Normal Reconstitution Research Articles

Impact of carbohydrate-reduced nutrition in septic patients on ICU: study protocol for a prospective randomised controlled trial

IntroductionSepsis is defined as detrimental immune response to an infection. This overwhelming reaction often abolishes a normal reconstitution of the immune cell homeostasis that in turn increases the risk for...

ICAM-1 Deficiency in the Bone Marrow Niche Impairs Quiescence andRepopulation of Hematopoietic Stem Cells.

SummaryThe bone marrow niche plays a critical role in controlling the fate of hematopoietic stem cells (HSCs) by integrating intrinsic and extrinsic signals. However, the molecular events in the HSC niche remain to be investigated. Here, we report that intercellular adhesion molecule-1 (ICAM-1) maintains HSC quiescence and repopulation capacity in the niche. ICAM-1-deficient mice (ICAM-1−/−) displayed significant expansion of phenotypic long-term HSCs with impaired quiescence, as well as favoring myeloid cell expansion. ICAM-1-deficient HSCs presented normal reconstitution capacity during serial transplantation; however, reciprocal transplantation experiments showed that ICAM-1 deficiency in the niche impaired HSC quiescence and repopulation capacity. In addition, ICAM-1 deletion caused failure to retain HSCs in the bone marrow and changed the expression profile of stroma cell-derived factors, possibly representing the mechanism for defective HSCs in ICAM-1−/− mice. Collectively, these observations identify ICAM-1 as a regulator in the bone marrow niche.

Early Outcomes of Vein Grafting for Reconstruction of Brachial Arterial Injuries in Children.

We postulated that, with microsurgical technique, vascular reconstruction with interpositional vein grafts in the pediatric population is safe and results in sustained vascular patency and excellent midterm outcomes. Twenty children with brachial artery injuries were treated with interpositional vein grafting at a tertiary pediatric hospital from 1995 to 2013. Medical records were evaluated for demographic, clinical, and radiographic data. Ten patients were available for longer-term follow-up. Pain at rest, pain with exercise, and temperature intolerance were assessed with visual analog scale (VAS) (range, 0-5). Functional outcomes were assessed using the Pediatric Outcomes Data Collection Instrument (PODCI) and Disabilities of the Arm, Shoulder, and Hand (DASH) questionnaires. Graft patency and flow were characterized via duplex sonography. Peak flow velocity and resistive index (RI) were compared with the contralateral extremity. Median patient age at the time of injury was 7.75 years (range, 4.6-11.5 years) and median follow-up was 1.75 years (range, 0.3-6.3 years). All patients had perfused hands with palpable radial pulses at follow-up. There were no clinically meaningful differences in motion, sensibility, or strength between affected and unaffected limbs. Mean VAS scores for pain at rest, pain with exercise, and temperature intolerance were 0.4, 0.4, and 0.6, respectively. Mean global PODCI and DASH scores were 98.0 and 5.1, respectively. Nine of 10 patients had patent arteries with normal flow patterns. In the patient with graft occlusion, there was collateralization around the elbow with normal reconstitution of the distal vessels. Mean peak flow velocity proximal and distal to the graft were 77.7 cm/s and 66.5 cm/s, respectively. Mean RI of the graft were 0.84 and 0.77, respectively. Brachial artery reconstruction using interpositional vein graft and microsurgical technique is safe and effective and results in excellent functional outcomes in children. Therapeutic V.

Aberrant Phosphorylation of MEF2C Is Dispensable for Hematopoiesis, and Induces Chemotherapy Resistance and Susceptibility to MARK Kinase Inhibition Therapy in Acute Myeloid Leukemia

For patients with acute myeloid leukemia (AML), failure to achieve complete remission after induction therapy or relapse after complete remission represents the major barrier to cure for both children and adults. Although genomics has revealed the mechanisms of pathogenesis of AML, the molecular mechanisms of chemotherapy resistance remain largely unknown. Using high-resolution mass spectrometry proteomics, we identified aberrant phosphorylation of MEF2C S222 in primary chemoresistant human AML specimens, and using capillary nanoimmunoassays, established its prevalence and prognostic significance in a cohort of 40 patients, spanning the major biologic subtypes of human AML, including refractory MLL-rearranged leukemias.MEF2C is a transcription factor required for hematopoietic cell fate determination. We found that expression of mutant S222A MEF2C that cannot be phosphorylated, but not phosphomimetic S222D, led to a leukemia stem cell defect in genetically-engineered MLL-AF9 mouse leukemias in vivo (Figure 1A). Similarly, loss of MEF2C phosphorylation impeded progression of human AML engrafted in immunodeficient mice in vivo. Using transcriptome and BH3 mitochondrial profiling, we found that loss of MEF2C phosphorylation induced intrinsic mitochondrial apoptosis and sensitization to both cytarabine and doxorubicin, due at least in part to the upregulation of pro-apoptotic factors BID and BMF.To determine the function of MEF2C phosphorylation in normal hematopoiesis, we used CRISPR/Cas9 genome engineering to generate transgenic mice with endogenous Mef2cS222A/S222A and S222D/S222D mutations. Both transgenic strains were born at Mendelian ratios, exhibited normal steady-state hematopoiesis, and normal reconstitution in competitive bone marrow transplants. In contrast, Mef2cS222A/S222A hematopoietic progenitor cells, transformed with MLL-AF9 oncogenes exhibited reduced colony forming capacity, as compared to transformed MEF2C wild-type litter-mate controls (Figure 1B). Thus, MEF2C S222 phosphorylation is selectively required for the survival of leukemia cells but is dispensable for normal hematopoietic function, establishing a therapeutic window for targeting MEF2C.To identify kinases that phosphorylate MEF2C and can serve as targets for therapeutic intervention, we screened recombinant serine/threonine kinases and identified the MARK kinase family as the specific enzymes that phosphorylate MEF2C S222. We found that MARK2 and MARK3 are highly overexpressed in the majority of human AML cell lines and primary patient specimens studied. We found that active MARK3, but not its inactive kinase mutants, can trans-activate MEF2C response elements using transcriptional reporter assays in a MEF2C S222-dependent manner. Importantly, MARK kinase inhibition by the ATP competitive inhibitor MRT199665, led to selective apoptosis of MEF2C-expressing human AML cell lines and primary AML patient samples, which conferred enhanced sensitivity to cytarabine (Figure 1C). Thus MEF2C phosphorylation is dispensable for normal hematopoiesis, enhances leukemia cell survival, and causes resistance to chemotherapy and susceptibility to MARK kinase inhibition. These findings establish a paradigm of rational functional therapy of AML chemoresistance. [Display omitted] DisclosuresHe:Foundation Medicine, Inc: Employment, Equity Ownership. Letai:Tetralogic: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding. Gray:Gatekeeper: Equity Ownership; Petra: Consultancy, Equity Ownership; C4: Consultancy, Equity Ownership; Syros: Consultancy, Equity Ownership. Armstrong:Epizyme, Inc: Consultancy.

Investigating The Role Of ARAP3 In The Regulation Of Hematopoietic Stem and Progenitor Cells

ARAP3 is a member of the dual Arf-and-Rho GTPase-activating proteins (GAP) family, functioning specifically to inactivate its substrates Arf6 and RhoA GTPases. ARAP3 is translocated to the plasma membrane after PIP3 binding to the first two of its five PH domains, facilitating its GAP activity in a PI3K-mediated manner. Rho family GTPases are found to play critical roles in many aspects of hematopoietic stem and progenitor cells (HSPCs), such as engraftment and migration, while a role for Arf family GTPases in hematopoiesis is less defined.Previous studies found that either exogenous ARAP3 expression in epithelial cells or RNAi-mediated ARAP3 depletion in endothelial cells disrupts F-actin or lamellipodia formation, respectively, resulting in a cell rounding phenotype and failure to spread. This implies that ARAP3 control of Arf6 and RhoA is tightly regulated, and maintaining precise regulation of ARAP3 levels is crucial to actin organization in the cell. Although ARAP3 was first identified in porcine leukocytes, its function in the hematopoietic system is incompletely understood.Germline deletion of Arap3 results in embryonic lethality due to angiogenic defects. Since endothelial cells are important for the emergence of HSCs during embryonic development, early lethality precludes further studying the role of ARAP3 in definitive hematopoiesis. Therefore, we generated several transgenic mouse models to manipulate ARAP3 in the hematopoietic compartment: (1) Arap3fl/fl;Vav-Cretg conditional knockout mice (CKO) deletes ARAP3 specifically in hematopoietic cells, (2) Arap3fl/fl;VE-Cadherin -Cretg CKO mice selectively deletes ARAP3 in embryonic endothelial cells and thereby hematopoietic cells, and (3) Arap3R302,3A/R302,3A germline knock-in mice (KI/KI) mutates the first PH domain to ablate PI3K-mediated ARAP3 activity in all tissues.We found an almost 100% and 90% excision efficiency in the Vav-Cretg- and VEC-Cretg- mediated deletion of ARAP3 in the bone marrow (BM), respectively. However, the CKO mice appear normal in steady-state hematopoiesis, showing normal peripheral blood (PB) counts and normal distributions of all lineages in the BM. Interestingly, we observed an expansion of the Lin-Scal+cKit+ (LSK) stem and progenitor compartment in the CKO mice. This is due to an increase in the multi-potent progenitor (MPP) fraction, but not the long-term or short-term HSC (LT- or ST-HSC) fractions. Although loss of ARAP3 does not alter the frequency of phenotypically-characterized HSCs, we performed competitive BM transplantation (BMT) studies to investigate the functional impact of ARAP3 deficiency. 500 LSK cells from Arap3 CKO (Arap3fl/fl;Vav-Cretg and Arap3fl/fl;VEC-Cretg) or Arap3fl/fl control littermate donors were transplanted with competitor BM cells into irradiated recipients. We observed similar donor-derived reconstitution and lineage repopulation in the mice transplanted with Arap3fl/fl and Arap3 CKO HSCs. Moreover, Arap3 CKO HSCs show normal reconstitution in secondary transplants.Arap3KI/KI mice are also grossly normal and exhibit an expanded MPP compartment. Importantly, Arap3KI/KI LSKs show impaired reconstitution compared to controls in the competitive BMT assays. Upon secondary and tertiary transplantation, reconstitution in both PB and BM diminished in the Arap3KI/KI groups, in contrast to sustained reconstitution in the control group. Additionally, we observed a marked skewing towards the myeloid lineage in Arap3KI/KI transplanted secondary and tertiary recipients. These data suggest a defect in HSC function in Arap3KI/KI mice. Myeloid-skewed reconstitution also points to the possibility of selection for “myeloid-primed” HSCs and against “balanced” HSCs, as HSCs exhaust during aging or upon serial transplantation.Taken together, our data suggest that ARAP3 plays a non-cell-autonomous role in HSCs by regulating HSC niche cells. Alternatively, the ARAP3 PH domain mutant that is incapable of locating to the plasma membrane in response to PI3K may exert a novel dominant negative function in HSCs. We are investigating mechanistically how ARAP3 controls HSC engraftment and self-renewal to elucidate the potential cell-autonomous and non-cell-autonomous roles of ARAP3 in HSCs. In summary, our studies identify a previously unappreciated role of ARAP3 as a regulator of hematopoiesis and hematopoietic stem and progenitor cell function. Disclosures:No relevant conflicts of interest to declare.

Sparc Is Dispensable for Murine Hematopoiesis, Despite Its Suspected Role in 5q- Myelodysplastic Syndrome

Abstract 4822Hematopoiesis is a complex process where a limited number of stem cells give rise to all mature blood cells. It involves interplay of several factors, many of which are yet to be identified. In a search for novel regulators of hematopoiesis, we chose to study SPARC (Secreted Protein Acidic and Rich in Cysteine, also known as Osteonection and BM40) because it is downregulated upon hematopoietic differentiation (Bruno et al., Mol Cell Biol, 2004) and might therefore play a role in the regulation of hematopoietic stem cells (HSC). SPARC is a matricellular protein that forms a major component of bone and is ubiquitously expressed in a variety of tissues. It is the founding member of a family of SPARC-like proteins. Several publications have indicated an important role for SPARC in hematopoiesis. In particular – knockdown of SPARC in zebrafish embryos resulted in an altered number of circulating blood cells, and a knockout mouse model showed thrombocytopenia and reduced erythroid colony formation.We carried out an in depth phenotypic and functional analysis of the hematopoietic system of SPARC knockout mice; using it as a model to gain insight into the role of SPARC in hematopoiesis. These mice are viable and fertile but show severe osteopenia and age-onset cataract at about six months of age. They also show an altered response to tumour growth and wound healing. We used mice (129SVJ background) (Gilmour et al. EMBO, 1998) that were less than six months old. These mice had normal peripheral blood counts and the bone marrow and spleen showed no alterations in morphology or cellularity. A detailed phenotypic analysis of precursors within the bone marrow showed no significant differences in myelo-erythroid precursors as compared to wild types (n=6). Though in vitro, the precursors showed lower ability to form BFU-E (n=5, p=0.048).In transplantations of lethally irradiated recipient mice, SPARC knockout cells gave rise to multi-lineage long-term reconstitution. Also, when competed with wild type cells, they provided reconstitution as well as their wild type counterparts. When SPARC knockout mice (n=8) were transplanted with wild type cells, there was normal reconstitution, indicating that a SPARC deficient niche can fully support normal hematopoiesis.We also tested if SPARC deficient mice respond differently to hematopoietic stress. We subjected mice (n=7) to sub lethal dose of irradiation and to experimentally induced anemia (n=7) and followed recovery by analyzing peripheral blood counts. In both SPARC knockouts and wild type mice, the blood counts recovered in a similar fashion.In conclusion, we find that SPARC is dispensable for murine hematopoiesis. It is possible that there are compensatory mechanisms involving other members of the SPARC family that ultimately lead to normal hematopoiesis in the murine model.In humans, SPARC maps to the deleted region in 5q MDS and has been reported to be 71 % down regulated in patient samples (Lehmann et al. Leukemia, 2007). It is the most prominent gene that is up regulated in response to lenalidomide, a drug that inhibits the malignant clone (Pellagatti et al. PNAS, 2007). SPARC is thus increasingly speculated to be involved in the pathophysiology of this hematopoetic disease. We analysed the expression levels of SPARC mRNA in the hematopoietic stem/progenitor cell compartment and found high expression levels in the CD34+ fraction of human cord blood cells. In contrast, there is very low level of SPARC expression in all compartments of murine HSCs. Therefore SPARC function may play a more important role in human hematopoiesis than in murine blood cell regulation. Disclosures:No relevant conflicts of interest to declare.

An RNAi Screen Reveals a Pattern of Addiction to Epigenetic Pathways That Distinguishes MLL-AF9 Leukemia From Normal Hematopoietic Cells

AbstractAbstract 65Epigenetic pathways act to control gene expression in a heritable fashion without altering DNA sequence, typically involving the control of chromatin structure. Several lines of evidence implicate the involvement of epigenetics in the pathogenesis of human cancer, however it remains uncertain to what extent manipulating epigenetic pathways can fully rectify malignant cellular states for therapeutic benefit. To systematically explore this issue, we have developed a novel RNAi screening pipeline that can distinguish the epigenetic requirements for normal and malignant hematopoiesis. This was achieved by first constructing a custom shRNA library targeting all known enzymatic complexes that regulate chromatin structure (1100 shRNAs in total). Next, each shRNA was delivered systematically to cells derived from a mouse model of chemotherapy-resistant acute myeloid leukemia driven by the oncogenes MLL-AF9 and NRAS, or to several non-transformed hematopoietic cell lines of different lineages. shRNAs were scored for their capacity to differentially inhibit growth of leukemic cells without influencing growth of non-leukemic cells. Each of the identified genes was then evaluated in vivo for its influence on normal reconstitution of the hematopoietic system following transplantation of shRNA-infected hematopoietic stem and progenitor cells into lethally irradiated recipient mice. Each gene was likewise suppressed in leukemia cells in vivo using both constitutive and conditional RNAi vectors. The net result of the in vivo testing was identification of 6 genes encoding different regulators of chromatin structure whose suppression provides therapeutic benefit in a mouse model of therapy-resistant AML, without significantly influencing the production of normal blood lineages. In support of the accuracy of our screening protocol, one of the identified genes from the screen encodes the protein Menin, a known MLL-AF9 cofactor essential for disease initiation and shown to be dispensable for steady-state hematopoiesis in knockout mice.Two of the identified hits in our screen are the genes Eed and Suz12, which encode two subunits of the PRC2 Polycomb complex (Eed and Suz12), which catalyzes histone H3K27 methylation to suppress gene expression. Inhibiting PRC2 function in MLL-AF9 leukemia cells leads to monocytic differentiation, as revealed by FACS, RT-qPCR, and cell morphology analysis. Microarray experiments coupled with chromatin immunoprecipitation identified a core program of myeloid fate determinants that are suppressed by PRC2 via H3K27 methylation. In addition, we observe that MLL-AF9 directly occupies several Polycomb gene promoters to upregulate their expression in leukemic cells. Our findings highlight an unexpected alliance between the MLL-AF9 oncogene (a Trithorax protein) and PRC2 (a Polycomb complex), which act together to block myeloid differentiation in AML. Our findings also highlight the utility of employing RNAi in vivo to identify novel therapeutic targets in otherwise chemotherapy-resistant disease models. Disclosures:No relevant conflicts of interest to declare.

Tumor Necrosis Factor Alpha Inhibits Olfactory Regeneration in a Transgenic Model of Chronic Rhinosinusitis–Associated Olfactory Loss

Olfactory loss is a debilitating symptom of chronic rhinosinusitis (CRS). Although olfactory sensory neurons (OSNs) are normally regenerated constantly in the olfactory epithelium (OE), a transgenic model of CRS-associated olfactory loss (inducible olfactory inflammation [IOI] mouse) shows that inflammation causes widespread OSN loss without progenitor cell proliferation. In this study, we further examine whether the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) inhibits olfactory regeneration. IOI mice underwent either unilateral bulbectomy or sham surgery and then were induced to express TNF-alpha in the OE for 1 week. After death, the mice were assessed histologically and with bromodeoxyuridine staining to determine the effect of TNF-alpha on olfactory regeneration. In the absence of TNF-alpha, bulbectomy was associated with death of OSNs, followed by robust proliferation of neural progenitors and regrowth of the OE. At 12 days postbulbectomy, OE thickness on the operated side had recovered to >80% of the unoperated side. In mice in which TNF-alpha expression was induced, significantly reduced proliferation was observed, associated with failure of normal reconstitution of OE thickness. The mechanism of olfactory dysfunction in CRS remains incompletely understood. Previous studies with a transgenic mouse model suggested that inflammation inhibits progenitor cell proliferation and olfactory regeneration. Here, the role of the CRS-associated cytokine TNF-alpha was investigated using surgical ablation of the olfactory bulb to stimulate synchronous OSN turnover. We find that TNF-alpha expression prevents normal OE recovery, supporting the role of suppressed olfactory regeneration in the pathophysiology of CRS-associated olfactory loss.

Anti-Vascular Endothelial Growth Factor Treatment in Combination with Chemotherapy Delays Hematopoietic Recovery Due to Decreased Proliferation of Bone Marrow Hematopoietic Progenitor Cells

Targeting of cancer by chemotherapy in combination with anti-vascular endothelial growth factor (VEGF) therapy has demonstrated not only the clinical efficacy but also a higher risk of serious hematologic complications including neutropenia. The purpose of the study was to elucidate the molecular mechanisms responsible for the development of neutropenia during the combination treatment. Mouse model and in vitro studies were undertaken to determine the effect of interference with VEGF signaling by VEGF-specific agents or a multitargeted VEGF receptor (VEGFR) tyrosine kinase inhibitor on proliferation of hematopoietic progenitor cell (HPC) and repopulation of the hematopoietic compartment after myeloablation. The studies demonstrated that blockage of VEGFR1 or VEGFR2 signaling decreased HPC proliferation and impaired repopulation of the hematopoietic compartment after myelosuppression by slowing the progression of HPC through the cell cycle. The combination of cytotoxic drugs and VEGFR tyrosine kinase inhibitor had an additive inhibitory effect and decreased proliferation of HPC significantly stronger than either agent alone. Signaling through both VEGFR1 and VEGFR2 is required for normal reconstitution of the hematopoietic compartment after cytotoxic chemotherapy.

Microenvironmental Cross-Talk of Wnt/Notch Signal in Hematopoietic Stem Cell

Microenvironment in the stem cell niche plays an important role to regulate self-renewal and differentiation of hematopoietic stem cells (HSCs). We previously showed opposing effects of b-catenin activation on HSC depending on target of activation in the bone marrow microenvironment. To further analyze the microenvironmental regulation of HSC by Wnt/b-catenin signal, we examined b-catenin activation mode in the trabecular bone marrows. In-situ immunohistochemistry of bone marrows revealed a compartmentalized enrichment of b-catenin in the spindle shaped, CD45(−) endosteal stroma of bone marrow (SNO cells) compared to CD45(+) hematopoietic cells. Receptors for canonical Wnt signals (Fz1, 2, 7, 8) or co-receptors (LRP5, 6) were also enriched in the CD45(−) mesenchymal stromal cells of bone marrows than hemapoietic cells. Moreover, accumulation of active form b-catenin was selectively observed in the “stimulated” bone marrows that had been irradiated or injected with Wnt 3a conditioned medium (Wnt 3a-CM), but not in the “steady state” bone marrows. To examine the effect of b-catenin activated stroma on HSCs, 5-FU bone marrow cells were co-cultured in-vitro for 5 days and transplanted into irradiated mice. A 3-fold higher expansion of primitive phenotype (Lin-Sca-1+c-kit+) cells were seen after culture without differences in cell cycle progression. Further, CRU analysis of the transplanted co-cultured cells displayed higher numbers of CRUs regenerated in the recipient bone marrows (65 CRU vs. 1155 CRUs for MIG vs. b-catenin MSC group, respectively). To directly test the effect of b-catenin activated stroma on HSC during normal reconstitution process, we compared HSC self-renewal in the marrows reconstituted with control or b-catenin activated MSCs; MIG or b-catenin transduced MSCs were directly injected into femur with bone marrow cells and each group mice marrows were then mixed (1:1) transplanted into secondary recipient mice for competitive CRU assay. A 3-fold higher CRU frequency was seen for the HSCs derived from marrows reconstituted with b-catenin/MSCs, indicating the physiological significance of b-catenin activation for in-vivo reconstitution. We next investigated the underlying mechanism for stromal b-catenin effects on HSCs. Expression analysis of b-catenin transduced or Wnt3a-stimulated MSCs revealed higher levels of notch ligands (jagged-1, dll-1), which was similarly observed in the trabecular endosteum of mice treated with Wnt3a-CM. A microarray-based expression analysis further supported up-regulation of notch ligands in b-catenin transduced MSCs, as evidenced by induction of dlk-1 and microfibrillar glycoprotein-2, a protein facilitating utilization of jagged-1. Importantly, induction of notch down-stream molecules (Hes-1 and Deltex-1) was demonstrated in the hematopoietic cells (Lin-Sca-1+) cells co-cultured on the b-catenin activated MSCs. Furthermore, enhancing effects of b-catenin/MSC for expanding undifferentiated cells were abrogated by treatment of gamma-secretase 2 inhibitors during the co-culture. These results show that b-catenin activated stromal cells activate notch signal in the contacting HSCs and that activated notch signal underlies the observed stimulatory effects of b-catenin activated stroma on HSCs. Taken together, Wnt/β-catenin activated stroma and the cross-talk with HSCs may function as a physiologically regulated microenvironmental cue for HSC self-renewal in the stem cell niche.

Development of mature cartilage constructs using novel three‐dimensional porous scaffolds for enhanced repair of osteochondral defects

In this study, we successfully developed two types of volume-reduced three-dimensional scaffolds, including cushion- and cylinder-shape scaffolds, fabricated from chitosan-based hyaluronic acid hybrid polymer fibers. Using these scaffolds combined with a bioreactor system, we regenerated histologically and mechanically mature cartilage constructs. The final goal of this study was to clarify the ability of this engineered cartilage construct to induce cartilage repair in osteochondral defects. The mature cartilage constructs regenerated with two types of scaffolds were implanted into 5-mm diameter osteochondral defects in the patellar groove of rabbits. At 12 weeks after implantation, the reparative tissues consisted of hyaline-like cartilage with evidence of stable fusion to adjacent native cartilage and normal reconstitution of subchondral bone. The histological score of these tissues significantly outranked the value of untreated tissue. Biomechanically, compression modulus of reparative tissue at 12 weeks postoperatively was comparative to that of normal articular cartilage. Our results indicate that the implantation of constructs with mature cartilage have potential as a better approach for joint resurfacing.

Plasmacytoid Dendritic Cell Reconstitution Following Bone Marrow Transplantation: Subnormal Recovery and Functional Deficit of IFN-α/β Production in Response to Herpes Simplex Virus

Infections with herpesviruses were frequent after bone marrow transplantation (BMT) before the preventive use of antiviral drugs, suggesting a deficit of innate immunity. A retrospective phenotypical and functional study was carried out on 25 patients 1-36 months after allogeneic BMT. Leukocyte counts followed a normal reconstitution, including natural killer (NK) cells and monocytes. Plasmacytoid dendritic cell (PDC) counts increased steadily, although they remained below normal values after 2 years. Most patients produced less interferon- alpha/beta (IFN-alphabeta) in vitro than healthy controls after infection with herpes simplex virus type 1 (HSV-1), whereas they responded normally to Sendai virus (SV). In addition, 6 patients had biologic signs of infection with herpesviruses, confirming a specific immunologic deficit against these viruses. IFN production was not correlated to PDC counts or to the occurrence of graft-versus-host disease (GVHD). Because all patients were under immunosuppressive treatment, we investigated the effect of drugs on IFN production by mononuclear cells. Glucocorticoids and cyclosporine A inhibited IFN production by infected leukocytes, with a predominant action on HSV-1-infected PDC. The inability of transplanted patients to mount an efficient immune response to herpesviruses may be partly related to drug toxicity toward cells of the innate immune system.

Reconstituição imunológica após transplante alogénico de células estaminais.

Patients submitted to allogeneic stem cell transplantation have a prolonged immunodeficiency, extending beyond one year post-transplant. The immune reconstitution after an allogeneic stem cell transplant is dependent upon several factors, such as the age of the recipient, the degree of in vivo or in vitro T-cell depletion, the emergence and eventual treatment of graft versus host disease. While neutrophils and NK cells recover normally within 2 months of transplant, the reconstitution of B- and T-cell immunity is significantly slower. In fact, it is impossible to dissociate the emergence of new B-lymphocytes from thymopoiesis, which appears to be determinant for the normal reconstitution of acquired immunity post-transplant. The serial analysis of T cell counts and its subsets, as well as T-cell function and, more recently, of T-cell receptor excision circles, show a delay in T-cell reconstitution in adult patients, patients with extensive chronic graft versus host disease and in the first 9 months post-transplant in recipients of T-cell depleted grafts. The incidence of opportunistic infections is higher in this population. Therefore, with the aim of reducing post-transplant mortality, the development of new strategies for the acceleration of immune reconstitution is crucial. One possibility is the use of adoptive cellular immunotherapy with donor leukocytes in order to boost the incipient donor's immune system in the receptor. Preliminary studies in animal models also suggest that interleukin-7 and keratinocyte growth factor improve thymopoiesis, thereby stimulating the immune reconstitution post-transplant.

Robust levels of long-term multilineage reconstitution in the absence of stem cell self-replication in W/Wv mice transplanted with purified stem cells.

Isolation of primitive blood stem cells by different methods results in cell populations with distinct biological activities. This study was aimed at resolving differences in the frequency of multilineage reconstituting cells (MRC) and their precursors (pMRC) in cell populations isolated by positive selection for Sca-1 compared to those isolated by negative selection for 15-1.1. Separation of wheat germ agglutinin-positive mouse bone marrow cells into 15-1.1neg or Sca-1+ subsets was performed by flow cytometry. The isolated cells were transplanted into W/Wv or normal irradiated recipient mice and reconstitution was evaluated over time. Sca-1+ cells were less frequent and contained more MRC than 15-1.1neg cells, while pMRC were found mainly among 15-1.1neg cells. MRC activity was exclusively contained within the Sca-1+ subpopulation of 15-1.1neg cells, but marrow from 7 robustly engrafted W/Wv mice did not contain donor-derived MRC, indicating that 15-1.1negSca-1+ cells contain low numbers of pMRC. Functional differences between 15-1.1neg and Sca-1+ cells were further confirmed by reverse transcriptase (RT)-PCR gene expression analysis. Early hematopoiesis-specific transcription factors (Scl, Gata-2, and Gata-1) were amplified from cDNA prepared from Sca-1+ but not 15-1.1neg cells. This study indicates that cell populations isolated as Sca-1+ are functionally distinct from those isolated as 15-1.1neg in that few pMRC are included among Sca-1+ cells and that MRC and pMRC are two distinct and separable cell populations.

Heterogeneous activation of the Fanconi anemia pathway by patient-derived FANCA mutants

Fanconi anemia (FA) is an autosomal recessive disorder of hematopoiesis characterized by hypersensitivity to DNA crosslinkers such as mitomycin C (MMC). There is growing evidence for a model of the FA pathway, wherein a nuclear multiprotein complex of five FA proteins (FANCA, C, E, F and G) regulates activation of FANCD2 into a monoubiquitinated form, which, collaborating with the BRCA1 machinery, affects cellular response to DNA damage. However, the role of the FA pathway in defective DNA damage response caused by various mutant forms of FA proteins has not been fully assessed. In the present study, 21 patient-derived FANCA mutants with a missense or a small in-frame deletion were expressed in FANCA-deficient fibroblasts and examined for complementation of MMC sensitivity and for reconstitution of the FA pathway: FANCA phosphorylation, interaction with FANCC, FANCF and FANCG and nuclear localization and FANCD2 monoubiquitination. The altered FANCA proteins complemented MMC sensitivity at different grades: five proteins (group I) behaved like wild-type FANCA, whereas the other proteins were either mildly (group II, n=4) or severely (group III, n=12) impaired. Group I proteins showed an apparently normal reconstitution of the FA pathway, thus they may be pathogenic by reducing endogenous expression or possibly benign polymorphisms. Reconstitution of the FA pathway by group II and III mutants closely correlated with cellular sensitivity to MMC. The different activation of the FA pathway may partly account for the phenotypic variation seen in FA patients.

Elucidation of a downstream boundary of the 3′ IgH regulatory region

Class switch recombination (CSR) changes the immunoglobulin heavy chain (IgH) constant region gene (C H) in B cells from IgM to IgG, IgA, or IgE, without modifying the variable region gene segment. This process requires transcription through switch (S) regions located upstream of the C H genes targeted for CSR, a process that relies on the activity of an uncharacterized regulatory region at the 3′ end of the C H locus (3′ IgH RR) that has been implicated via the effects of pgk- neo cassettes inserted into the locus. The 30 kb region just downstream of the most 3′ C H gene (Ca) contains four known enhancer elements including HS3a, HS1,2, HS3b, and HS4. Replacement of either of the proximal two enhancer elements (HS3a or HS1,2) with a pgk- neo gene cassette disrupted germline transcription of and CSR to most C H genes. However, replacement of either of the enhancers with a loxP sequence had no effect on CSR indicating that these elements are not critical for CSR. Insertion of a pgk- neo cassette at various sites within the C H locus inhibited CSR to upstream, but not downstream C H genes, supporting the notion that the pgk- neo cassette insertion into the locus short-circuits the ability of the 3′ RR to facilitate CSR of dependent C H genes upstream of the insertion. These analyses also indicated that the key elements of the 3′ IgH RR were downstream from HS1,2. In this study, we have sought to localize the 3′ IgH RR by defining its 3′ boundary. For this purpose, a pgk- neo gene cassette was targeted 2 kb downstream of the HS4 element in ES cells that had normal ability to undergo CSR. We then employed Rag-2 deficient blastocyst complementation to generate chimeric mice that harbored B cells homozygous for this mutation. Such chimeras exhibited normal reconstitution of the splenic compartment and had normal serum immunoglobulin levels. Upon in vitro activation, transcription from the pgk- neo cassette was induced in B cells, however, CSR to all measured IgH isotypes occurred at normal levels. These findings, coupled with previous pgk- neo insertion studies, suggest that key elements of the 3′ IgH RR lie within a 17 kb region between HS1,2 and 2 kb downstream of HS4.

Mice reconstituted with DNA polymerase beta-deficient fetal liver cells are able to mount a T cell-dependent immune response and mutate their Ig genes normally.

The ubiquitously expressed, error-prone DNA polymerase beta (polbeta) plays a role in base excision repair, and the involvement of this molecule in the nonhomologous end joining (NHEJ) process of DNA repair has recently been demonstrated in yeast. Polbeta-deficient mice are not viable, and studies on conditional mutants revealed a competitive disadvantage of polbeta(-/-) vs. wild-type cells. We show here that polbeta-deficient mice survive up to day 18.5 postcoitum, but die perinatally; a circumstance that allowed the investigation of a potential role of polbeta in lymphocyte development by transfer of fetal liver cells (FLC) derived from polbeta(-/-) embryos into lethally irradiated hosts. FLC transfers using mutant cells lead to an almost normal reconstitution of the lymphocyte compartment, indicating that polbeta-deficiency does not prevent V(D)J recombination, which is known to employ factors of the NHEJ pathway. Mice reconstituted with polbeta(-/-) FLC mount a normal T cell-dependent immune response against the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP). Moreover, germinal center B cells from NP-immunized reconstituted mice show normal levels and patterns of somatic point mutations in their rearranged antibody genes, demonstrating that polbeta is not critically involved in somatic hypermutation.

Bone Marrow Transplantation in Patients with Fanconi Anemia: Experience with Cyclophosphamide and Total Body Irradiation Conditioning Regimen

Eleven patients with Fanconi anemia (FA) underwent bone marrow transplantation (BMT) between March 1985 and May 1990 in a single institution. Ten patients received bone marrow from healthy full human leukocyte antigen (HLA) matched siblings and one patient from her father (one antigen mismatch). Ten patients were conditioned with cyclophosphamide (Cy) at a dose of 5 mg/kg per day for 4 days followed by total body irradiation (TBI) for a total of 600 cGy over 3 days. Six of the 11 patients are alive and have normal reconstitution of their bone marrow. Median follow-up was 72 months (range 42–84). Three of the 10 patients who received Cy and TBI (two HLA compatible, one antigen mismatch) had graft failure. Five patients developed at least grade III acute graft-versus-host disease (GVHD). The rates of graft failure and GVHD are, however, still significantly high. Modification of the conditioning regimen and GVHD prophylaxis is needed to improve the outcome.

Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate.

The noncatalytic beta-subunit is responsible for the latency of casein kinase 2 (CK2) activity toward calmodulin. Twenty-one mutants of the beta-subunit bearing either deletions or Ala substitutions for charged residues in the 5-6, 55-70, and 171-178 sequences have been assayed for their ability to substitute for wild-type beta-subunit as a suppressor of activity toward calmodulin. The only mutations that reduced the ability of the beta-subunit to suppress calmodulin phosphorylation activity, though being compatible with normal reconstitution of CK2 holoenzyme, were those affecting Asp55, Glu57 and the whole triplet Glu59-Asp-Glu61. The activity of CK2 holoenzyme, either native or reconstituted, toward calmodulin can be elicited by a variety of polybasic effectors, including polylysine, polyarginine, salmine, and histones H4, H3, and, to a lesser extent, H2a and H2b. Histone H1 and polyamines are conversely ineffective. The latent "calmodulin kinase" activity of CK2 can also be specifically unmasked by a peptide (alpha[66-86]) reproducing a basic insert of the catalytic subunit. This effect is reversed by equimolar addition of a peptide (beta[55-71]) including the 55-64 acidic stretch of the beta-subunit. Comparable polylysine stimulation was observed with the holoenzymes reconstituted with either beta wt or the beta mutants capable of assembling with the alpha-subunit, with the notable exception of those bearing Ala substitutions for acidic residues at positions 55, 57, and 59-61. These were nearly insensitive to 42 nM polylysine, which conversely promotes a more than 10-fold increase of calmodulin phosphorylation with wild-type beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone marrow transplantation with interleukin-2-activated bone marrow followed by interleukin-2 therapy for acute myeloid leukemia in mice

We have investigated approaches to induce graft-versus-leukemia (GVL) effect in autologous bone marrow transplantation (ABMT) without graft- versus-host disease to improve survival and cure in leukemia. The present study shows that bone marrow transplantation (BMT) using syngeneic bone marrow activated with interleukin-2 (ABM) for 24 hours in vitro, followed by interleukin-2 (IL-2) therapy, was superior to BMT with fresh, syngeneic bone marrow (FBM) in terms of survival and cure in mice with acute myeloid leukemia (P less than .001) and led to normal hematopoietic reconstitution. Addition of IL-2 therapy after BMT with FBM did not improve the results over BMT with FBM alone (P = .98). These results suggest that the GVL effect of ABMT can be enhanced by using ABM for BMT followed by IL-2 therapy without compromising engraftment.