scholarly journals Exosomes from primed MSCs can educate monocytes as a cellular therapy for hematopoietic acute radiation syndrome

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Matthew H. Forsberg ◽  
John A. Kink ◽  
Anna S. Thickens ◽  
Bryson M. Lewis ◽  
Charlie J. Childs ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Radiation ◽  
Prolonged Survival ◽  
Human Monocytes ◽  
Acute Radiation Syndrome ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Hematopoietic Recovery ◽  
Lethal Irradiation

Abstract Background Acute radiation syndrome (ARS) is caused by acute exposure to ionizing radiation that damages multiple organ systems but especially the bone marrow (BM). We have previously shown that human macrophages educated with exosomes from human BM-derived mesenchymal stromal cells (MSCs) primed with lipopolysaccharide (LPS) prolonged survival in a xenogeneic lethal ARS model. The purpose of this study was to determine if exosomes from LPS-primed MSCs could directly educate human monocytes (LPS-EEMos) for the treatment of ARS. Methods Human monocytes were educated by exosomes from LPS-primed MSCs and compared to monocytes educated by unprimed MSCs (EEMos) and uneducated monocytes to assess survival and clinical improvement in a xenogeneic mouse model of ARS. Changes in surface molecule expression of exosomes and monocytes after education were determined by flow cytometry, while gene expression was determined by qPCR. Irradiated human CD34+ hematopoietic stem cells (HSCs) were co-cultured with LPS-EEMos, EEMos, or uneducated monocytes to assess effects on HSC survival and proliferation. Results LPS priming of MSCs led to the production of exosomes with increased expression of CD9, CD29, CD44, CD146, and MCSP. LPS-EEMos showed increases in gene expression of IL-6, IL-10, IL-15, IDO, and FGF-2 as compared to EEMos generated from unprimed MSCs. Generation of LPS-EEMos induced a lower percentage of CD14+ monocyte subsets that were CD16+, CD73+, CD86+, or CD206+ but a higher percentage of PD-L1+ cells. LPS-EEMos infused 4 h after lethal irradiation significantly prolonged survival, reducing clinical scores and weight loss as compared to controls. Complete blood counts from LPS-EEMo-treated mice showed enhanced hematopoietic recovery post-nadir. IL-6 receptor blockade completely abrogated the radioprotective survival benefit of LPS-EEMos in vivo in female NSG mice, but only loss of hematopoietic recovery was noted in male NSG mice. PD-1 blockade had no effect on survival. Furthermore, LPS-EEMos also showed benefits in vivo when administered 24 h, but not 48 h, after lethal irradiation. Co-culture of unprimed EEMos or LPS-EEMos with irradiated human CD34+ HSCs led to increased CD34+ proliferation and survival, suggesting hematopoietic recovery may be seen clinically. Conclusion LPS-EEMos are a potential counter-measure for hematopoietic ARS, with a reduced biomanufacturing time that facilitates hematopoiesis.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Evaluation of Patient-Derived Xenografts for Modeling Outcome of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3759-3759
Author(s):  
Abdulmohsen M Alruwetei ◽  
Hernan Carol ◽  
Rosemary Sutton ◽  
Glenn M Marshall ◽  
Richard B Lock
Keyword(s):  
Gene Expression ◽  
Drug Resistance ◽  
Clinical Outcome ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Nsg Mice ◽  
Relapse Prediction ◽  
Pediatric All ◽  
Selection Of

Abstract Introduction: Children with acute lymphoblastic leukemia (ALL) are stratified at diagnosis based on molecular/cytogenetic characteristics and their response to initial treatment to receive risk-adapted multi-agent chemotherapy. The majority of ALL patients are stratified as Intermediate Risk (IR) and present with moderate levels of minimal residual disease (MRD<5x104) after receiving induction therapy, although an unacceptably high proportion of these patients relapse. The lack of specific prognostic features makes it difficult to predict the response of IR patients to treatment. The early identification of patients who are destined to relapse would facilitate improvements in tailored treatments for IR ALL patients. Recent progress in the development of patient-derived xenografts (PDXs) in immune-deficient mice represents an opportunity to improve relapse prediction in ALL patients. The aims of this study were to: (1) optimize the engraftment conditions of IR pediatric ALL samples to predict patient response to treatment; and, (2) to assess the development and mechanisms of therapy-induced drug resistance. Methods: Two pairs of IR pediatric ALL patients were matched based on clinical and genetic features, except that one patient from each pair relapsed early while the other remains relapse-free (ALL-Rel and ALL-CR1, respectively). Three parameters were varied in establishing PDXs by inoculating one million bone marrow (BM) derived biopsy cells collected at diagnosis into groups of 4 mice: (1) mouse strain (NOD/SCID vs. NSG); (2) site of inoculation (intravenous vs. intra-femoral); and (3) early treatment of mice with a 2-week induction chemotherapy regimen of vincristine, dexamethasone, and L-asparaginase (VXL). Leukemia engraftment was monitored weekly based on the proportion of human versus mouse CD45+ cells in the murine PB, and the median times to engraftment were compared according to patient outcome. The median time to engraft was also compared between the VXL-treated and non-treated groups. PDXs harvested from mice were compared for ex vivo sensitivity to single agent vincristine, dexamethasone and L-asparaginase. PDX gene expression profiles were also compared to identify pathways associated with evasion of VXL treatment in vivo. Results: The efficiency of engraftment was greater for NSG mice (29/32 mice engrafted) versus NOD/SCID mice (20/32 mice), and primary ALL cells also engrafted significantly faster in NSG mice (median time to engraft 71.1 days) compared with NOD/SCID mice (83.5 days) (P < 0.01), with no apparent difference associated with clinical outcome. Intrafemoral inoculation did not improve the efficiency or speed of engraftment compared with intravenous inoculation, nor predicted clinical outcome. However, PDX responses to VXL induction chemotherapy reflected the clinical outcome of the patients from whom they were derived; those derived from the 2 ALL-Rel patients exhibited in vivo drug resistance (leukemia growth delay of 1 and 6.2 days) compared with those derived from the 2 ALL-CR1 patients (34.7 and >119.8 days). Further, ex vivo analysis showed that the PDXs derived from the ALL-Rel patients exhibited resistance to vincristine or L-asparaginase compared with those derived from the ALL-CR1 cases. Moreover, the in vivo VXL treatment of an ALL-CR1 PDX resulted in selection of cells that exhibited vincristine resistance. Gene expression profiling revealed significant up-regulation of microtubule associated proteins (MAPs) and tubulin isotypes (alpha and beta) in vincristine-resistant PDXs. Genes that were significantly upregulted in vincristine resistant PDXs with a false discovery rate (FDR) < 0.05 and P value < 0.02 include TUBB6, TUBA1A, TUBA1B, MAP1S, TUBA3D and TBCA. The increased expression of genes that affect microtubule functions suggest that changes in microtubule dynamics and/or stability led to decreased sensitivity to antimicrotubule agents. Conclusions: In vivo selection of PDXs with an induction-type regimen of chemotherapeutic drugs may lead to improved relapse prediction and identify novel mechanisms of drug resistance in IR pediatric ALL. Support: Steven Walter Foundation; NHMRC Australia, APP1057746 Disclosures No relevant conflicts of interest to declare.

scholarly journals Identifying a Diagnostic Window for the Use of Gene Expression Profiling to Predict Acute Radiation Syndrome

2020 ◽  
Vol 195 (1) ◽  
Author(s):  
Patrick Ostheim ◽  
Omoleye Coker ◽  
Simone Schüle ◽  
Cornelius Hermann ◽  
Stephanie E. Combs ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Acute Radiation ◽  
Acute Radiation Syndrome ◽  
Diagnostic Window

scholarly journals Macrophages Educated with Exosomes from Primed Mesenchymal Stem Cells Treat Acute Radiation Syndrome by Promoting Hematopoietic Recovery

2019 ◽  
Vol 25 (11) ◽  
pp. 2124-2133 ◽  
Author(s):  
John A. Kink ◽  
Matthew H. Forsberg ◽  
Sofiya Reshetylo ◽  
Soroush Besharat ◽  
Charlie J. Childs ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Acute Radiation ◽  
Acute Radiation Syndrome ◽  
Hematopoietic Recovery

Self-Renewal and Differentiation in Hematopoietic Stem and Progenitor Cells Is Controlled By the APC/C Coactivator Cdh1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2370-2370
Author(s):  
Daniel Ewerth ◽  
Stefanie Kreutmair ◽  
Birgit Kügelgen ◽  
Dagmar Wider ◽  
Julia Felthaus ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Research Funding ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Introduction: Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously renew the hematopoietic system by differentiation into mature blood cells. The process of differentiation is predominantly initiated in G1 phase of the cell cycle when stem cells leave their quiescent state. During G1 the anaphase-promoting complex or cyclosome (APC/C) associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate proliferation. In addition, Cdh1 has been shown to control terminal differentiation in neurons, muscle cells or osteoblasts. Here we show that Cdh1 is also a critical regulator of human HSPC differentiation and self-renewal. Methods: Human CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokine combinations. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with flow cytometric detection of CD34 expression. The knockdown and overexpression of Cdh1 was achieved by lentiviral delivery of suitable vectors into target cells. After cell sorting transduced (GFP+) CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice. Results: G-CSF mobilized CD34+ cells showed effective differentiation into granulocytes (SCF, G-CSF), erythrocytes (SCF, EPO) or extended self-renewal (SCF, TPO, Flt3-L) when stimulated in vitro. The differentiation was characterized by a fast downregulation of Cdh1 on protein level, while Cdh1 remained expressed under self-renewal conditions. A detailed analysis of different subsets, both in vitro and in vivo, showed high Cdh1 level in CD34+ cells and low expression in myeloid cells. Analysis of proliferation revealed lowest division rates during self-renewal, accompanied by higher frequency of CD34+ cells. The fastest proliferation was found after induction of erythropoiesis. These experiments also showed a more rapid decrease of HSPCs' colony-forming ability and of CD34+ cells during granulopoiesis after 2-3 cell divisions in contrast to a moderate decline under self-renewal conditions. The depletion of Cdh1 (Cdh1-kd) had no effect on total cell numbers or proliferation detected by CFSE during differentiation and self-renewal, but showed an increase in S phase cells. These results were confirmed at the single cell level by measuring the cell cycle length of individual cells. Independent of cell cycle regulation, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of Glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies compared to control cells. These results suggest that the majority of cells reside in a more undifferentiated state due to Cdh1-kd. The overexpression of Cdh1 showed reversed results with less S phase cells and tendency to increased differentiation in liquid culture and CFU assays. To further validate our results in vivo, we have established a NSG xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Preliminary data in murine hematopoiesis support our hypothesis showing enhanced PB chimerism upon Cdh1-kd. Looking for a mediator of these effects, we found the Cdh1 target protein TRRAP, a cofactor of many HAT complexes, increased upon Cdh1-kd under self-renewal conditions. We use currently RT-qPCR to determine, if this is caused by a transcriptional or post-translational mechanism. Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells, represses erythropoiesis in vitro and facilitates engraftment capacity and B cell development of human HSPCs in vivo. This work was supported by Josè Carreras Leukemia Foundation grant DCJLS R10/14 (to ME+RW) Disclosures Ewerth: Josè Carreras Leukemia Foundation: Research Funding. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Distal elements are critical for human CD34 expression in vivo

Blood ◽  
2002 ◽  
Vol 100 (13) ◽  
pp. 4420-4426 ◽  
Author(s):  
Yutaka Okuno ◽  
Claudia S. Huettner ◽  
Hanna S. Radomska ◽  
Victoria Petkova ◽  
Hiromi Iwasaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Functional Analysis ◽  
Transgenic Mice ◽  
Flanking Sequence ◽  
Hematopoietic Stem ◽  
Critical Elements ◽  
Human Cd34 ◽  
Stem And Progenitor Cells ◽  
Cd34 Expression

The elements regulating gene expression in hematopoietic stem cells are still poorly understood. We previously reported that a 141-kilobase (kb) human CD34 transgene confers properly regulated human CD34 expression in transgenic mice. A construct with only the human CD34 promoter and 3′ enhancer region is not sufficient, suggesting that critical distal elements are necessary for expression of the human CD34 gene. To further localize such elements, we analyzed deletion constructs of the human CD34 gene and evaluated their function in transgenic mice. Constructs harboring as little as 18 kb of 5′ and 26 kb of 3′ human CD34 flanking sequence conferred human expression in tissues of transgenic mice with a pattern similar to that of the 141-kb human transgene. In contrast, a construct harboring 10 kb of 5′ and 17 kb of 3′ human CD34 flanking sequence gave no expression. These data demonstrate that regions between 10 to 18 kb upstream and/or 17 to 26 kb downstream of the human CD34 gene contain critical elements for human CD34 expression in vivo. Further functional analysis of these regions in transgenic mice will be crucial for understanding CD34 gene expression in hematopoietic stem and progenitor cells.

scholarly journals A Novel Combination Therapy for Paediatric T Cell Acute Lymphoblastic Leukaemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3767-3767
Author(s):  
Ben Christopher Ede ◽  
Paraskevi Diamanti ◽  
Charlotte V. Cox ◽  
Allison Blair
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Combination Therapy ◽  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
Nsg Mice ◽  
Gene And Protein Expression ◽  
Apoptotic Proteins

Abstract T cell acute lymphoblastic leukaemia (T-ALL) is a rare form of leukaemia that accounts for approximately 15% of paediatric ALL cases. Unfortunately, approximately 20% of patients do not achieve long term remission as a result of failure of therapy to eradicate the disease. T-ALL is a highly heterogeneous disease that displays a spectrum of immunophenotypes, chromosomal aberrations and gene expression profiles. This heterogeneity has prompted research into more targeted therapies, with the aim of overcoming drug resistance often found with standard chemotherapeutic regimens. Here, we build upon use of the drug Parthenolide (PTL), which has shown promise in treatment of T-ALL and other leukaemias such as BCP-ALL and AML, in combination with ABT-263, a BCL-2 family antagonising agent. Bone marrow samples from 10 T-ALL cases, taken at diagnosis, were treated with PTL in vitro for 24 hours then viability was assessed using the annexin V / PI flow cytometric assay. Variable cytotoxic effects were observed in samples treated with PTL (1-10µM), with half maximal inhibitory concentrations ranging from 2.6-10 µM. At the highest dose tested, the proportion of surviving cells ranged from 5.79-56% (median 35.33%). BM from 5 of these samples was used for whole genome microarray (WGA) analysis. We compared gene expression in bulk ALL and in specific subpopulations, known to have leukaemia initiating capacity in vivo; CD34+/CD7+, CD34+/CD7-, CD34-/CD7+ and CD34-/CD7- cells. WGA data demonstrated that CD34+/CD7- was the only subpopulation to express significantly lower levels (5.38 fold) of the pro-apoptotic gene Bcl-2L11 (BIM) compared to the unsorted bulk T-ALL cells, p=0.006. Interestingly, we have previously shown that CD34+/CD7- cells from a few patients were resistant to PTL treatment in vivo compared to unsorted cells. To validate these results, mRNA and relative protein quantification was performed by qPCR and western blotting in bulk material from 8 of the 10 samples, 3 of which had been analysed by microarray for BIM expression. We found that the gene and protein expression levels of BIM were negatively correlated with PTL resistance in vitro, p≤0.0001 and p=0.049 respectively. This suggests that reduced BIM expression is related to PTL resistance. We next evaluated the effects of combining PTL and ABT-263 on T-ALL cells in vitro. ABT-263 is a BH3 protein mimetic, like BIM it promotes apoptosis by blocking the inhibitory effects that BCL-2 anti-apoptotic proteins have on pro-apoptotic proteins. The effects of combining the drugs were assessed in 7 of the original 10 samples. Unsorted ALL cells were incubated with PTL and ABT-263 for 24 hours, before viability was analysed by flow cytometry and drug synergy was calculated via the Chou Talalay method. This drug combination showed enhanced cytotoxicity to T-ALL cells compared to PTL (p=0.0282) or ABT-263 (p=0.0358) alone. Moreover, the highest combined dose tested (2.5µM PTL with 0.25µM ABT-263) killed 86.1±9% cells cf 71.8±18% with ABT-263 alone and only 21.7±11% with PTL alone. The combination also showed synergism with a combination index value below 1 in all doses tested. Previous findings in our laboratory have shown that in vivo PTL treatment eliminated childhood leukaemia in NOD/LtSz-scid IL-2Rγc null (NSG) mice, in most cases tested. It may be possible to further enhance this toxicity using ABT-263 alone or in combination with PTL. NSG mice were inoculated with unsorted T-ALL cells and leukaemia was allowed to establish until levels in peripheral blood (PB) exceeded 0.1%. NSG mice were subsequently treated orally for 21 days with 100mg/kg of ABT-263 or placebo and leukaemia burden was monitored weekly in PB aspirates. Twenty-eight days following commencement of treatment, leukaemia burden in the placebo treated group was 80.73±2.94% and the animals were electively culled. In contrast, disease burden was significantly lower in the treated animals at this stage (35.2±2.1%, p=0.004). ABT-263 treatment has significantly improved survival of all xenografts to date, (P<0.014). In summary, we have shown that PTL resistance is related to the expression of BIM. By combining PTL with ABT-263, which mimics the pro-apoptotic action of BIM, the drugs work synergistically to enhance T-ALL cytotoxicity in vitro. Ongoing in vivo studies will assess the full potential of this combination therapy for paediatric T-ALL. Disclosures No relevant conflicts of interest to declare.

Abstract 328: Viral Anti-Atherogenic Proteins Alter Monocyte and Neutrophil Invasion in Mice and Bag3 Gene Expression

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Jennifer Davids ◽  
Sami Saikaly ◽  
Alexandra Lucas
Keyword(s):  
Gene Expression ◽  
Knockout Mice ◽  
Granzyme B ◽  
Nod Mice ◽  
Response To Treatment ◽  
Mouse Strains ◽  
Human Monocytes ◽  
Caspase 1 ◽  
Apoptotic Pathways

Aim Atherosclerosis is characterized by chronic inflammation and cell death (apoptosis). Serine protease inhibitors, or serpins, regulate inflammatory, thrombotic and apoptotic pathways. Poxviruses encode cross-class serpins that prevent host cell apoptosis. Serp-2, from Myxoma, reduces plaque, inflammation and apoptosis in animal models; CrmA, from Vaccinia, does not. Both serpins target Caspase 1 and Granzyme B, but the reasons for the differing effects in vivo are unknown. In prior research three Myxoma viral proteins, including Serp-2, reduced monocyte invasion, plaque growth, and aneurysm formation in ApoE-/- mice after angioplasty. These same proteins alter expression of a shared cohort of 48 apoptosis-related genes in human monocytes treated with camptothecin. This study assesses the effects of Serp-2 and CrmA on apoptotic gene expression in a mouse peritoneal inflammation model. Methods Mouse strains deficient for Granzyme B (GzmB, N=13) and Caspase 1 (Casp1, N=15) were compared to their respective background mice (C57Bl/6 and Nod, N=15 each) 18 hours after treatment with PMA and either Serp-2 or CrmA. RNA was isolated and analyzed by RT-PCR and normalized to GAPDH, then to the PMA-only treated control. Results Compared to human monocytes, mouse peritoneal exudates from knockout mice displayed differential alteration of BCL2-associated athanogene 3 (BAG3) in response to treatment with Serp-2 or CrmA. Serp-2 treatment reduced expression levels compared to CrmA treatment in GzmB (p=0.0267) and C57Bl/6 mice (p=0.0280). Casp1-/- mice treated with Serp-2 downregulate BAG3, expressing 9.7-fold less than Nod mice (p=0.0006), but CrmA had no significant effect in either strain. An associated differential migration of Ly6Chi and Ly6Ghi cells was also discovered in knockout mice with serpin treatments. Discussion One candidate gene found in human monocytes, BAG3, has reduced gene expression after Serp-2 treatment in mouse peritoneal exudates but increased by CrmA. BAG3 is known to alter cell migration and apoptosis, important to atherosclerotic progression. BAG3 is regulated by 3 myxomaviral proteins in human and mouse cells, underscoring a potential role as a lynchpin in viral protein anti-inflammatory and anti-apoptotic pathways.

“Antitumor Activity of Novel Anti-MM Agents and Combinations, the Proteasome Inhibitor Bortezomib and Multikinase Inhibitor Sorafenib, Both Applied as Monotherapy and in Combination in NOD/SCID-IL2-Receptor-Gamma-chain−/− (NSG) Mice Using a Intratibial Tumor Dissemination approach”.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4912-4912
Author(s):  
Julia Schüler ◽  
Dagmar Wider ◽  
Dietmar Pfeifer ◽  
Josefina Udi ◽  
Heinz-Herbert Fiebig ◽  
...  
Keyword(s):  
Gene Expression ◽  
Primary Tumor ◽  
Combined Therapy ◽  
Control Group ◽  
Gamma Chain ◽  
Multikinase Inhibitor ◽  
Tumor Inhibition ◽  
Nsg Mice ◽  
C Value

Abstract Abstract 4912 Since novel treatment options are needed in multiple myeloma (MM), novel anti-MM agents and combinations are eagerly pursued to further improve the prognosis for MM patients. For potentially novel therapeutic agents, functional in vivo models are highly valuable. We have established a cell line-based, disseminated MM model in NOD/SCID-IL2-receptor-gamma-chain−/− (NSG) mice. In our current analysis, the multikinase inhibitor sorafenib was validated alone and in combination with the well-established anti-MM agent bortezomib in 6 independent experiments. Optimized dose and schedule were determined as follows: 1. sorafenib (100mg/kg/d; d0-11) alone, 2. bortezomib (0.7mg/kg/day (d); d0,4,11) alone, 3. both in combination with the respective doses and schedules compared to 4. a control group. L363 cells were injected intratibialy into NSG mice and respective therapies were started 7 days after L363-injection (d0). Tumor growth was monitored with daily monitoring of MM-symptoms, flow-cytometry (FACS) and fluorescence-based in vivo imaging (FI). Tumor inhibition was calculated as the proportional reduction of mean MM-cell-infiltration at the respective compartment of the test- compared to the control-group (optimal T/C in %). Furthermore, hollow bones of the injected mice were retrieved when mice were sacrificed, cells flushed out and MM cells purified by MACS microbeads. Total RNA was isolated from these cells and gene expression profiles analyzed using the HG-U133 Plus 2.0 array (Affymetrix) and the Expressionist software (Genedata AG, Basel). L363 engrafted reliably (take rate=100%) at the injection site and in distant organs, such as bone marrow (BM; 100%), spleen (38%) and rarely liver (8%); in the latter organs as previously reported. Control mice developed MM symptoms, such as hind limb pareses, weight loss and osteolyses. At the respective doses and schedules, the examined compounds were well tolerated in tumor-bearing mice. No acute toxicity could be observed and maximal body weight loss was 4% with mono- and 11% with combined therapy. Primary tumor development was markedly reduced by sorafenib (optimal T/C of 11% on d11), as well as with bortezomib, albeit to a lesser extend (optimal T/C: 22% on d5). BM metastases were also significantly reduced by sorafenib with an optimal T/C value of 21% on d11. Bortezomib reduced BM infiltration to an optimal T/C value of 46% on d5 as compared to the control. Combined therapy of sorafenib and bortezomib showed most pronounced anti-tumor and anti-metastatic effects, inducing T/C values of 17% (primary tumor) and 7% (BM) on day 11, respectively. Table 1. Antitumor effect of Sorafenib and Bortezomib in mono- and combined therapy in the L363-xenograft model Compound Side effects Primary tumor Bone marrow Dose Mortality Max. median bwc1 FI2 tumor inhibition FI2 tumor inhibition [mg/kg/d] [n] [%] [%] [%] Sorafenib 100 0 / 5 96 11 21 Bortezomib 0.7 0 / 5 97 22 46 Soraf. / Bortez. 100 / 0.7 0 / 5 89 17 7 1 bwc=body weight changes 2 Tumor inhibition was calculated as the median % of MM cells determined by FI at respective compartments of the test vs. control group multiplied by 100 (optimal test/control (T/C) in %) L363 engraftment into NSG is a valuable in vivo MM model which exhibits high reproducibility, take- and metastases-rates and closely mimics the clinical situation. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor MM growth. Sorafenib showed promising results in our MM model, in particular in combination with bortezomib. Amongst others, a detailed characterization of the anti-tumor activity of both compounds will be provided by gene expression analysis of L363 cells isolated from untreated vs. treated mice. Further investigations to validate other innovative anti-MM agents as well as their combinations are currently also pursued. Disclosures No relevant conflicts of interest to declare.

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