Sibling Cells Research Articles

The Impact of Graft-versus-Host Disease on the Relapse Rate in Patients with Lymphoma Depends on the Histological Subtype and the Intensity of the Conditioning Regimen

The purpose of this study was to analyze the impact of graft-versus-host disease (GVHD) on the relapse rate of different lymphoma subtypes after allogeneic hematopoietic cell transplantation (allo-HCT). Adult patients with a diagnosis of Hodgkin lymphoma, diffuse large B cell lymphoma, follicular lymphoma (FL), peripheral T cell lymphoma, or mantle cell lymphoma (MCL) undergoing HLA-identical sibling or unrelated donor hematopoietic cell transplantation between 1997 and 2009 were included. Two thousand six hundred eleven cases were included. A reduced-intensity conditioning (RIC) regimen was used in 62.8% of the transplantations. In a multivariate analysis of myeloablative cases (n = 970), neither acute (aGVHD) nor chronic GVHD (cGVHD) were significantly associated with a lower incidence of relapse/progression in any lymphoma subtype. In contrast, the analysis of RIC cases (n = 1641) showed that cGVHD was associated with a lower incidence of relapse/progression in FL (risk ratio [RR], .51; P = .049) and in MCL (RR, .41; P = .019). Patients with FL or MCL developing both aGVHD and cGVHD had the lowest risk of relapse (RR, .14; P = .007; and RR, .15; P = .0019, respectively). Of interest, the effect of GVHD on decreasing relapse was similar in patients with sensitive disease and chemoresistant disease. Unfortunately, both aGVHD and cGVHD had a deleterious effect on treatment-related mortality and overall survival (OS) in FL cases but did not affect treatment-related mortality, OS or PFS in MCL. This study reinforces the use of RIC allo-HCT as a platform for immunotherapy in FL and MCL patients.

Numb family proteins: novel players in cardiac morphogenesis and cardiac progenitor cell differentiation

Vertebrate heart formation is a spatiotemporally regulated morphogenic process that initiates with bilaterally symmetric cardiac primordial cells migrating toward the midline to form a linear heart tube. The heart tube then elongates and undergoes a series of looping morphogenesis, followed by expansions of regions that are destined to become primitive heart chambers. During the cardiac morphogenesis, cells derived from the first heart field contribute to the primary heart tube, and cells from the secondary heart field, cardiac neural crest, and pro-epicardial organ are added to the heart tube in a precise spatiotemporal manner. The coordinated addition of these cells and the accompanying endocardial cushion morphogenesis yield the atrial, ventricular, and valvular septa, resulting in the formation of a four-chambered heart. Perturbation of progenitor cells' deployment and differentiation leads to a spectrum of congenital heart diseases. Two of the genes that were recently discovered to be involved in cardiac morphogenesis are Numb and Numblike. Numb, an intracellular adaptor protein, distinguishes sibling cell fates by its asymmetric distribution between the two daughter cells and its ability to inhibit Notch signaling. Numb regulates cardiac progenitor cell differentiation in Drosophila and controls heart tube laterality in Zebrafish. In mice, Numb and Numblike, the Numb family proteins (NFPs), function redundantly and have been shown to be essential for epicardial development, cardiac progenitor cell differentiation, outflow tract alignment, atrioventricular septum morphogenesis, myocardial trabeculation, and compaction. In this review, we will summarize the functions of NFPs in cardiac development and discuss potential mechanisms of NFPs in the regulation of cardiac development.

Reactivated CD4+Tm cells of T1D patients and siblings display an exaggerated effector phenotype with heightened sensitivity to activation-induced cell death.

Dysfunction in effector memory has been proposed to contribute to autoimmunity in type 1 diabetes (T1D). Using a unique cohort of age- and sex-matched T1D patients, nonaffected siblings, and unrelated control children, we undertook a detailed analysis of proliferation, activation, effector responses, and apoptosis in reactivated CD4(+)Tm cells during T-cell receptor stimulation. Across cohorts, there was no difference in the proliferation of reactivated CD4(+)Tm cells. In T1D patients and siblings, CD4(+)Tm cells easily acquired the activated CD25(+) phenotype and effectively transitioned from a central (CD62L(+)Tcm) to an effector memory (CD62L(-)Tem) phenotype with an elevated cytokine "signature" comprising interferon (IFN)-γ and interleukin-10 in T1D patients and IFN-γ in siblings. This amplified Tem phenotype also exhibited an exaggerated immune shutdown with heightened sensitivity to activation-induced cell death and Fas-independent apoptosis. Apoptosis resulted in the elimination of one-half of the effector memory in T1D patients and siblings compared with one-third of the effector memory in control subjects. These data suggest genetic/environment-driven immune alteration in T1D patients and siblings that manifests in an exaggerated CD4(+)Tem response and shutdown by apoptosis. Further immunological studies are required to understand how this exaggerated CD4(+)Tem response fits within the pathomechanisms of T1D and how the effector memory can be modulated for disease treatment and/or prevention.

Axogenesis in the antennal nervous system of the grasshopper Schistocerca gregaria revisited: the base pioneers.

The antennal nervous system of the grasshopper Schistocerca gregaria comprises two parallel pathways projecting to the brain, each pioneered early in embryogenesis by a pair of sibling cells located at the antennal tip. En route, the growth cones of pioneers from one pathway have been shown to contact a guidepost-like cell called the base pioneer. Its role in axon guidance remains unclear as do the cellular guidance cues regulating axogenesis in the other pathway supposedly without a base pioneer. Further, while the tip pioneers are known to delaminate from the antennal epithelium into the lumen, the origin of this base pioneer is unknown. Here, we use immunolabeling and immunoblocking methods to clarify these issues. Co-labeling against the neuron-specific marker horseradish peroxidase and the pioneer-specific cell surface glycoprotein Lazarillo identifies not only the tip pioneers but also a base pioneer associated with each of the developing antennal pathways. Both base pioneers co-express the mesodermal label Mes3, consistent with a lumenal origin, whereas the tip pioneers proved Mes3-negative confirming their affiliation with the ectodermal epithelium. Lazarillo antigen expression in the antennal pioneers followed a different temporal dynamic: continuous in the tip pioneers, but in the base pioneers, only at the time their filopodia and those of the tip pioneers first recognize one another. Immunoblocking of Lazarillo expression in cultured embryos disrupts this recognition resulting in misguided axogenesis in both antennal pathways.

The structural basis of the Tle4-Tli4 complex reveals the self-protection mechanism of H2-T6SS in Pseudomonas aeruginosa.

The type VI secretion system (T6SS) has recently been demonstrated to mediate interbacterial competition and to discriminate between self and nonself. T6SS(+) bacteria employ toxic effectors to inhibit rival cells and concurrently use effector cognate immunity proteins to protect their sibling cells. The effector and immunity pairs (E-I pairs) endow the bacteria with a great advantage in niche competition. Tle4-Tli4 (PA1510-PA1509) is a newly identified E-I pair that is controlled by H2-T6SS in Pseudomonas aeruginosa. Tle4 exhibits phospholipase activity, which destroys the cell membrane of rival cells, and the periplasm-located Tli4 in donor cells eliminates this toxic effect of Tle4. In this paper, the structure of the Tle4-Tli4 complex is reported at 1.75 Å resolution. Tle4 consists of two domains: a conserved α/β-hydrolase domain and an unusual cap domain in which two lid regions (lid1 and lid2) display a closed conformation that buries the catalytic triad in a deep funnel. Tli4 also displays a two-domain structure, in which a large lobe and a small lobe form a crab claw-like conformation. Tli4 uses this crab claw to grasp the cap domain of Tle4, especially the lid2 region, which prevents the interfacial activation of Tle4 and thus causes enzymatic dysfunction of Tle4 in sister cells.

Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis.

Bilaterian neurogenesis is characterized by the generation of diverse neural cell types from dedicated neural stem/progenitor cells (NPCs). However, the evolutionary origin of NPCs is unclear, as neurogenesis in representatives of the bilaterian sister group, the Cnidaria, occurs via interstitial stem cells that also possess broader, non-neural, developmental potential. We address this question by analysing neurogenesis in an anthozoan cnidarian, Nematostella vectensis. Using a transgenic reporter line, we show that NvSoxB(2) - an orthologue of bilaterian SoxB genes that have conserved roles in neurogenesis - is expressed in a cell population that gives rise to sensory neurons, ganglion neurons and nematocytes: the three primary neural cell types of cnidarians. EdU labelling together with in situ hybridization, and within the NvSoxB(2)::mOrange transgenic line, demonstrates that cells express NvSoxB(2) before mitosis and identifies asymmetric behaviours of sibling cells within NvSoxB(2)(+) lineages. Morpholino-mediated gene knockdown of NvSoxB(2) blocks the formation of all three neural cell types, thereby identifying NvSoxB(2) as an essential positive regulator of nervous system development. Our results demonstrate that diverse neural cell types derive from an NvSoxB(2)-expressing population of mitotic cells in Nematostella and that SoxB genes are ancient components of a neurogenic program. To our knowledge this is the first description of a lineage-restricted, multipotent cell population outside the Bilateria and we propose that neurogenesis via dedicated, SoxB-expressing NPCs predates the split between cnidarians and bilaterians.

Specification of neuronal subtypes by different levels of Hunchback.

During the development of the central nervous system, neural progenitors generate an enormous number of distinct types of neuron and glial cells by asymmetric division. Intrinsic genetic programs define the combinations of transcription factors that determine the fate of each cell, but the precise mechanisms by which all these factors are integrated at the level of individual cells are poorly understood. Here, we analyzed the specification of the neurons in the ventral nerve cord of Drosophila that express Crustacean cardioactive peptide (CCAP). There are two types of CCAP neurons: interneurons and efferent neurons. We found that both are specified during the Hunchback temporal window of neuroblast 3-5, but are not sibling cells. Further, this temporal window generates two ganglion mother cells that give rise to four neurons, which can be identified by the expression of empty spiracles. We show that the expression of Hunchback in the neuroblast increases over time and provide evidence that the absolute levels of Hunchback expression specify the two different CCAP neuronal fates.

A genetic screen in Myxococcus xanthus identifies mutants that uncouple outer membrane exchange from a downstream cellular response.

Upon physical contact with sibling cells, myxobacteria transiently fuse their outer membranes (OMs) and exchange OM proteins and lipids. From previous work, TraA and TraB were identified to be essential factors for OM exchange (OME) in donor and recipient cells. To define the genetic complexity of OME, we carried out a comprehensive forward genetic screen. The screen was based on the observation that Myxococcus xanthus nonmotile cells, by a Tra-dependent mechanism, block swarm expansion of motile cells when mixed. Thus, mutants defective in OME or a downstream responsive pathway were readily identified as escape flares from mixed inocula seeded on agar. This screen was surprisingly powerful, as we found >50 mutants defective in OME. Importantly, all of the mutations mapped to the traAB operon, suggesting that there may be few, if any, proteins besides TraA and TraB directly required for OME. We also found a second and phenotypically different class of mutants that exhibited wild-type OME but were defective in a responsive pathway. This pathway is postulated to control inner membrane homeostasis by covalently attaching amino acids to phospholipids. The identified proteins are homologous to the Staphylococcus aureus MprF protein, which is involved in membrane adaptation and antibiotic resistance. Interestingly, we also found that a small number of nonmotile cells were sufficient to block the swarming behavior of a large gliding-proficient population. This result suggests that an OME-derived signal could be amplified from a few nonmotile producers to act on many responder cells.

Analytical model for macromolecular partitioning during yeast cell division.

BackgroundAsymmetric cell division, whereby a parent cell generates two sibling cells with unequal content and thereby distinct fates, is central to cell differentiation, organism development and ageing. Unequal partitioning of the macromolecular content of the parent cell — which includes proteins, DNA, RNA, large proteinaceous assemblies and organelles — can be achieved by both passive (e.g. diffusion, localized retention sites) and active (e.g. motor-driven transport) processes operating in the presence of external polarity cues, internal asymmetries, spontaneous symmetry breaking, or stochastic effects. However, the quantitative contribution of different processes to the partitioning of macromolecular content is difficult to evaluate.ResultsHere we developed an analytical model that allows rapid quantitative assessment of partitioning as a function of various parameters in the budding yeast Saccharomyces cerevisiae. This model exposes quantitative degeneracies among the physical parameters that govern macromolecular partitioning, and reveals regions of the solution space where diffusion is sufficient to drive asymmetric partitioning and regions where asymmetric partitioning can only be achieved through additional processes such as motor-driven transport. Application of the model to different macromolecular assemblies suggests that partitioning of protein aggregates and episomes, but not prions, is diffusion-limited in yeast, consistent with previous reports.ConclusionsIn contrast to computationally intensive stochastic simulations of particular scenarios, our analytical model provides an efficient and comprehensive overview of partitioning as a function of global and macromolecule-specific parameters. Identification of quantitative degeneracies among these parameters highlights the importance of their careful measurement for a given macromolecular species in order to understand the dominant processes responsible for its observed partitioning.

Trophectoderm DNA fingerprinting by quantitative real-time PCR successfully distinguishes sibling human embryos.

To validate a novel and more practical system for trophectoderm DNA fingerprinting which reliably distinguishes sibling embryos from each other. In this prospective and blinded study two-cell and 5-cell samples from commercially available sibling cell lines and excess DNA from trophectoderm biopsies of sibling human blastocysts were evaluated for accurate assignment of relationship using qPCR-based allelic discrimination from 40 single nucleotide polymorphisms (SNPs) with low allele frequency variation and high heterozygosity. Cell samples with self relationships averaged 95.1 ± 5.9 % similarity. Sibling relationships averaged 57.2 ± 5.9 % similarity for all 40 SNPs, and 40.8 ± 8.2 % similarity for the 25 informative SNPs. Assignment of relationships was accomplished with 100 % accuracy for cell lines and embryos. These data demonstrate the first trophectoderm qPCR-based DNA fingerprinting technology capable of unequivocal discrimination of sibling human embryos. This methodology will empower research and development of new markers of, and interventions that influence embryonic reproductive potential.

Stretched cell cycle model for proliferating lymphocytes.

Stochastic variation in cell cycle time is a consistent feature of otherwise similar cells within a growing population. Classic studies concluded that the bulk of the variation occurs in the G1 phase, and many mathematical models assume a constant time for traversing the S/G2/M phases. By direct observation of transgenic fluorescent fusion proteins that report the onset of S phase, we establish that dividing B and T lymphocytes spend a near-fixed proportion of total division time in S/G2/M phases, and this proportion is correlated between sibling cells. This result is inconsistent with models that assume independent times for consecutive phases. Instead, we propose a stretching model for dividing lymphocytes where all parts of the cell cycle are proportional to total division time. Data fitting based on a stretched cell cycle model can significantly improve estimates of cell cycle parameters drawn from DNA labeling data used to monitor immune cell dynamics.

Morphology and ultrastructure of the germarium in panoistic ovarioles of a basal “apterygotous” insect, Thermobia domestica

It has been shown that in Drosophila the germline stem cells (GSCs), similar to the germline and non-germline stem cells of other species, develop and function in specialized microenvironments formed by somatic cells, referred to as the niches. In the fruit fly ovaries, the female GSCs divide asymmetrically to produce new GSCs and the progenitor cells, the cystoblasts (Cbs). Each Cb then divides to generate the cyst composed of 16 interconnected sibling cells, the cystocytes. After cyst formation, specific molecules are transferred to one of the cystocytes which differentiates into the oocyte, whereas the other 15 cystocytes become the nurse cells. We have studied morphology and ultrastructure of the germaria in the ovarioles (ovaries) of a basal “apterygotous” insect, the firebrat (Thermobia domestica). Our analyses have revealed that in this insect, putative GSCs are present along the anterior apex of the germarium. These cells are separated from each other and from the basement lamina covering the ovariole by characteristic somatic cells, termed the apical somatic cells (ASCs), or their elongated processes. We believe that all the ASCs of a given ovariole constitute a “dispersed” niche in which putative GSCs are anchored. Our analyses have additionally shown that in Thermobia, both the Cbs and young (meiotic) oocytes are always individual and never form syncytial cysts. These findings indicate that in certain basal insects the syncytial phase of oogenesis has been eliminated during evolution. Finally, we show that in the early meiotic oocytes of Thermobia, during the so-called bouquet stage, prominent Balbiani bodies (Bbs) are formed. Analysis of serial micrographs indicates that the Bbs invariably arise next to the segment of the nuclear envelope to which the telomeres of the bouquet chromosomes are attached. We suggest, in the light of these data, that the localization of the Bb together with the polar attachment of the bouquet chromosomes play a crucial role in the early asymmetrization of Thermobia oocytes.

CLoNe is a new method to target single progenitors and study their progeny in mouse and chick.

Cell lineage analysis enables us to address pivotal questions relating to: the embryonic origin of cells and sibling cell relationships in the adult body; the contribution of progenitors activated after trauma or disease; and the comparison across species in evolutionary biology. To address such fundamental questions, several techniques for clonal labelling have been developed, each with its shortcomings. Here, we report a novel method, CLoNe that is designed to work in all vertebrate species and tissues. CLoNe uses a cocktail of labelling, targeting and transposition vectors that enables targeting of specific subpopulations of progenitor types with a combination of fluorophores resulting in multifluorescence that describes multiple clones per specimen. Furthermore, transposition into the genome ensures the longevity of cell labelling. We demonstrate the robustness of this technique in mouse and chick forebrain development, and show evidence that CLoNe will be broadly applicable to study clonal relationships in different tissues and species.

Pilot trial of risk‐adapted cyclophosphamide intensity based conditioning and HLA matched sibling and unrelated cord blood stem cell transplantation in newly diagnosed pediatric and adolescent recipients with acquired severe aplastic anemia

Cyclophosphamide-based conditioning regimens and allogeneic hematopoietic stem cell transplantation (AlloHSCT) from matched related donors (MRD) has resulted in the highest survival rates in children and adolescents with acquired severe aplastic anemia (SAA). Time to transplant has consistently been associated with decreased overall survival. Reduced toxicity conditioning and AlloHSCT has been used successfully in other pediatric non-malignant diseases. We piloted a risk-adapted AlloHSCT approach, using fludarabine and anti-thymocyte globulin based conditioning with high (200 mg/kg) and low (60 mg/kg) dose cyclophosphamide as upfront treatment in newly diagnosed pediatric patients with acquired SAA incorporating alternative donor sources, including cord blood. Average risk for non-engraftment patients with <10 transfusions received low dose cyclophosphamide (60 mg/kg); High Risk, those with ≥10 transfusions received conditioning regimen with higher intensity cyclophosphamide (200 mg/kg). Seventeen patients were enrolled and underwent AlloHSCT including 12 males and 5 females with mean age of 8 years (range 3-16), and median follow-up time of 39 months (range 1-135). Donor sources included MRD BM (6/6 [n = 9], 5/6 [n = 2]) and unrelated CB (5/6 [n = 4], 4/6 [n = 2]). Five year OS was 67.6% (37.9-85.4). Three secondary graft failures (17.6%) occurred in the low dose cyclophosphamide arm. Upfront treatment with risk-adapted cyclophosphamide conditioning AlloSCT is well tolerated for the management of newly diagnosed pediatric and adolescent patients with acquired SAA. However, the increased risk of graft rejection in the lower dose arm warrants additional research regarding the optimal intensity of cyclophosphamide-based conditioning regimen to reduce toxicity without increasing graft failure.

A novel method to study aging in bacteria that reproduce by morphologically symmetric fission

The study of aging in fission bacteria has been especially difficult because of the challenge in identifying older sibling cells due to their morphological similarity to younger sibling cells. This study develops a generic method solely based on the analysis of cell growth rate (rate of cell volume increase). The proposed method does not require any special assumptions or measurements regarding the physical features of older siblings, such as older cell poles or slightly greater cell sizes. Therefore, the proposed method is applicable to the study of far more types of bacteria than those of existing methods. Bacteria that can be examined using this method include, but are not limited to, the following categories: (1) cocci bacteria in which tracking a larger number of old pole cells through successive generations is formidable, (2) possible bacteria where cell poles are either weakly correlated or uncorrelated to aging, and (3) bacteria that reproduce by multiple-fission. This new method provides a useful tool to study the relationship between cell poles and aging in bacteria.

Successful Stem Cell Tranplant Following HLA Identical Sibling Selection Using PGD Combined With HLA Typing In a Case Of CD40 Ligand Deficiency

Pre-Implantation genetic diagnosis and pre implantation Human Leukocyte Antigen (HLA) typing has been successfully used to conceive unaffected HLA identical siblings to provide stem cells for Hematopoietic Stem Cell Transplant (HSCT) (Goussetis, Evgenios, et al. Biology of Blood and Marrow Transplantation 16.3 (2010): 344-349). This technique has been effective in treatment of disease such as Leukemia, Diamond-Blackfan anemia, Thallasemia etc. With improvement of molecular techniques it has been possible to do gene testing from a single cell. We report first case of X-linked Hyper CD40 ligand deficiency where preimplantation genetic diagnosis (PGD) and pre implantation HLA typing were used to achieve successful HSCT from the resulting HLA identical sibling donor. We used dual hematopoietic stem cell sources from cord blood and bone from the nine month old sibling. Currently, patient is 34 months post transplantation doing well with full immunologic reconstitution. Patient was a 14 month old male who presented with a 2 month history of intermittent fever &amp; poor oral intake. He was drooling excessively and tired on appearance. He had also developed mouth sores and later worsening stridor. Chest x-ray revealed right-sided patchy infiltrates. A laryngoscopy was performed that showed supraglottic stenosis. He was transferred to the PICU where he was intubated, ventilated and treated with 10 days of Meropenem. He was later extubated when it was confirmed that there was healing of the supraglottic area. Patient had Leukopenia and received GCSF. Labs also showed decreased level of IgG, IgA and elevated IgM with low titers to tetanus, H flu and diphtheria vaccines. HIV test was negative. His CD40 ligand test revealed deletion of Exon 2 (Figure 1) confirming the diagnosis. Patient's mother was also confirmed to be a carrier for the mutation. He was started on IVIG supplementation and later subcutaneous Ig. When the patient was diagnosed, he had no siblings but his mother was in the first trimester of a pregnancy. HLA typing was done utilizing chorionic villus sampling. Unfortunately, the fetus was found not to be a match and efforts were directed at finding an unrelated donor through multiple donor drives. A well matched unrelated donor was not available after multiple attempts and donor drives for two years. Cord blood transplant and mismatched unrelated donor transplant were considered. Transplants utilizing mismatched unrelated donors for CD40 ligand deficiency are rare and are associated with an increased risk of complications as well as lower overall survival rate. Due to these factors, the parents declined these options for transplant. Parents then decided to pursue PGD with Preimplantation HLA Testing. After PGD and combined HLA testing an unaffected embryo that was a HLA match to the patient was selected after multiple attempts (Figure 2). The mother underwent IVF with the selected embryo and delivered a male sibling. The genetic testing for CD40 ligand deficiency and HLA typing was repeated on the proposed donor baby after birth. The repeat tests confirmed that the proposed donor baby was unaffected and a 10 of 10 HLA match for the patient. The patient was subjected to a conditioning regimen consisting of Busulfan, Cytoxan and GVH prophylaxis with Methotrexate and Tacrolimus. Following the regimen, the patient was infused with stem cell dose consisting of fresh bone marrow (1.86x108 TNC/Kg) harvested from the matched sibling donor and cryopreserved cord blood (0.29x108 TNC/Kg) obtained during the sibling donor's birth. The patient was found to be well engrafted (98% on day 18, 100% at 9 months) post transplant. The CD40L test showed 60% CD40L on T cells after stimulation with ICOS (inducible co-stimulator) increased from 0% pre-transplant. Most patients with hyper CD40 ligand deficiency inherit the causative gene mutation from one of their parents. The average family size in the US is 3.14 (Daphne Lofquist, et al. Households and Families: 2010 Census Briefs).This results in a low probability of finding a sibling donor that is both unaffected and a HLA match. Undergoing multiple pregnancies with low probabilities of conceiving an unaffected HLA matched sibling is stressful, time consuming and ineffective. PGD combined with HLA typing provides an effective way of choosing an embryo that result in a matched HLA donor sibling and subsequent successful stem cell transplant. Disclosures: No relevant conflicts of interest to declare.

Bfr (bendamustine, fludarabine, rituximab) Nonmyeloablative Allogeneic Conditioning: A Novel Regimen Inducing Immunosuppression Without Myelosuppression

BackgroundWe previously reported the use of BFR, with phase 1 escalating daily doses of 70, 90, 110, or 130 mg/m2 of bendamustine daily x 3 days prior to transplantation without dose-limiting toxicity (Khouri et al. ASH 2011, Abstract # 894). In this study, we report results of an expanded phase 2 trial. MethodsBendamustine 130 mg/m2 IV daily on days -5 to - 3 prior to transplantation , together with 30 mg/m2 IV of fludarabine given on the same days. Rituximab was given at a dose of 375 mg/m2 IV on day –13 and 1000 mg/m2 on days -6, +1, and +8, as previously described. Rituximab was omitted in patients with T-cell diseases. Tacrolimus and mini-methotrexate were used for GVHD prophylaxis. In addition, thymoglobulin 1 mg/kg IV was given on days -2, and -1 in patients receiving an unrelated donor transplant. ResultsThe analysis included all 56 patients who received BFR (48 from the expanded phase 2 trial, and 8 from the phase 1 part). Histologies included: mantle cell (n=16), CLL (n=15) (3 with 17p- and 2 Richter's), follicular (n=13), Diffuse large cell (n=9), peripheral T-cell lymphoma (n=3). Median age was 56 (range, 59-70) years. Median prior treatments was 3 (range, 1-7); 7 (12%) had failed a prior autotransplant. At study entry,27 (48%) patients were in CR/CRu, 23 (41%) in PR, and 6 (11%) had refractory disease. Thirty (54%) received their transplants from HLA-compatible siblings and 26 (46%) from unrelated donors. Median number of CD34-positive cells infused was 5.58 x 106/kg. Neutrophil counts recovered to > 0.5 x 109/L a median of 6 days after transplantation (range, 0-16 days). The median number of days on G-CSF was 1.5 days (range, 0-8 days). Thirteen patients (23%) did not require G-CSF as they never experienced an ANC < 0.5 x 109/L. Forty-nine patients (87.5%) did not require platelet transfusions. Platelet counts recovered to > 20 x 109/L in the remaining 7 patients after a median of 11 days (range, 10-19 days). All patients engrafted donor cells. By day 30, median donor myeloid and T-cells were 85% and 97%, respectively. Both increased to 100% by day 90. Recovery of donor cells was similar in both sibling and unrelated donor transplantation. Acute grade 2-4 GvHD and chronic extensive GvHD were observed in 7 (12.5%) and 8 (14%) of patients respectively. Treatment-related mortality at 1-year was 9%. Six patients died: 2 of progression, 2 of infection and 2 of GVHD complications. With a median follow-up time of 12 months (range, 2-37 months), the OS and PFS rates were 89% and 80%, respectively. ConclusionsOur results show that the BFR regimen with bendamustine at a dose level of 130 mg/m2 daily x 3 days, constitutes a well-tolerated nonmyeloablative allogeneic conditioning for lymphoid malignancies. It allows engraftment of both sibling and unrelated donor cells with minimal myelosuppression. This regimen can be used as a platform for allogeneic transplantation in the outpatient setting. Disclosures:Khouri:Cephalon: Research Funding. Off Label Use: Bendamustine in transplantation.

Abstract B14: Deciphering individual roles of p53 and NF1 in suppressing gliomagenesis

Abstract Cancer is difficult to treat because it is a disease caused by multiple genetic alterations. Understanding how individual mutations contribute to pathogenesis and how they cooperate to aid malignancy offers deep insights of tumorigenic mechanisms that will facilitate the design of effective treatment. However, conventional methods, including most mouse cancer models, do not offer the temporal and spatial resolution required to study tumorigenesis in great details, especially because single gene mutations rarely lead to detectable malignancy. To resolve this issue, our lab uses a novel mouse model termed MADM (Mosaic Analysis with Double Markers) to analyze the contribution of individual mutations to aberrant behaviors of tumor cell of origin during pre-transforming stages. Starting with a mouse heterozygous for a TSG, MADM can generate sparse mutant cells that are null for the TSG via Cre-loxP mediated inter-chromosomal mitotic recombination. Concurrently, MADM unambiguously labels mutant cells with GFP and their wild-type sibling cells with RFP, allowing us to precisely analyze behavioral changes of green mutant cells, using their sibling red cells as the internal control. Using MADM to model glioblastoma, the most common and aggressive malignant primary brain tumor, our lab previously demonstrated that oligodendrocyte precursor cell (OPC) is the cell of origin. Using MADM to generate p53 and NF1 double-null OPCs, we found that mutant OPCs dramatically over-expanded at the pre-transforming stage compared to WT sibling cells. Further analysis indicated that mutant OPCs manifest augmented proliferative activities and halted differentiation processes. However, it is still unknown what is the individual contribution of the loss of p53 and NF1 and how their cooperativity leads to malignant transformation. Utilizing MADM, we generated genetic mosaic mice with individual loss of either p53 or NF1 in OPCs and determined the cellular property changes of mutant cells, including proliferative rate, differentiation ability, overall expansion, and malignant transformation. We found that, while the mosaic loss of NF1 alone is sufficient to lead to increased proliferation and impaired differentiation of mutant cells, it is insufficient to lead to malignancy even though mutant cells overwhelm the mouse brain, suggesting that p53 plays critical gatekeeping roles in gliomagenesis. On the other hand, when we examined brains containing p53-null OPCs, we were surprised to find that the loss of p53 alone had no detectable effect on cell number, proliferation and differentiation of mutant cells. We reasoned that the tumor suppressing functions of p53 may only be manifested in an NF1 mutant background, and thus generated a new mosaic model in which all OPCs are NF1-null while green cells are p53 null and red ones are p53 WT. Surprisingly, we still found no differences in cell number, proliferation, or differentiation between green and red cells, suggesting that p53 suppress gliomagenesis through some unknown mechanisms. In summary, our data suggested that NF1 loss leads to glioma initiation by promoting proliferation and inhibiting differentiation of OPCs while p53 loss only contributes to later stage of glioma progressions, and that an effective treatment paradigm would need to address all these mechanisms to halt malignancy. Citation Format: Phillippe Gonzalez, Chong Liu, Hui Zong. Deciphering individual roles of p53 and NF1 in suppressing gliomagenesis. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B14.

P120RasGAP mediates ephrin/Eph-dependent attenuation of FGF/ERK signals during cell fate specification in ascidian embryos

ERK1/2 MAP kinase exhibits a highly dynamic activation pattern in developing embryos, which largely depends on fibroblast growth factor (FGF) signals. In ascidian embryos, FGF-dependent activation of ERK1/2 occurs differentially between sister cells during marginal zone and neural lineage patterning. Selective attenuation of FGF signals by localised ephrin/Eph signals accounts for this differential ERK activation, which controls the binary fate choice of each sibling cell pair. Here, we show that p120 Ras GTPase-activating protein (p120RasGAP) is a crucial mediator of these ephrin/Eph signals. First, inhibition of p120RasGAP has a similar effect to inhibition of ephrin/Eph function during marginal zone and neural patterning. Second, p120RasGAP acts epistatically to ephrin/Eph signals. Third, p120RasGAP physically associates with Eph3 in an ephrin-dependent manner. This study provides the first in vivo evidence that the functional association between Eph and RasGAP controls the spatial extent of FGF-activated ERK.

Single-cell dynamics of the chromosome replication and cell division cycles in mycobacteria

During the bacterial cell cycle, chromosome replication and cell division must be coordinated with overall cell growth in order to maintain the correct ploidy and cell size. The spatial and temporal coordination of these processes in mycobacteria is not understood. Here we use microfluidics and time-lapse fluorescence microscopy to measure the dynamics of cell growth, division and chromosome replication in single cells of Mycobacterium smegmatis. We find that single-cell growth is size-dependent (large cells grow faster than small cells), which implicates a size-control mechanism in cell-size homoeostasis. Asymmetric division of mother cells gives rise to unequally sized sibling cells that grow at different velocities but show no differential sensitivity to antibiotics. Individual cells are restricted to one round of chromosome replication per cell division cycle, although replication usually initiates in the mother cell before cytokinesis and terminates in the daughter cells after cytokinesis. These studies reveal important differences between cell cycle organization in mycobacteria compared with better-studied model organisms.