BM did not abrogate rejection in primed recipients. Imaging studies using B6 GFP+ BM indicated that antibody-mediated BM rejection was complete by 18 hrs in B6-primed BALB/c mice. In contrast, T cell-mediated rejection in non-primed mice took 〉6 days with donor BM first eliminated from lymphoid organs and later, from the BM cavity and parenchymal organs. We hypothesized that mega-BM doses might overcome primed rejection however, increasing the BM cell dose 10-fold was sufficient to overcome T cell priming in B cell-deficient muMT mice but not antibody-mediated rejection in WT mice. Despite the formidable barrier to engraftment, high donor chimerism levels were achieved in a proportion of primed mice by the combination of high dose murine immunoglobulin (mIg) infusion pre-BMT, in vivo T cell-depletion peri-BMT and mega-BM dose. The addition of splenectomy pre-BMT to this protocol only modestly added to the efficacy of this combination strategy. Although mIg, especially in combination with splenectomy, reduced antibody levels, the reduction was unlikely to be of sufficient magnitude to account for the increased survival and engraftment seen in mice when used in conjunction with in vivo TCD. The efficacy of mIg was more likely due to inhibition of Fc receptor-mediated clearance of antibody-bound cells. The heretofore unappreciated rapidity of antibody-mediated BM rejection in allosensitized recipients indicates there is no window of opportunity for clinical intervention after BMT and dictates the need for greater patient evaluation and therapeutic strategies prior to BMT.

Description: Cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODNs) are synthetic ODNs with unmethylated DNA sequences that mimic viral and bacterial DNA and protect against infectious agents and tumor challenge. We show that CpG ODNs markedly accelerated graft-versus-host disease (GVHD) lethality by Toll-like receptor 9 (TLR9) ligation of host antigen-presenting cells (APCs), dependent upon host IFNγ but independent of host IL-12, IL-6, or natural killer (NK) cells. Imaging studies showed significantly more green fluorescent protein–positive (GFP+) effector T cells in lymphoid and nonlymphoid organs. In engraftment studies, CpG ODNs promoted allogeneic donor bone marrow (BM) rejection independent of host IFNγ, IL-12, or IL-6. During the course of these studies, we uncovered a previously unknown and critical role of donor BM APCs in modulating the rejection response. CpG ODNs promoted BM rejection by ligation of donor BM, but not host, TLR9. CpG ODNs did not impair engraftment of TLR9−/− BM unless wild-type myeloid (CD11b+) but not B-lineage (CD19+) BM cells were added to the donor inoculum. The importance of donor BM APCs in modulating the strength of the host antidonor rejection response was underscored by the finding that B7-1/B7-2−/− BM was less likely than wild-type BM to be rejected. Collectively, these data offer new insight into the mechanism of alloresponses regulating GVHD and BM rejection.