scholarly journals Murine transfer of a human gene variant associated with exceptional longevity displays senolytic effects both in immune compartment and endothelium of aged mice

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
E Ciaglia ◽  
F Montella ◽  
M Malavolta ◽  
A Carrizzo ◽  
V Lopardo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Human Gene ◽  
Control Group ◽  
Lymphoid Tissues ◽  
Funding Source ◽  
M2 Macrophages ◽  
Private Company ◽  
Hematopoietic Stem ◽  
Exceptional Longevity ◽  
Age Related

Abstract   The persistence and accumulation of senescent cells has been shown to potentially play a role in the pathophysiology of age-related cardiovascular diseases. Indeed with time, a decline in immune efficacy, termed immunesenescence, and a deleterious secretory phenotype of senescent cells (SASP) generate that inflammatory background mainly mediating the elderly cardiovascular phenotype. Long Living Individuals (LLI) which delay aging, represent a model of positive biology and an exceptional resource to study and find a way to improve general public health. Previous studies from our group have shown that a human gene associated with exceptional longevity (LAV-BPIFB4) was able to block the atherosclerotic process in ApoE−/− mice by conferring the animals with a pro-resolving M2 macrophages profile. Furthermore, LAV-BPIFB4 promotes the recruitment of hematopoietic stem cells, reparative vascularization and frailty reduction. Here we sought to underpinn the role of LAV-BPIFB4 in counteracting the age-related remodeling of the immune responses. The effects of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer on the immune dynamics in old mice have been investigated by an extensive flow cytometric approach in lymphoid tissues (bone marrow, spleen and peripheral blood). C57BL/6J mice were assigned to two age-matched experimental groups: a treatment group (AAV-LAV-BPIFB4; N=6 mice, aged 18–23 months and a control group (AAV-GFP; N=6 mice, aged 18–23 months. 30th and 60th day since the beginning of the infection, SA-beta Gal substrate has been used to identify CD45+ senescent cells in freshly isolated PBMC, splenocytes, bone marrow (BM)-derived cells. As expected, we monitored an increase in SA-betaGal activity in blood. This increase is most significant in CD11b+ myeloid cells, whithout affecting neither CD3+T neither NK1.1+Natural Killer (NK) cell compartment. Notably 30 days AAV-LAV-BPIFB4 infection and to a more the 60 days-treatment, resulted in a significant decrease in senescent pool of peripheral immune cells and a concomitant enrichment of senescent cells in spleen. Concomitantly, aorta from AAV-LAV treated mice showed significant reduction in SA-beta Gal positive area. Furthermore a LAV-BPIFB4 induction of pro-resolving M2 macrophages compared to control group was documented in the main haemocateretic organ. As consequence the senolytic effect of LAV-BPIFB4 gene-therapy well correlated with the rescue of proliferative index of splenocytes upon PHA stimulation, and their functional protective response to lipopolysaccharide (LPS) in term of IL-6 and TNF-alpha secretion. The restoration of a protective and balanced immune response finally reflected the reduction of senescent phenopype acquired by mouse aortic endothelial cells during the aging process in vivo. A better underpinning of the senolytic action of LAV-BPIFB4 may offer a valuable therapeutic tool to reverse aging phenotype causing most of cardiac diseases Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Cariplo Foundation (n.2016-0874) to AAP and CV

scholarly journals Silent cerebral embolisation during TAVI: evolution, predictors and neurocognitive effects

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
M De Carlo ◽  
R Liga ◽  
G.M Migaleddu ◽  
M Scatturin ◽  
C Spaccarotella ◽  
...  
Keyword(s):  
White Matter ◽  
Median Number ◽  
Funding Source ◽  
Private Company ◽  
Diffuse White Matter ◽  
Transcatheter Aortic Valve ◽  
Age Related ◽  
Cerebral Mri ◽  
Neurocognitive Evaluation

Abstract Background Most patients undergoing transcatheter aortic valve implantation (TAVI) develop silent cerebral ischemic lesions (SCIL) detectable at magnetic resonance imaging (MRI). The natural history and clinical relevance of SCIL are not well established. We aimed to assess the characteristics, predictors, evolution, and neurocognitive effects of SCIL. Methods Cerebral MRI was performed within 7 days before TAVI to assess baseline status and age-related white matter changes (ARWMC) score. MRI was repeated postoperatively to assess the occurrence, location, number and dimensions of SCIL. Patients developing SCIL underwent a third MRI at 3–5 months follow-up. A neurocognitive evaluation was performed before TAVI, at discharge and at 3-month follow-up. Results Of the 117 patients enrolled, 96 underwent a postprocedural MRI; SCIL were observed in 76% of patients, distributed in all vascular territories, with a median number of 2 lesions, median diameter 4.5 mm, and median total volume 140 mm3. Independent predictors of SCIL occurrence were a higher baseline ARWMC score and the use of self-expanding or mechanically-expanded bioprostheses. Among 47 patients who underwent follow-up MRI, only 26.7% of postprocedural SCIL evolved into a gliotic scar. SCIL occurrence was associated with a more pronounced transient neurocognitive decline early after TAVI and with a lower recovery at follow-up. Conclusions SCIL occur in the vast majority of patients undergoing TAVR and are predicted by a more diffuse white matter damage at baseline and by the use of non-balloon-expandable prostheses. Although most SCIL disappear within months, their occurrence has a limited but significant impact on neurocognitive function. Figure 1 Funding Acknowledgement Type of funding source: Private company. Main funding source(s): unrestricted grants from Edwards Lifesciences SA, Nyon, Switzerland, and from Medtronic Italia SpA, Milan, Italy

scholarly journals Metabolic changes following transcatheter bariatric embolotherapy for weight loss in obesity: secondary outcomes from a prospective RCT

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
M Fried ◽  
V.Y Reddy ◽  
P Neuzil ◽  
R Rosen ◽  
P Sramkova ◽  
...  
Keyword(s):  
Weight Loss ◽  
Metabolic Changes ◽  
Exclusion Criterion ◽  
Control Group ◽  
Oral Glucose ◽  
Diabetic Patients ◽  
Funding Source ◽  
Post Intervention ◽  
Private Company ◽  
C Peptide

Abstract Background/Introduction Obesity and its comorbid conditions (i.e. type II diabetes mellitus, atrial fibrillation, coronary artery disease, hypertension, etc...) is a growing burden globally, however, the current treatments (i.e. bariatric surgery, intragasrtic balloons and/or pharmaceutical therapy) pose substantial risks or are contraindicated for various populations. Transcatheter bariatric embolotherapy of left gastric artery by reducing “hunger” hormones from the gastric fundus is a procedure for weight loss that has been growing in prominence over the last several years, however, to date no randomized-controlled trial has been conducted until our study. We studied TBE in a double-blind, sham procedure, first in human RCT of patients (pts) with obesity. Purpose The purpose of this study was to assess the safety and efficacy of TBE for weight loss in obese patients as well as to evaluate metabolic changes. Methods After IV propofol, eligible pts (age 21–60; BMI 35–50 kg/m2) were randomized 1:1 to Sham (skin nick & 1 hr wait) or TBE. All pts received Lifestyle Therapy (behavioral and diet education). Study staff following the pts were also blinded to treatment. Blood samples for gastrointestinal hormones were collected in EDTA tubes containing a protease inhibitor cocktail and frozen per local laboratory standards. All collected samples were assessed together in two batches at the end of the study. The hormones analyzed included ghrelin, GIP, GLP-1, Visfatin, resistin, PAI-1 (total), Leptin, and C-Peptide. An Oral Glucose Tolerance Test (OGTT) and a diabetes assay was performed at baseline and at 6- and 12-months post-intervention. Note, while diabetes was an exclusion criterion for this study, pre-diabetes was not. Results 44 pts were enrolled, of which 40 pts were randomized equally to the groups, with no major complications in either group. TBE demonstrated superior weight loss over the control group at 6- and 12-months post-intervention in both intention-to-treat and per-protocol analyses. At 6 and 12 months, the TBE group demonstrated a clinically meaningful decrease in glucose 1-hour post-fasting by OGTT. GIP levels in the TBE group increased at a mean of 21%, indicative of an improvement in pre-diabetic milieu. Circulating plasma visfatin levels decreased 20% at 6 months and 26% at 12 months in the TBE group indicating a decrease in body fat. C-Peptide levels were noticeably increased in the TBE group at 6 months possibly indicating improvements in insulin sensitivity and beta-cell function. Conclusion(s) TBE is safe and results in clinically significant weight loss and demonstrated a positive effect on glucose homeostasis in pre-diabetic patients. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Endobar Solutions, LLC

Study on mechanism of Tim-3 on immune escape in benzene-induced acute myeloid leukemia

2020 ◽  
Author(s):  
qiong Ning ◽  
xiangxin li ◽  
Xiangdong Jian ◽  
Xiaopeng He
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Immune Escape ◽  
Serum Levels ◽  
Control Group ◽  
M2 Macrophages ◽  
Experimental Group ◽  
Group Serum ◽  
Acute Myeloid

Abstract To study the mechanism of Tim-3 on immune escape in benzene-induced acute myeloid leukemia (AML), to provide potential targets of clinical monitoring and intervention of hematological toxicity in benzene-induced AML . C3H/He mice were randomly divided into control group and experimental group. Serum levels of IL-12 in the experimental group were significantly lower than that in the control group. Serum levels of TGF-β1 in the experimental group were significantly higher than that in the control group( p <0.05). The proportion of Tim-3 positive CD14 + monocytes of bone marrow and spleen in the experimental group were both significantly higher than that in the control group ( p <0.05) by Flow cytometry (FCM). Compared with the control group, the expression of Tim-3 on (M1+M2) macrophages of bone marrow in the experimental group significantly increased by immunofluorescence assay. The expression of type M2 macrophages in (M1+M2) macrophages of bone marrow and spleen tissues in the experimental group were both higher than that in the control group. The expression levels of p-PI3K, p-AKT and p-mTOR in the experimental group were all significantly higher than that in the control group. Tim-3 was highly expressed in macrophages in benzene-induced AML. It promoted the activation of PI3K/AKT/mTOR signaling pathway, stimulated the secretion of anti-inflammatory cytokines, and inhibited the secretion of pro-inflammatory cytokines. High expression of Tim-3 changed the phenotype and function of macrophages by promoting the macrophages polarization, thus inducing negative immune response in the tumor microenvironment and tumor immune escape.

scholarly journals Inflammatory bone marrow microenvironment

Hematology ◽  
2019 ◽  
Vol 2019 (1) ◽  
pp. 294-302 ◽  
Author(s):  
Nils B. Leimkühler ◽  
Rebekka K. Schneider
Keyword(s):  
Bone Marrow ◽  
Defense Mechanism ◽  
Clonal Selection ◽  
Low Grade ◽  
Hematopoietic Stem ◽  
Bm Niche ◽  
Age Related ◽  
Stem And Progenitor Cells ◽  
Potentially Malignant ◽  
Stress Signals

Abstract Self-renewing hematopoietic stem cells and their progeny, lineage-specific downstream progenitors, maintain steady-state hematopoiesis in the bone marrow (BM). Accumulating evidence over the last few years indicates that not only primitive hematopoietic stem and progenitor cells (HSPCs), but also cells defining the microenvironment of the BM (BM niche), sense hematopoietic stress signals. They respond by directing and orchestrating hematopoiesis via not only cell-intrinsic but also cell-extrinsic mechanisms. Inflammation has many beneficial roles by activating the immune system in tissue repair and as a defense mechanism. However, chronic inflammation can have detrimental effects by stressing HSPCs, leading to cell (DNA) damage resulting in BM failure or even to leukemia. Emerging data have demonstrated that the BM microenvironment plays a significant role in the pathogenesis of hematopoietic malignancies, in particular, through disrupted inflammatory signaling, specifically in niche (microenvironmental) cells. Clonal selection in the context of microenvironmental alterations can occur in the context of toxic insults (eg, chemotherapy), not only aging but also inflammation. In this review, we summarize mechanisms that lead to an inflammatory BM microenvironment and discuss how this affects normal hematopoiesis. We pay particular attention to the process of aging, which is known to involve low-grade inflammation and is also associated with age-related clonal hematopoiesis and potentially malignant transformation.

scholarly journals Expression of human adenosine deaminase in mice after transplantation of genetically-modified bone marrow

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1733-1741 ◽  
Author(s):  
M Kaleko ◽  
JV Garcia ◽  
WR Osborne ◽  
AD Miller
Keyword(s):  
Bone Marrow ◽  
Adenosine Deaminase ◽  
Dna Sequences ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Helper Virus ◽  
Lymphoid Tissues ◽  
Hematopoietic Stem ◽  
Wide Range

Abstract A high titer retroviral vector was used to transfer a human adenosine deaminase (h-ADA) cDNA into murine bone marrow cells in vitro. The h- ADA cDNA was linked to the retroviral promoter, and the vector also contained a neomycin phosphotransferase gene as a selectable marker. Infected marrow was transplanted into syngeneic W/Wv recipients, and h- ADA expression was monitored for 5.5 months. Several weeks after transplantation, h-ADA was detected in the erythrocytes of all nine recipients, eight of which expressed levels equal to the endogenous enzyme. This level of expression persisted in two of six surviving mice, while expression in three others stabilized at lower, but readily detectable, levels. Only one mouse had no detectable h-ADA after 5.5 months. Vector DNA sequences with common integration sites were found in hematopoietic and lymphoid tissues of the mice at 5.5 months, providing evidence that hematopoietic stem cells had been infected. Furthermore, all mice transplanted with marrow that had been selected in G418 before infusion had multiple vector copies per genome. While this category included the two highest h-ADA expressors, it also included the negative mouse. Thus, multiple copies of the vector were not sufficient to guarantee long-term h-ADA expression. Mice were monitored for “helper virus” infections with an assay designed to detect a wide range of replication-competent retroviruses, including those endogenous to the mouse genome. No helper virus was detected in the two highest h-ADA expressors, ruling out helper-assisted vector spread as a cause of the high h-ADA expression. These results help provide a foundation for the development of somatic gene therapy techniques to be used in the treatment of human disease.

Grb10 Mediated Akt Activation Is Required for Induction of CML Like Myeloproliferative Disease in Mice by BCR-ABL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1012-1012
Author(s):  
Corinna Albers ◽  
Anna L. Illert ◽  
Cornelius Miething ◽  
Christian Peschel ◽  
Justus Duyster
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinases ◽  
Chromosomal Translocation ◽  
Control Group ◽  
Myeloproliferative Disease ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Vitro Analysis ◽  
The Impact

Abstract Chronic myelogenous leukaemia (CML) results from the neoplastic transformation of hematopoietic stem cells (HSC) and is characterized by a chromosomal translocation t(9;22)(q34;q11). This aberration leads to the expression of the oncogenic tyrosine kinase BCR-ABL, which mediates signals for proliferation, transformation and anti-apoptosis via various signalling pathways. Grb10, a member of the growth factor bound proteins, is known to bind activated tyrosine kinases like BCR-ABL and might be involved in the activation of the Akt signalling pathway. Here we report the impact of Grb10 for BCR-ABL mediated transformation. We exerted a siRNA based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both a Grb10 microRNA and the BCR-ABL oncogene on a single construct. This approach ensured knockdowns of more than 90% in every BCR-ABL transformed cell. Methylcellulose assays demonstrated that bone marrow coexpressing Grb10 microRNA and BCR-ABL had a 4-fold decreased colony forming ability compared to control cells. We then transduced bone marrow (BM) with retrovirus coexpressing Grb10 microRNA and p185 BCR-ABL and transplanted lethally irradiated recipient Balb/C mice. The onset and progression of leukaemia was significantly delayed in mice transplanted with Grb10 microRNA and BCR-ABL compared with the BCR-ABL transduced control microRNA group. However, we were not able to completely avoid the development of leukaemia by Grb10 knockdown. Mice transplanted with the Grb10 knockdown construct showed a delayed lymphoblastic disease, positive for B220, whereas the control group developed a rapid myeloproliferative disease, characterized by CD11b and Gr-1. In vitro analysis of BaF/3 and 32D cells showed that Grb10 knockdown in combination with BCR-ABL expression leads to a reduced phosphorylation of Akt. Taken together our data demonstrate that Grb10 is required for the development of a myeloproliferative disease by BCR-ABL in mice. Hereby, Grb10 seems to be critical for the BCR-ABL induced activation of the Akt pathway. In addition, this study describes a novel approach to express an oncogene and a microRNA using a single retroviral construct. This tool can be used to systematically screen for drugable signalling targets involved in oncogenesis.

Limited Regeneration but Functional Preservation of Hematopoietic Stem Cells in the Mice Developing Leukemia via Constitutive Expression of Notch1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 204-204 ◽  
Author(s):  
Xiaoxia Hu ◽  
Hongmei Shen ◽  
Hui Yu ◽  
Feng Xu ◽  
Jianmin Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Molecular Mechanisms ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Cells ◽  
Constitutive Expression ◽  
Hematopoietic Progenitor Cell ◽  
Control Group ◽  
Extrinsic Factors ◽  
Hematopoietic Stem ◽  
The Impact

Abstract Leukemia development is a complex process involving both intrinsic and extrinsic factors. While many environmental factors have been studied, the impact of leukemic environment on normal hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) has not been definitively investigated. In this study, we have formally addressed this important issue by examining the potential functional alterations of HSC and HPC in the mice bearing Notch1-induced T acute lymphoblastic leukemia (T-ALL). The MSCV retrovirus vector containing cDNA encoding oncogenic intracellular domain of Notch1 (ICN1) pseudotyped with VSV-G was used to infect Lin−Sca-1+ cells in order to induce leukemic development. Normal hematopoietic cells from the B6.SJL strain (CD45.1+) were co-transplanted with Notch1 transduced Lin−Sca-1+ cells (CD45.2+) into lethally irradiated recipients. In this robust leukemia model with 100% penetrance, the normal hematopoietic cell compartment marked by CD45.1 in the leukemic marrow was sorted for phenotypic analyses and functional assays at different time points. Same numbers of the normal hematopoietic cells without Notch1-transduced cells were transplanted into the irradiated recipients as controls. As expected, progressive hematopoietic suppression was observed at both HSC and HPC levels in the leukemic mice. The frequency of HSC enriched population (Lin−c-Kit+Sca-1+, LKS) in the leukemic group was 7 times lower than that in the control at the 4th week of leukemogensis. When normalized to the bone marrow cellularity, the absolute yield of each population was 246 times lower in the leukemic group than that in the control group. These data were highly consistent with significantly lower yields of colony forming unit (CFU) and cobblestone area forming cell (CAFC). To measure the long-term engraftment of HSCs from leukemic environment, we performed the competitive bone marrow transplantation (cBMT), in which equal numbers of CD45.1+ cells isolated from leukemic or control mice and competitor cells (CD45.1/.2) at the 2nd week of leukemogenesis were co-transplanted into lethally irradiated C57BL/6J recipients. Unexpectedly, the multilineage engraftment of the hematopoietic cells isolated from the leukemic mice was 3 times more than that of the control group. Moreover, HSCs from the leukemic environment remained functional in serial transplant recipients. Finally, to explore the underlying molecular mechanisms for the enhanced function of normal HSC in the cBMT model, we examined a number of cell cycle and self-renewal regulators in HSC and HPC from leukemic marrow and control group at the time of harvest prior to transplantation by qRT-PCR. There was a significant decrease in p18 expression when compared with the control, whereas p21 expression was significantly increased. Notch1, Gfi1 and c-myc signalings were also elevated in the HSCs from leukemic environment. In summary, our current work provides the first definitive evidence for the reversible inhibition of normal HSC growth by the leukemic environment, thereby having important implications for HSC transplantation as well as leukemogenesis.

Modeling Anemia of Aging in Inbred Mouse Strains

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3444-3444
Author(s):  
Luanne L. Peters ◽  
Shirng-wern Tsaih ◽  
Rong Yuan
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Kidney Function ◽  
Inbred Mouse ◽  
Mouse Strains ◽  
Inbred Mouse Strains ◽  
Hematopoietic Stem ◽  
Age Related ◽  
Mouse Phenome Database

Abstract Anemia of aging is now recognized as a significant medical problem. The National Health and Nutrition Examination Survey (NHANES III) revealed a steady increase in anemia in both males and females after the age of 50. Based upon the WHO definition of anemia (<13 g/dL hemoglobin (Hgb) in men; <12 g/dL in women), ~10% of the community dwelling population ≥ 65 years of age are anemic. Underlying causes fall into three broad groups, each representing ~1/3 of cases: nutritional deficits/blood loss; inflammation, kidney disease and myelodysplasia; and unexplained anemia. Although anemia of aging is usually mild, it is no longer considered a normal part of aging. It is associated with poor health and increased vulnerability to adverse outcomes in a multitude of circumstances, placing an enormous burden on the healthcare system that will only grow as the population continues to age. As part of The Jackson Laboratory Aging Center (http://agingmice.jax.org/), we are performing an extensive phenotypic analysis of multiple traits related to aging in 32 inbred mouse strains. All data are, or will be upon completion, publicly available via the Mouse Phenome Database (MPD, www.jax.org/phenome). Complete blood counts were obtained at 6, 12, 18, and 24 months of age in 30 strains. Two-way ANOVA reveals that both strain and age significantly impact Hgb in mice. A highly significant strain-by-age interaction is also seen. Substantial inter-strain and within strain sex variability in the decline in Hgb levels with age is seen among the strains analyzed, suggesting genetic influences. Significant declines in Hgb levels in females at 18 and/or 24 months vs. 6 months occurred in 21 of the 30 strains and, in males, 17 strains. Haplotype association mapping (HAM) using a dense SNP panel identified multiple distinct, age-related loci influencing Hgb levels. For example, a locus on chromosome (Chr) 13 significantly associated with Hgb levels at 12 months of age in males was not detected even at the suggestive level at 18 months of age where two new highly significant loci emerged (Chrs 14, 17). Only two strains show a statistically significant increase in percent circulating reticulocytes with age, indicative of a proliferative anemia. Failure of a significant reticulocyte response in all other strains suggests that an age-related compromise in bone marrow function (hematopoiesis-restricted anemia) predominates in aged, anemic mice. The ratio of urinary albumin to creatinine (ACR) is commonly used as an indicator of kidney damage in mice. In females, the ACR is stable and does not rise significantly with age in the majority of strains, suggesting that declining kidney function is not a major cause of anemia of aging in female inbred mice. Significant increases in IL-6 and TNFα are seen in strains 129SvImJ, C3H/HeJ, and DBA/2J, suggesting a pro-inflammatory state. From this preliminary analysis of a large ongoing project, we can conclude: Hgb levels in mice vary significantly by strain and sex, and decline significantly with age in many strains. Other baseline hematological traits (e.g., red blood cell counts, platelet counts) likewise vary by strain, age and sex. These data are available via the Mouse Phenome Database (project Peters4). The anemia of aging seen in most strains correlates most closely with restricted hematopoiesis, as indicated by the failure of the reticulocyte count to increase in response to declining Hgb levels. There is growing evidence that decrements in hematopoietic stem cell number and function play a role in the aging process in humans. Notably, hematopoietic stem cell numbers and bone marrow cellularity data will be available on the MPD as these analyses are completed. HAM analysis suggests that distinct age-related loci influence Hgb levels in mice. In a small subset of strains, anemia of aging may reflect declining kidney function, as occurs in humans. Preliminary data suggests an increase in cytokine levels in some strains, again mimicking the aging human population. Increased IL-6 levels as a cause of anemia of aging is of particular interest due to its inhibition of hepcidin and thus iron availability. Overall, the data indicate that anemia of aging occurs in mice and models that seen in elderly human populations. Additional data including iron levels, T4, BUN, and more on aging inbred mouse strains will be posted to the MPD in the near future.

Safe and Efficient Expansion of Human HSC with Sendai Virus Vector Expressing HoxB4 in Fetal Sheep.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 693-693
Author(s):  
Shigeo Masuda ◽  
Tomoyuki Abe ◽  
Makoto Inoue ◽  
Mamoru Hasegawa ◽  
Satoshi Hayashi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Insertional Mutagenesis ◽  
Sendai Virus ◽  
Retroviral Vectors ◽  
Transduction Efficiency ◽  
Control Group ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Colony Pcr ◽  
Cd34 Cells

Abstract Abstract 693 Background: Homeobox B4 (HoxB4) has been shown to be a potent stem cell self-renewal gene, especially in hematopoietic stem cells (HSC). Accumulating evidence from murine studies indicates that the overexpression of HoxB4 enhances in vivo and ex vivo expansion of HSC. Although no leukemia has been observed after transplantation of HoxB4-transduced cells in murine models, the study using large animals such as dogs and non-human primates with retroviral vectors expressing HoxB4 showed the frequent development of leukemia. Regarding retroviral vectors expressing HoxB4, there is another concern, that is, insertional leukemogenesis, which has been elucidated in the hematopoietic stem cell gene therapy for X-SCID. To avoid the insertional mutagenesis, other vectors may be considered, including Epstein-Barr nuclear antigen (EBNA)-1 based episomal vectors or the transposon; however, problems are left, i.e. low transduction efficiency with EBNA vectors and unclear safety with transposon vectors. To avoid both the persistent HoxB4 expression and insertional mutagenesis leading to leukemogenesis, we have developed a new type of Sendai virus (SeV) vector; it lacks the polymerase gene, namely P-defective SeV (SeV/δP) vector. SeV is an enveloped virus with a non-segmented, negative-stranded RNA genome. SeV-based vectors are non-integrating, cytoplasmic vectors. They replicate exclusively in the cytoplasm of transduced cells, and do not go through a DNA phase; therefore, there is no concern about the unwanted integration of foreign sequences into chromosomal DNA of the host. We have previously shown that the transduction efficiency of human CD34+ cells with the SeV vector was very high; around 70% (100 multiplicity of infections). On the other hand, SeV/δP vectors are incapable of self-replication, thus enabling transient gene expression without spoiling their ability to efficiently transduce CD34+ cells. Here, using the SeV/δP vector expressing HoxB4 (SeV/δP/HoxB4 vector), we examined the effectiveness and safety of human HSC expansion after in utero transplantation to fetal sheep. Methods: After enrichment of CD34+ cells from cryopreserved human umbilical cord blood, these cells were repeatedly exposed to SeV/δP/HoxB4 vector every 24 h for 4 days. The transduced cells (3.2–11.7 × 105) were transplanted into the abdominal cavity of fetal sheep at 45–50 gestational days (full term, 147 days) that have premature immune system (HoxB4 group, n = 4; control group, n = 4). The engraftment of hematopoietic cells derived from human HSC in the lambs after birth was quantitatively evaluated by colony PCR of the bone marrow. The development of leukemia was assessed by regular sampling of peripheral blood and bone marrow. Results: The human–sheep chimeric ratio in the bone marrow of HoxB4 group was calculated 4.8-times higher than that of control group after birth, as assessed by colony PCR. The SeV genome was no longer detectable in the bone marrow and peripheral blood of lambs as assessed by RNA-PCR, confirming the SeV vectors were cleared. No leukemia developed in any of the sheep in either group at present (at 12 months after transplantation). Conclusion: The SeV/δP vector would be suitable for transient expression of HoxB4 in human CD34+ cells, enabling 4.8-times expansion of human HSC as assessed by their repopulating ability in sheep. The expansion of human HSC with the SeV vector was comparable to that with HoxB4-retroviral vectors. In addition, the SeV/δP vector is free of concern about transgene-related and insertional leukemogenesis and should be safer than retroviral vectors. Disclosures: No relevant conflicts of interest to declare.

Neutropenia Does Not Delay Lymphopoietic Regeneration in NODSCIDcγ−/− Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4831-4831
Author(s):  
Stefanie Bugl ◽  
Stefan Wirths ◽  
R Müller Martin ◽  
Märklin Melanie ◽  
Tina Wiesner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Early Event ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Lymphoid Progenitors ◽  
Fate Decision

Abstract Abstract 4831 Introduction: Previously it was demonstrated that lymphopoiesis is rapidly established after transplantation of wild type stem cells into lymphopenic NODSCIDcγ−/− mice. These data were interpreted as evidence for an “empty” preformed lymphopoietic niche being replenished by lymphoid progenitors. We hypothesized that antibody-induced neutropenia might influence early post transplant fate decision to myeloid rather than lymphoid differentiation resulting in delayed lymphoid reconstitution. Materials and Methods: 25,000 flow sorted CD45.2-expressing wild type Lin-/Sca1+/c-Kit+ (LSK) cells from C57BL/6 mice were transplanted into sublethally irradiated B-/T-/NK-cell deficient NODSCIDcγ−/− mice (CD45.1). Three groups of n = 7 mice received anti-Gr1 or anti-1A8 i.p. every 48 h to induce continuous antibody-mediated neutropenia vs. PBS as control. Blood was harvested at regular intervals to monitor the engraftment. After 16, 22, and 34 days, animals were sacrificed and underwent blood and bone marrow analysis. Results: Hematopoietic regeneration started with the emergence of donor-derived monocytes in all groups as well as neutrophils in the control group as early as 9 days after transplantation. On day 14, B cells were to be detected for the first time, followed by T lymphocytes approximately 20 days after transplantation. Besides the fact that neutrophils were undetectable in the antibody treated groups, the peripheral blood revealed no significant changes between the neutropenic mice and the control group at any point of time. At the bone marrow level, an increase of LSK and granulocyte-macrophage progenitors (GMPs) at the expense of megakaryocyte erythrocyte progenitor cells (MEPs) was found in neutropenic mice. Common lymphoid progenitors (CLPs), however, were not significantly different. Conclusions: The engraftment of wild type donor cells after hematopoietic stem cell transplantation into NODSCIDcγ−/− mice started with the production of monocytes and neutrophils. B-lymphocytes were detectable by day 14 after transplantation. The production of T-cells started around day 20. Continuous antibody-mediated neutropenia did not significantly delay lymphoid regeneration. Although the marrow of neutropenic mice displayed increased proliferation of granulocyte progenitors, CLPs were unchanged. We conclude that the detection of donor-derived lymphocytes in the host peripheral blood is a relatively early event after LSK transplantation. Moreover, antibody induced neutropenia is not sufficient to induce sustainable changes in early hematopoietic fate decisions on the bone marrow level. Disclosures: No relevant conflicts of interest to declare.

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