Vascular Endothelial Cells Produce Soluble Factors That Mediate the Recovery of Human Hematopoietic Stem Cells after Radiation Injury

AbstractThe risk of terrorism with nuclear or radiologic weapons is considered to be high over the coming decade. Ionizing radiation can cause a spectrum of hematologic toxicities, from mild myelosuppression to myeloablation and death. However, the potential regenerative capacity of human hematopoietic stem cells (HSCs) after radiation injury has not been well characterized. In this study, we sought to characterize the effects of ionizing radiation on human HSCs and to determine whether signals from vascular endothelial cells could promote the repair of irradiated HSCs. Exposure of human bone marrow CD34+ cells to 400 cGy caused a precipitous decline in hematopoietic progenitor cell content and primitive cells capable of repopulating nonobese diabetic/severe combined immunodeficient mice (SCID-repopulating cells), which was not retrievable via treatment with cytokines. Conversely, culture of 400 cGy–irradiated bone marrow CD34+ cells with endothelial cells under noncontact conditions supported the differential recovery of both viable progenitor cells and primitive SCID-repopulating cells. These data illustrate that vascular endothelial cells produce soluble factors that promote the repair and functional recovery of HSCs after radiation injury and suggest that novel factors with radiotherapeutic potential can be identified within this milieu

Donor Allospecific CD44high Central Memory T Cells Have Decreased Ability to Mediate Graft-vs.-Host Disease

Data from both animal models and humans have demonstrated that effector memory T cells (TEM) and central memory T cells (TCM) from unprimed donors have decreased ability to induce graft-vs-host disease (GVHD). Allospecific TEM from primed donors do not mediate GVHD. However, the potential of alloreactive TCM to induce GVHD is not clear. In this study, we sought to answer this question using a novel GVHD model induced by T cell receptor (TCR) transgenic OT-II T cells. Separated from OT-II mice immunized with OVA protein 8 weeks earlier, the allospecific CD44high TCM were able to mediate skin graft rejection after transfer to naive mice, yet had dramatically decreased ability to induce GVHD. We also found that these allospecific CD44high TCM persisted in GVHD target organs for more than 30 days post-transplantation, while the expansion of these cells was dramatically decreased during GVHD, suggesting an anergic or exhausted state. These observations provide insights into how allospecific CD4+ TCM respond to alloantigen during GVHD and underscore the fundamental difference of alloresponses mediated by allospecific TCM in graft rejection and GVHD settings

Nonmyeloablative regimen preserves "niches" allowing for peripheral expansion of donor T-cells

AbstractT-cell recovery following myeloablative preparatory regimens and cord blood transplantation in adult patients gen erally occurs between 1 and 3 years following allogeneic bone marrow transplantation. T-cell reconstitution may involve thymic education of donor-derived precursors or peripheral expansion of mature T-cells transferred in the graft. We measured quantitative and qualitative immunologic reconstitution, T-cell receptor spectratyping, and T-cell receptor excision circle (TREC) levels in adult recipients of umbilical cord blood transplants following a novel nonmyeloablative regimen. These results were compared to previously published results of similar patients receiving a myeloablative regimen and cord blood stem cells. With small numbers of patients treated so far, T-cells (CD3+) reached normal levels in adults 6 to 12 months following nonmyeloablative transplantation compared with 24 months in adults receiving a myeloablative regimen. At 12 months after transplantation, the numbers of phenotypically naive (CD45RA) T-cells were higher in those receiving the nonmyeloablative regimen. The T-cell repertoire in cord blood recipients treated with a nonmyeloablative regimen was markedly more diverse and robust compared with the repertoire in those receiving the myeloablative regimen at similar time points. TRECs (which are generated within the thymus and identify new thymic emigrants and those that have not divided) were detected 12 months after transplantation in the nonmyeloablative recipients, whereas TRECs were not detected in adults until 18 to 24 months in those receiving myeloablative regimens. Thus, in adults receiving a nonmyeloablative preparatory regimen, the quantitative and qualitative recovery of T-cells occurs through rapid peripheral expansion. The ability of patients receiving a nonmyeloablative regimen to recover within a few months suggests that the peripheral niches in which T-cells can proliferate are preserved in these patients compared to those receiving ablative regimens. Moreover, the presence of TREC-positive cells within 1 year suggests that thymic recovery is likewise accelerated in non myeloablative compared to myeloablative regimens.Biol Blood Marrow Transplant 2002;8(5):249-56

Iterated Conformal Dynamics and Laplacian Growth

The method of iterated conformal maps for the study of Diffusion Limited Aggregates (DLA) is generalized to the study of Laplacian Growth Patterns and related processes. We emphasize the fundamental difference between these processes: DLA is grown serially with constant size particles, while Laplacian patterns are grown by advancing each boundary point in parallel, proportionally to the gradient of the Laplacian field. We introduce a 2-parameter family of growth patterns that interpolates between DLA and a discrete version of Laplacian growth. The ultraviolet putative finite-time singularities are regularized here by a minimal tip size, equivalently for all the models in this family. With this we stress that the difference between DLA and Laplacian growth is NOT in the manner of ultraviolet regularization, but rather in their deeply different growth rules. The fractal dimensions of the asymptotic patterns depend continuously on the two parameters of the family, giving rise to a "phase diagram" in which DLA and discretized Laplacian growth are at the extreme ends. In particular we show that the fractal dimension of Laplacian growth patterns is much higher than the fractal dimension of DLA, with the possibility of dimension 2 for the former not excluded.Comment: 13 pages, 12 figures, submitted to Phys. Rev.

On two problems in graph Ramsey theory

We study two classical problems in graph Ramsey theory, that of determining the Ramsey number of bounded-degree graphs and that of estimating the induced Ramsey number for a graph with a given number of vertices. The Ramsey number r(H) of a graph H is the least positive integer N such that every two-coloring of the edges of the complete graph $K_N$ contains a monochromatic copy of H. A famous result of Chv\'atal, R\"{o}dl, Szemer\'edi and Trotter states that there exists a constant c(\Delta) such that r(H) \leq c(\Delta) n for every graph H with n vertices and maximum degree \Delta. The important open question is to determine the constant c(\Delta). The best results, both due to Graham, R\"{o}dl and Ruci\'nski, state that there are constants c and c' such that 2^{c' \Delta} \leq c(\Delta) \leq 2^{c \Delta \log^2 \Delta}. We improve this upper bound, showing that there is a constant c for which c(\Delta) \leq 2^{c \Delta \log \Delta}. The induced Ramsey number r_{ind}(H) of a graph H is the least positive integer N for which there exists a graph G on N vertices such that every two-coloring of the edges of G contains an induced monochromatic copy of H. Erd\H{o}s conjectured the existence of a constant c such that, for any graph H on n vertices, r_{ind}(H) \leq 2^{c n}. We move a step closer to proving this conjecture, showing that r_{ind} (H) \leq 2^{c n \log n}. This improves upon an earlier result of Kohayakawa, Pr\"{o}mel and R\"{o}dl by a factor of \log n in the exponent.Comment: 18 page

A cardinal role for cathepsin D in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci

The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP) and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D-/- hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function

Selective Enhancement of Donor Hematopoietic Cell Engraftment by the CXCR4 Antagonist AMD3100 in a Mouse Transplantation Model

The interaction between stromal cell-derived factor-1 (SDF-1) with CXCR4 chemokine receptors plays an important role in hematopoiesis following hematopoietic stem cell transplantation. We examined the efficacy of post transplant administration of a specific CXCR4 antagonist (AMD3100) in improving animal survival and in enhancing donor hematopoietic cell engraftment using a congeneic mouse transplantation model. AMD3100 was administered subcutaneously at 5 mg/kg body weight 3 times a week beginning at day +2 post-transplant. Post-transplant administration of AMD3100 significantly improves animal survival. AMD3100 reduces pro-inflammatory cytokine/chemokine production. Furthermore, post transplant administration of AMD3100 selectively enhances donor cell engraftment and promotes recovery of all donor cell lineages (myeloid cells, T and B lymphocytes, erythrocytes and platelets). This enhancement results from a combined effect of increased marrow niche availability and greater cell division induced by AMD3100. Our studies shed new lights into the biological roles of SDF-1/CXCR4 interaction in hematopoietic stem cell engraftment following transplantation and in transplant-related mortality. Our results indicate that AMD3100 provides a novel approach for enhancing hematological recovery following transplantation, and will likely benefit patients undergoing transplantation

Alphavirus Replicon Particle Vaccine Breaks B Cell Tolerance and Rapidly Induces IgG to Murine Hematolymphoid Tumor Associated Antigens

De novo immune responses to myeloid and other blood-borne tumors are notably limited and ineffective, making our ability to promote immune responses with vaccines a major challenge. While focus has been largely on cytotoxic cell-mediated tumor eradication, B-cells and the antibodies they produce also have roles in anti-tumor responses. Indeed, therapeutic antibody-mediated tumor cell killing is routinely employed in patients with hematolymphoid cancers, but whether endogenous antibody responses can be incited to blood-born tumors remains poorly studied. A major limitation of immunoglobulin therapies is that cell surface expression of tumor-associated antigen (TAA) targets is dynamic and varied, making promotion of polyclonal, endogenous B cell responses appealing. Since many TAAs are self-antigens, developing tumor vaccines that enable production of antibodies to non-polymorphic antigen targets remains a challenge. As B cell responses to RNA vaccines are known to occur, we employed the Viral Replicon Particles (VRP) which was constructed to encode mouse FLT3. The VRP-FLT3 vaccine provoked a rapid IgG B-cell response to this self-antigen in leukemia and lymphoma mouse models. In addition, IgGs to other TAAs were also produced. Our data suggest that vaccination with RNA viral particle vectors incites a loss of B-cell tolerance that enables production of anti-tumor antibodies. This proof of principle work provides impetus to employ such strategies that lead to a break in B-cell tolerance and enable production of broadly reactive anti-TAA antibodies as potential future therapeutic agents for patients with hematolymphoid cancers

OSL-thermochronometry of feldspar from the KTB borehole, Germany

The reconstruction of thermal histories of rocks (thermochronometry) is a fundamental tool both in Earth science and in geological exploration. However, few methods are currently capable of resolving the low-temperature thermal evolution of the upper ∼2 km of the Earth's crust. Here we introduce a new thermochronometer based on the infrared stimulated luminescence (IRSL) from feldspar, and validate the extrapolation of its response to artificial radiation and heat in the laboratory to natural environmental conditions. Specifically, we present a new detailed Na-feldspar IRSL thermochronology from a well-documented thermally-stable crustal environment at the German Continental Deep Drilling Program (KTB). There, the natural luminescence of Na-feldspar extracted from twelve borehole samples (0.1–2.3 km depth, corresponding to 10–70 °C) can be either (i) predicted within uncertainties from the current geothermal gradient, or (ii) inverted into a geothermal palaeogradient of 29±2 °C km−1, integrating natural thermal conditions over the last ∼65 ka. The demonstrated ability to invert a depth–luminescence dataset into a meaningful geothermal palaeogradient opens new venues for reconstructing recent ambient temperatures of the shallow crust (200 °C Ma−1 range). Although Na-feldspar IRSL is prone to field saturation in colder or slower environments, the method's primary relevance appears to be for borehole and tunnel studies, where it may offer remarkably recent (<0.3 Ma) information on the thermal structure and history of hydrothermal fields, nuclear waste repositories and hydrocarbon reservoirs

CpG methylation potentiates pixantrone and doxorubicin-induced DNA damage and is a marker of drug sensitivity

DNA methylation is an epigenetic modification of the mammalian genome that occurs predominantly at cytosine residues of the CpG dinucleotide. Following formaldehyde activation, pixantrone alkylates DNA and particularly favours the CpG motif. Aberrations in CpG methylation patterns are a feature of most cancer types, a characteristic that may determine their susceptibility to specific drug treatments. Given their common target, DNA methylation may modulate the DNA damage induced by formaldehyde-activated pixantrone. In vitro transcription, mass spectrometry and oligonucleotide band shift assays were utilized to establish that pixantrone–DNA adduct formation was consistently enhanced 2–5-fold at discrete methylated CpG doublets. The methylation-mediated enhancement was exquisitely sensitive to the position of the methyl substituent since methylation at neighboring cytosine residues failed to confer an increase in pixantrone–DNA alkylation. Covalent modification of DNA by formaldehyde-activated doxorubicin, but not cisplatin, was augmented by neighbouring CpG methylation, indicating that modulation of binding by CpG methylation is not a general feature of all alkylators. HCT116 colon cancer cells vastly deficient in CpG methylation were 12- and 10-fold more resistant to pixantrone and doxorubicin relative to the wild-type line, suggesting that these drugs may selectively recognize the aberrant CpG methylation profiles characteristic of most tumour types