Atomic force microscopy and single-molecule force spectroscopy were combined to image and manipulate purple membrane patches from Halobacterium salinarum. Individual bacteriorhodopsin molecules were first localized and then extracted from the membrane; the remaining vacancies were imaged again. Anchoring forces between 100 and 200 piconewtons for the different helices were found. Upon extraction, the helices were found to unfold. The force spectra revealed the individuality of the unfolding pathways. Helices G and F as well as helices E and D always unfolded pairwise, whereas helices B and C occasionally unfolded one after the other. Experiments with cleaved loops revealed the origin of the individuality: stabilization of helix B by neighboring helices.

Cw and pulsed high-field EPR (95 GHz, 3.4 T) are performed on site-directed spin labeled bacteriorhodopsin (BR) mutants. The enhanced Zeeman splitting leads to spectra with resolved g-tensor components of the nitroxide spin label. The g(xx) component shift determined for 10 spin labels located in the cytoplasmic loop region and in the protein interior along the BR proton channel reveals a maximum close to position 46 between the proton donor D96 and the retinal. A plot of g(xx) versus A(zz) of the nitrogen discloses grouping of 12 spin labeled sites in protic and aprotic sites. Spin labels at positions 46, 167 and 171 show the aprotic character of the cytoplasmic moiety of the proton channel whereas nitroxides at positions 53, 194 and 129 reveal the protic environment in the extracellular channel. The enhanced sensitivity of high-field EPR with respect to anisotropic reorientational motion of nitroxides allows the characterization of different motional modes for spin labels bound to positions 167 and 170. The motional restriction of the nitroxide at position 167 of the double mutant V167C/D96N is decreased in the M(N) photo-intermediate. An outward shift of the cytoplasmic moiety of helix F in the M(N) intermediate would account for the high-field EPR results and is in agreement with diffraction and recent X-band EPR data.

Immune recovery was retrospectively analyzed in a cohort of 41 patients with acute leukemia, myelodysplastic syndrome and nonmalignant diseases, who received αβ T-and B-cell-depleted allografts from haploidentical family donors. Conditioning regimens consisted of fludarabine or clofarabine, thiotepa, melphalan and serotherapy with OKT3 or ATG-Fresenius. Graft manipulation was carried out with anti-TCRαβ and anti-CD19 Abs and immunomagnetic microbeads. The γδ T cells and natural killer cells remained in the grafts. Primary engraftment occurred in 88%, acute GvHD (aGvHD) grades II and III-IV occurred in 10% and 15%, respectively. Immune recovery data were available in 26 patients and comparable after OKT3 (n = 7) or ATG-F (n = 19). Median time to reach 4100 CD3+ cells/μL, 4200 CD19+ cells/μL and 4200 CD56+ cells/μL for the whole group was 13, 127 and 12.5 days, respectively. Compared with a historical control group of patients with CD34+ selected grafts, significantly higher cell numbers were found for CD3+ at days +30 and +90 (267 vs 27 and 397 vs 163 cells/μL), for CD3+4+ at day +30 (58 vs 11 cells/μL) and for CD56+ at day +14 (622 vs 27 cells/μL). The clinical impact of this accelerated immune recovery will be evaluated in an ongoing prospective multicenter trial.Bone Marrow Transplantation (2015) 50, S6-S10; doi:10.1038/bmt.2015.87 INTRODUCTIONTransplantation of haploidentical stem cells has become an accepted option for pediatric patients and adults with high-risk malignancies who lack a matched related or unrelated donor.1 In recent years, the majority of pediatric transplant centers chose the CD34+ selection of peripheral stem cells, which allowed minimizing GvHD by effective reduction of T cells in the graft. Together with myeloablative, TBI or busulfan-based conditioning regimens, acceptable hematopoietic engraftment and survival rates have been observed in both adult and pediatric patients with acute leukemia and nonmalignant diseases.2,3 However, infectious complications caused by delayed immune recovery were a major reason for transplant-related mortality, and rates between 5 and 37% 4-8 have been described in pediatric cohorts. To improve the immune recovery, we have established a new T-cell depletion method that removes αβ+ T lymphocytes via a biotinylated antiTcRαβ Ab followed by an anti-biotin Ab conjugated to magnetic microbeads while retaining γδ+ T lymphocytes, natural killer (NK) cells and other cells in the graft. In addition, CD19+ B lymphocytes were concomitantly depleted for the prevention of posttransplant EBV-associated lymphoproliferative disease. The conditioning regimens consisted of fludarabine or clofarabine, thiotepa, melphalan and serotherapy. Our aim was to reduce the conditioning toxicity without affecting engraftment rates and to accelerate immune recovery. Here, we report immune reconstitution data of a cohort of pilot patients transplanted with this approach. PATIENTS AND METHODSWe report a retrospective analysis of a cohort of 41 pediatric patients who received TCRαβ/CD19-dep...

SummaryNeonates show an impaired anti-microbial host defence, but the underlying immune mechanisms are not understood fully. Myeloid-derived suppressor cells (MDSCs) represent an innate immune cell subset characterized by their capacity to suppress T cell immunity. In this study we demonstrate that a distinct MDSC subset with a neutrophilic/granulocytic phenotype (Gr-MDSCs) is highly increased in cord blood compared to peripheral blood of children and adults. Functionally, cord blood isolated Gr-MDSCs suppressed T cell proliferation efficiently as well as T helper type 1 (Th1), Th2 and Th17 cytokine secretion. Beyond T cells, cord blood Gr-MDSCs controlled natural killer (NK) cell cytotoxicity in a cell contact-dependent manner. These studies establish neutrophilic Gr-MDSCs as a novel immunosuppressive cell subset that controls innate (NK) and adaptive (T cell) immune responses in neonates. Increased MDSC activity in cord blood might serve as key fetomaternal immunosuppressive mechanism impairing neonatal host defence. Gr-MDSCs in cord blood might therefore represent a therapeutic target in neonatal infections.

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