Sublethal Irradiation Research Articles

Long-Lasting Impact of Neonatal Exposure to Total Body Gamma Radiation on Secondary Lymphoid Organ Structure and Function.

The acute period after total body irradiation (TBI) is associated with an increased risk of infection, principally resulting from the loss of hematopoietic stem cells, as well as disruption of mucosal epithelial barriers. Although there is a return to baseline infection control coinciding with the apparent progressive recovery of hematopoietic cell populations, late susceptibility to infection in radiation-sensitive organs such as lung and kidney is known to occur. Indeed, pulmonary infections are particularly prevalent in hematopoietic cell transplant (HCT) survivors, in both adult and pediatric patient populations. Preclinical studies investigating late outcomes from localized thoracic irradiation have indicated that the mechanisms underlying pulmonary delayed effects are multifactorial, including exacerbated and persistent production of pro-inflammatory molecules and abnormal cross-talk among parenchymal and infiltrating immune and inflammatory cell populations. However, in the context of low-dose TBI, it is not clear whether the observed exacerbated response to infection remains contingent on these same mechanisms. It is possible instead, that after systemic radiation-induced injury, the susceptibility to infection may be independently related to defects in alternative organs that are revealed only through the challenge itself; indeed, we have hypothesized that this defect may be due to radiation-induced chronic effects in the structure and function of secondary lymphoid organs (SLO). In this study, we investigated the molecular and cellular alterations in SLO (i.e., spleen, mediastinal, inguinal and mesenteric lymph nodes) after TBI, and the time points when there appears to be immune competence. Furthermore, due to the high incidence of pulmonary infections in the late post-transplantation period of bone marrow transplant survivors, particularly in children, we focused on outcomes in mice irradiated as neonates, which served as a model for a pediatric population, and used the induction of adaptive immunity against influenza virus as a functional end point. We demonstrated that, in adult animals irradiated as neonates, high endothelial venule (HEV) expansion, generation of follicular helper T cells (TFH) and formation of splenic germinal centers (GC) were rapidly and, more importantly, persistently impaired in SLO, suggesting that the early-life exposure to sublethal radiation had long-lasting effects on the induction of humoral immunity. Although the neonatal TBI did not affect the overall outcome from influenza infection in the adults at the earlier time points assessed, we believe that they nonetheless contribute significantly to the increased mortality observed at subsequent late time points. Furthermore, we speculate that the detrimental and persistent impact on the induction of CD4 T- and B-cell responses in the mediastinal lymph nodes will decrease the animals' ability to respond to other aerial pathogens. Since many of these pathogens are normally cleared by antibodies, our findings provide an explanation for the susceptibility of survivors of childhood HCT to life-threatening respiratory tract infections. These findings have implications regarding the need for increased monitoring in pediatric hematopoietic cell transplant patients, since they indicate that there are ongoing and cumulative defects in SLO, which, importantly, develop during the immediate and early postirradiation period when patients may appear immunologically competent. The identification of changes in immune-related signals may offer bioindicators of progressive dysfunction, and of potential mechanisms that could be targeted so as to reduce the risk of infection from extracellular pathogens. Furthermore, these results support the potential susceptibility of the pediatric population to infection after sublethal irradiation in the context of a nuclear or radiological event.

Induction of tolerance and prolongation of islet allograft survival by syngeneic hematopoietic stem cell transplantation in mice

BackgroundsSyngeneic or autologous hematopoietic stem cells transplantation (HSCT) has been proposed to treat autoimmune diseases because of its immunosuppressive and immunomodulatory effects, which can also contribute to posttransplant antirejection therapy. In this study, we explored the tolerogenic effect of syngeneic HSCT on prolonging islet allograft survival. MethodsC57BL/6 mice received syngeneic HSCT plus preconditioning with sublethal irradiation. Then islets of BALB/c mice were transplanted into the renal subcapsular of C57BL/6 mice after chemically induced into diabetes. ResultsHSCT mice exhibited improved islet allograft survival and increased serum insulin compared to control mice. Islet allografts of HSCT mice displayed lower level lymphocyte infiltration and stronger insulin staining than control mice. T cells of HSCT mice proliferated poorly in response to allogeneic splenocytes compared to control mice. Mice appeared reversed interferon-γ (IFN-γ)/interleukin-4 (IL-4) ratio to a Th2 immune deviation after syngeneic HSCT. The percentage of CD8+ T cells was lower, while percentage of CD4+CD25+Foxp3+ T regulatory cells (Tregs) was higher in HSCT mice than control mice. HSCT mice showed higher percentage of CTLA-4+ T cells and expression of CTLA-4 mRNA than control mice. Targeting of CTLA-4 by intraperitoneal injection of anti-CTLA-4 mAb abrogated the effect of syngeneic HSCT on prolonging islet allograft survival, inhibiting activity of T cells in response to alloantigen, promoting Th1 to Th2 immune deviation and up regulating CD4+CD25+Foxp3+ Tregs. ConclusionsSyngeneic HSCT plus preconditioning of sublethal irradiation induces tolerance and improves islet allograft survival in fully mismatched mice model. Th1 to Th2 immune deviation, increased CD4+CD25+Foxp3+ Tregs and up-regulation of CTLA-4 maybe contribute to the tolerogenic effect induced by syngeneic HSCT.

Anti-mouse CD122 antibody promotes the hematopoietic repopulating capacity of cord blood CD34⁺ cells in NOD/SCID mice

The study was aimed to investigate the effect of anti-mouse CD122 antibody on the hematopoietic repopulating capacity of cord blood CD34⁺ cells in a humanized murine model-non obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After sublethal irradiation with γ-ray, NOD/SCID mice were intraperitoneally injected with 200 µg mouse isotype control antibody or anti-mouse CD122 antibody. Human cord blood CD34⁺ cells or phosphate-buffered saline (PBS) were injected via the tail vein at 6-8 hours later. Cohort of the mice injected with anti-mice CD122 antibody or control antibody alone were sacrificed at different time point (at week 2, 3, and 4 weeks) after the injection, and the percentage of NK cells in the peripheral blood was analyzed by flow cytometry. To evaluate the effect of anti-mouse CD122 antibody on the repopulating capacity of cord blood CD34⁺ cells in the recipient mice, phenotype analysis was performed in the bone marrow at 6 and 8 weeks after the transplantation. The results showed that the proportion of NK cells in the peripheral blood were (4.6 ± 0.6)% and (5.7 ± 1.7)% at week 2 and 3 after anti-CD122 antibody injection respectively,which decreased by 60%, compared with the mice injected with isotype control antibody. After 6 and 8 weeks of cord blood CD34⁺ cell transplantation,the percentage of human CD45⁺ in the bone marrow of the recipient mice treated with anti-mice CD122 antibody was (63.0 ± 12.2)% and (53.2 ± 16.3)%,respectively,which were dramatically higher than that in the mice treated with isotype control antibody (7.7 ± 3.6)% and (6.1 ± 2.4)%. Moreover,at 8 weeks after transplantation,human CD34⁺ cells appeared significantly in the recipients treated with anti-CD122 antibody. It is concluded that the anti-mouse CD122 antibody enhances the hematopoietic repopulating capacity of cord blood CD34⁺ cells in the NOD/SCID mice through decreasing the proportion of NK cells.

Adequate stimulation of hematopoietic stem cell proliferation by a polyherbal formulation (EMSA eritin) leading to lymphocyte differentiation in BALB/c mice after radiation

Radiotherapy is still an essential option for treatment of various stages of cancer. However, the irradiation administered would deplete the hematopoietic stem cells in the bone marrow, which would eventually decrease the number of lymphocytes and erythrocytes in the circulatory system. EMSA eritin is a polyherbal formulation that has the ability to stimulate erythropoiesis in mice after radiation therapy. Erythropoiesis is a complex mechanism involving the interaction of dozens of genes that may also be involved in other cellular process, such as lymphopoiesis. In this study, we elucidated the potential of EMSA eritin to stimulate other mechanisms, in addition to being an inducer of erythrocyte production. Bioinformatic analysis results indicated that the EMSA eritin formulation contains multiactive compounds that synergistically drive hematopoiesis and trigger lymphocyte differentiation. We then tested this prediction using an in vivo cell-culture system in a mice model. Experimental results suggested that EMSA eritin is able to induce hematopoietic stem cell proliferation and differentiate these cells into lymphocytes in BALB/c mice after sublethal radiation therapy. Moreover, the polyherbal formulation increased proliferation and survival of B and T lymphocytes in a dose-dependent manner. The results indicate that the polyherbal formulation is adequate to ameliorate both erythropoiesis and lymphopoiesis. The formulation thus appears very promising for treatment of patients following radiation therapy. However, this warrants further investigation.

Preconditioning allows engraftment of mouse and human embryonic lung cells, enabling lung repair in mice.

Repair of injured lungs represents a longstanding therapeutic challenge. We show that human and mouse embryonic lung tissue from the canalicular stage of development (20-22 weeks of gestation for humans, and embryonic day 15-16 (E15-E16) for mouse) are enriched with progenitors residing in distinct niches. On the basis of the marked analogy to progenitor niches in bone marrow (BM), we attempted strategies similar to BM transplantation, employing sublethal radiation to vacate lung progenitor niches and to reduce stem cell competition. Intravenous infusion of a single cell suspension of canalicular lung tissue from GFP-marked mice or human fetal donors into naphthalene-injured and irradiated syngeneic or SCID mice, respectively, induced marked long-term lung chimerism. Donor type structures or 'patches' contained epithelial, mesenchymal and endothelial cells. Transplantation of differentially labeled E16 mouse lung cells indicated that these patches were probably of clonal origin from the donor. Recipients of the single cell suspension transplant exhibited marked improvement in lung compliance and tissue damping reflecting the energy dissipation in the lung tissues. Our study provides proof of concept for lung reconstitution by canalicular-stage human lung cells after preconditioning of the pulmonary niche.

EMSA Eritin Drives Expansion of Regulatory T Cells and Promotes T Cells Differentiation in Irradiated Mice.

Sublethal irradiation therapy in cancer treatment causes generalized immunosuppression, which results in a range of DNA damage. We examined the significance of a polyherbal medicine called "EMSA Eritin" on immunological responses in sublethally irradiated mice focusing on the involvement of Treg, naïve T cell, and also the development and differentiation of T cells in thymus. Normal BALB/c mice were sublethally irradiated with dose of 600 rad. The irradiated mice were then orally administered by EMSA Eritin once a day at different doses: 1.04, 3.12, 9.37 mg/g body weight. The treatment was performed for 14 days. On day 15, immunological responses were observed by analyzing the status of Treg and differentiation of T cells in thymus. The administration of EMSA Eritin to irradiated mice resulted in a significant increase of pre T cells, Treg cells, and naïve T cells, which in general could maintain and normalize healthy condition in mice.

Hhex is Required at Multiple Stages of Adult Hematopoietic Stem and Progenitor Cell Differentiation.

Hhex encodes a homeodomain transcription factor that is widely expressed in hematopoietic stem and progenitor cell populations. Its enforced expression induces T-cell leukemia and we have implicated it as an important oncogene in early T-cell precursor leukemias where it is immediately downstream of an LMO2-associated protein complex. Conventional Hhex knockouts cause embryonic lethality precluding analysis of adult hematopoiesis. Thus, we induced highly efficient conditional knockout (cKO) using vav-Cre transgenic mice. Hhex cKO mice were viable and born at normal litter sizes. At steady state, we observed a defect in B-cell development that we localized to the earliest B-cell precursor, the pro-B-cell stage. Most remarkably, bone marrow transplantation using Hhex cKO donor cells revealed a more profound defect in all hematopoietic lineages. In contrast, sublethal irradiation resulted in normal myeloid cell repopulation of the bone marrow but markedly impaired repopulation of T- and B-cell compartments. We noted that Hhex cKO stem and progenitor cell populations were skewed in their distribution and showed enhanced proliferation compared to WT cells. Our results implicate Hhex in the maintenance of LT-HSCs and in lineage allocation from multipotent progenitors especially in stress hematopoiesis.

279. Efficient and Safe Lentiviral Vector-Mediated Hematopoietic Stem Cell Gene Therapy in MNGIE Mice

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients are deficient in thymidine phosphorylase(TP) resulting in systemic thymidine(Thd) and deoxyuridine(dUrd) accumulation affecting mtDNA replication and causing mitochondrial dysfunction. Common symptoms are gastrointestinal dysmotility, progressive ophthalmoplegia and leukoencephalopathy. Allogenic hematopoietic stem cell (HSC) transplantation has been shown to reduce disease symptoms, but is not well tolerated due to the inherent toxicity of the procedure. Therefore, syngeneic ex vivo lentiviral vector HSC gene therapy overexpressing the native cDNA or the codon optimized (TPco) sequence driven by the phosphoglycerate kinase (PGK) or spleen focus forming virus (SFFV) promoters in Tp−/-Upp−/- double knockout mice, a model for MNGIE disease, was investigated. At 1 month post transplantation after sublethal total body irradiation, very low TP activity was detected in blood of control wild type mice (0.07±0.03nmoles/h/mg), but enzyme activities in PGK treated mice were at least 90-fold higher (PGK-TP = 150±4 and PGK-TPco = 96±4 nmoles/h/mg), and in SFFV recipient mice 400-fold higher (450±5 nmoles/h/mg). Consequently, a significant reduction of plasma and urine Thd and dUrd levels was observed. Long-term follow up (14 months) showed on average 1.2-fold wild type TP activity levels increase in LV-PGK-TP and LV-PGK-TPco and 36-fold in SFFV-TPco treated mice. This was sufficient for sustained reduction of plasma and urine nucleoside levels, which was achieved at 76.5±8.2% donor chimerism levels with low LV vector copy numbers (1.0±1.1VCN/donor cell). The LV integration profile in bone marrow cells of primary recipients was analyzed; LVs displayed the expected tendency to integrate within highly expressed genes and the integration pattern did not differ from that of other SIN-LV vectors in other disease models (primary immune deficiencies and lysosomal enzyme storage disorders). Overall, stem cell gene therapy provided stable TP expression and long-term biochemical correction in MNGIE mice without genotoxicity or apparent phenotoxicity, which will be further evaluated for somatic and neurological phenotype correction and optimized to develop a clinical protocol to treat MNGIE patients.

OP45 – 3024: Efficient lentiviral vector-mediated hematopoietic stem cell gene therapy in MNGIE mice

Objective Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients are deficient in thymidine phosphorylase (TP) resulting in systemic thymidine (Thd) and deoxyuridine (dUrd) accumulation affecting mtDNA replication and causing mitochondrial dysfunction. Common symptoms are gastrointestinal dysmotility, progressive ophthalmoplegia and leukoencephalopathy. Allogeneic hematopoietic stem cell (HSC) transplantation has been shown to reduce disease symptoms, but is not well tolerated due to the inherent toxicity of the procedure. Application of lentiviral vector therapy. Methods Therefore, syngeneic ex vivo lentiviral vector HSC gene therapy overexpressing the native cDNA or the codon optimized (TPco) sequence driven by the phosphoglycerate kinase (PGK) or spleen focus forming virus (SFFV) promoters in Tp-/-Upp-/- double knockout mice, a model for MNGIE disease, was investigated. Results At 1 month post transplantation after sublethal total body irradiation, very low TP activity was detected in blood of control wild type mice (0.07±0.03nmoles/h/mg), but enzyme activities in PGK treated mice were at least 90-fold higher (PGK-TP = 150±4 and PGK-TPco=96±4 nmoles/h/mg), compared to a 400-fold increase observed in SFFV recipient mice (450±5) and consequently, a significant reduction of plasma and urine Thd and dUrd levels was observed. Long-term follow up (14 months post treatment) showed on average 1.2-fold TP wild type activity levels in LV-PGK-TP and LV-PGK-TPco and 36-fold in SFFV-TPco treated mice, sufficient for sustained reduction of plasma and urine nucleoside levels, which was achieved at 76.5±8.2% donor chimerism levels with low LV vector copy numbers (1.0±1.1VCN/donor cell). Conclusion Overall, stem cell gene therapy provided stable TP expression and long-term biochemical correction in MNGIE mice without genotoxicity or apparent phenotoxicity, which will be further evaluated for somatic and neurological phenotype correction and optimized to develop a clinical protocol to treat MNGIE patients.

Total body sub-lethal irradiation accelerates thymic senescence by reducing lymphoid progenitors in the thymus (HEM7P.236)

Abstract Total Body Irradiation (TBI) dramatically damages the BM and thymus organs, however the long-term effects of irradiation and aging on both BM cells and thymocytes are still unclear. In this study, we described that although the kinetics of thymocyte restoration was varied with the sex, dose and exposure age, a long-term and dose dependent reduction of DN1a,b progenitors and a transient over-response peak of thymocytes around 5-6 weeks were shown unanimously in all groups after irradiation, which led to the recovery of thymus maintained in a lower level of total thymocyte number through life. Such events were primarily due to the reduction of functional LSK progenitors in BM rather than the changes to the thymic environment. The damage of irradiation in BM LSK cells was dose dependent and 0.5 Gy was able to result in a significant long-term reduction of LSK progenitors. Responding to the reduction of thymocytes, both DN3 and DN4 subsets played a critical role on the compensation of the reduction of thymocytes with a periodical profile after irradiation, but the recovery potential was dramatically reduced at the age of 12 months. This study provided the evidence that the capability of LSK cells for lymphocyte production in BM is limited and can be dramatically damaged after a single dose of relatively low TBI, which consequently accelerates premature thymic aging and senescence. Our results may also be useful for the evaluation and therapeutic intervention of human TBI events.

MRNA and microRNA expression profiles of radioresistant NCI-H520 non-small cell lung cancer cells.

To elucidate the mechanism of radioresistance in non-small cell lung cancer (NSCLC) cells and to identify key molecules conferring radioresistance, the radioresistant subclone NCI-H520/R, derived from the NCI-H520 NSCLC cell line, was established with eight rounds of sublethal irradiation. The radioresistant features were subsequently assessed using a clonogenic assay, analysis of apoptosis and an MTT assay, the gene expression levels were examined using an Agilent Whole Human Genome 4×44 k Oligo microarray and Agilent Human miRCURY™ LNA array, and confirmed by reverse transcription-quantitative polymerase chain reaction. Pathway analysis and Gene Ontology (GO) analysis were performed to determine the biological functions of the subset of differentially expressed genes. miRNA-mRNA correlation analysis between the expression levels of each miRNA and all its predicted target genes was performed to further understand the radioresistance in the NCI-H520 cells. Following eight rounds of sublethal irradiation, a total of 2,862 mRNAs were significantly differentially expressed in the NCI-H520/R cells, including 893 upregulated genes and 1,969 downregulated genes. A total of 162 upregulated miRNAs and 274 downregulated miRNAs were significantly deregulated in the NCI-H520/R cells. Multiple core regulatory processes and signaling pathways were identified as being of likely relevance to radioresistance in NCI-H520/R cells, including the mitogen-activated protein kinase signaling pathway and neurotrophin signaling pathway. The expression of genes associated with radioresistance reflects the complex biological processes involved in clinical cancer cell eradication and requires further investigation for future enhancement of therapy.

Radiation treatment and cancer stem cells.

Radiotherapy is a standard treatment for many cancers and is frequently used as primary or adjuvant therapy, often in combination with surgery or chemotherapy or both. However, locoregional recurrence or metastatic spread still occurs in a high proportion of patients after radiotherapy. In this regard, emerging evidences suggest that sublethal radiation paradoxically promotes expansion of cancer stem cell population that is highly tumorigenic and is reminiscent of non-neoplasm stem cells. In this review, we discussed recent findings that demonstrate the increase in cancer stem cells after irradiation, and the possible cellular mechanisms with a perspective of tumor microenvironment. A further understating on the mechanistic mechanisms underlying radiation-enhanced malignant phenotypes might increase the efficacy of radiotherapy for cancer treatment.

Combined immune checkpoint protein blockade and low dose whole body irradiation as immunotherapy for myeloma.

BackgroundMultiple myeloma is characterized by the presence of transformed neoplastic plasma cells in the bone marrow and is generally considered to be an incurable disease. Successful treatments will likely require multi-faceted approaches incorporating conventional drug therapies, immunotherapy and other novel treatments. Our lab previously showed that a combination of transient lymphodepletion (sublethal whole body irradiation) and PD-1/PD-L1 blockade generated anti-myeloma T cell reactivity capable of eliminating established disease. We hypothesized that blocking a combination of checkpoint receptors in the context of low-dose, lymphodepleting whole body radiation would boost anti-tumor immunity.MethodsTo test our central hypothesis, we utilized a 5T33 murine multiple myeloma model. Myeloma-bearing mice were treated with a low dose of whole body irradiation and combinations of blocking antibodies to PD-L1, LAG-3, TIM-3, CD48 (the ligand for 2B4) and CTLA4.ResultsTemporal phenotypic analysis of bone marrow from myeloma-bearing mice demonstrated that elevated percentages of PD-1, 2B4, LAG-3 and TIM-3 proteins were expressed on T cells. When PD-L1 blockade was combined with blocking antibodies to LAG-3, TIM-3 or CTLA4, synergistic or additive increases in survival were observed (survival rates improved from ~30% to >80%). The increased survival rates correlated with increased frequencies of tumor-reactive CD8 and CD4 T cells. When stimulated in vitro with myeloma cells, CD8 T cells from treated mice produced elevated levels proinflammatory cytokines. Cytokines were spontaneously released from CD4 T cells isolated from mice treated with PD-L1 plus CTLA4 blocking antibodies.ConclusionsThese data indicate that blocking PD-1/PD-L1 interactions in conjunction with other immune checkpoint proteins provides synergistic anti-tumor efficacy following lymphodepletive doses of whole body irradiation. This strategy is a promising combination strategy for myeloma and other hematologic malignancies.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-014-0043-z) contains supplementary material, which is available to authorized users.

C-Met signalling is required for efficient postnatal thymic regeneration and repair.

We have reported that in vivo administration of the hybrid cytokine rIL-7/HGFβ or rIL-7/HGFα, which contains interleukin-7 (IL-7) and the β- or α-chain of hepatocyte growth factor (HGF), significantly enhances thymopoiesis in mice after bone marrow transplantation. We have shown that the HGF receptor, c-Met, is involved in the effect of the hybrid cytokines. To address the role of c-Met signalling in thymocyte development and recovery, we generated conditional knockout (cKO) mice in which c-Met was specifically deleted in T cells by crossing c-Met(ft/ft) mice with CD4-Cre transgenic mice. We show here that although the number of total thymocytes and thymocyte subsets in young c-Met cKO mice is comparable to age-matched control (Ctrl) mice, the cKO mice were more susceptible to sub-lethal irradiation and dexamethasone treatment. This was demonstrated by low recovery in thymic cellularity in c-Met cKO mice after insult. Furthermore, the number of total thymocytes and thymocyte subsets was markedly reduced in 6- to 12-month-old cKO mice compared with age-matched Ctrl mice, and the thymic architecture of 12-month-old cKO mice was similar to that of 20-month-old wild-type mice. In addition, c-Met deficiency reduced cell survival and the expression of Bcl-xL in double-positive thymocytes, and decreased cell proliferation and the expression of cyclin E and cyclin-dependent kinase 5 in single-positive thymocytes. Our data indicate that c-Met signalling plays an important role in thymic regeneration after thymic insult. In addition, T-cell-specific inactivation of c-Met accelerates age-related thymic involution.

Interleukin-12 preserves the cutaneous physical and immunological barrier after radiation exposure.

The United States continues to be a prime target for attack by terrorist organizations in which nuclear detonation and dispersal of radiological material are legitimate threats. Such attacks could have devastating consequences to large populations, in the form of radiation injury to various human organ systems. One of these at risk organs is the cutaneous system, which forms both a physical and immunological barrier to the surrounding environment and is particularly sensitive to ionizing radiation. Therefore, increased efforts to develop medical countermeasures for treatment of the deleterious effects of cutaneous radiation exposure are essential. Interleukin-12 (IL-12) was shown to elicit protective effects against radiation injury on radiosensitive systems such as the bone marrow and gastrointestinal tract. In this article, we examined if IL-12 could protect the cutaneous system from a combined radiation injury in the form of sublethal total body irradiation and beta-radiation burn (β-burn) directly to the skin. Combined radiation injury resulted in a breakdown in skin integrity as measured by transepidermal water loss, size of β-burn lesion and an exacerbated loss of surveillant cutaneous dendritic cells. Interestingly, intradermal administration of IL-12 48 h postirradiation reduced transepidermal water loss and burn size, as well as retention of cutaneous dendritic cells. Our data identify IL-12 as a potential mitigator of radiation-induced skin injury and argue for the further development of this cytokine as a radiation countermeasure.

The Complex of Tests for the Quantitative Evaluation of the Effects of Radiation on Laboratory Animals

Exposure to ionizing radiation leads to exhaustion of the pool of stem cells, increases differentiated cell stress, and activated reparation and apoptosis processes. Apart from well-documented acute effects, radiotherapy leads to delayed effects manifesting in few years after successful treatment. The aim of our study was the development of a test battery for evaluation of irradiation aftereffects in organisms characterized by different radiosensitivity. We chose C57BL mice as the ideal strain, because according to published data, they are characterized by medium radiosensitivity. At the preliminary stage, we have to assess informative value of some methods for the sublethal irradiation dose of 750 R. For evaluation of the damaging effects of radiation (lymphocyte count and maturity), we used, along with classical criteria, some complex parameters (body weight, serum homeostasis, behavior) characterizing general status of the organism. We also performed histological analysis of the liver, spleen, and pancreas and ranged the injuries by their severity.

Establishment of a Novel Bladder Cancer Xenograft Model in Humanized Immunodeficient Mice

Background/Aims: The aim of this study was to develop a novel model by transplanting human bladder cancer xenografts into humanized immunodeficient mice (SCID). Methods: The animals first underwent sublethal irradiation and then were subjected to simultaneous transplantation of human lymphocytes (5 × 10⁷ cells/mouse i.p.) and human bladder cancer cells (3 × 10⁶ cells/mouse s.c.). Results: The xenografts developed in all 12 mice that had received bladder cancer BIU-87 cells, and the tumor specimens were evaluated histologically. All 6 model mice expressed human CD3 mRNA and/or protein in the peripheral blood, spleens and xenografts. The mean proportion of human CD3⁺ cells was 19% with a level of human IgG 532.4µ/ml in the peripheral blood at Week 6 after transplant inoculation. The re-constructed human immune system in these mice was confirmed to be functional by individual in vitro testing of their proliferative, secretory and cytotoxic responses. Conclusion: The successful engraftment of the human bladder cancer xenografts and the establishment of the human immune system in our in vivo model described here may provide a useful tool for the development of novel therapeutic strategies targeting at bladder cancer.

Sub-lethal ionizing radiation alters Foxp3 expression in CD4+ T regulatory (Treg) cells in vitro and in vivo

Meeting abstracts The use of sub-lethal radiation has been shown to alter cell phenotype and gene expression. T regulatory (Treg) cells are phenotypically defined as being CD4+CD25+ Foxp3+ T cells and are known to suppress the function of CD8+ cytotoxic T lymphocytes (CTLs). Inhibiting the

Dasatinib Shows Therapeutic Potential in the Murine Xenograft Model for TCF3 rearranged Acute Lymphoblastic Leukemia

Transcription Factor 3 (TCF3) rearrangements are a recurring chromosomal abnormality in B-cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) occurring in approximately five percent of pediatric ALL. Historically, the majority of these patients carried a poor prognosis, but advances with more intensive cytotoxic chemotherapy have improved the survival rate while exposing patients to increased short and long-term toxicities. Two genetic rearrangements produce the chimeric transcription factors, TCF3-PBX1 t(1;19)(q23;p13) and a much rarer TCF3-HLF t(17;19)(q22;p13). Sadly, TCF3-HLF remains an extremely difficult disease to treat with few, if any known survivors. Although it is unknown how these translocations lead directly to disease, it is established that they do result in diseases arrested in a later stage of B-cell differentiation and pre-B cell receptor (pre-BCR) dependence. Recently, we highlighted the concept of targeting the pre-BCR pathway for therapeutic potential using dasatinib (Sprycel). Here, we further examine dasatinib effectiveness in the murine xenograft model for TCF3-rearranged ALL. Methods: Primary patient samples were obtained with written informed consent approved by the Institutional Review Board of Oregon Health and Science University and processed. Mononuclear cells were separated by Ficoll and exposed to increasing concentrations of dasatinib. Inhibitory Concentration of fifty-percent viability (IC50) was calculated for each sample. The median IC50 for over four hundred acute leukemic samples interrogated by this assay was calculated to approximately 100nM. Samples with IC50 values below 30nM were deemed hypersensitive to dasatinib. For xenografts, frozen viable primary patient samples were thawed and grafted via tail-vein into NOD/SCID/IL-2rgnull(NSG) mice 24 hours after sub-lethal irradiation with 200 cGy. Upon engraftment, and in vivo expansion, animals were euthanized and leukemic cells recovered from the spleen were then injected in secondary recipients. One week after injection the mice were divided into two groups and treated by oral gavage with dasatinib at 50mg/kg/dose daily or citrate control for 5 days per week. Treatment continued until the day of sacrifice (4-20 weeks). Peripheral blood engraftment was monitored weekly starting on week 3 by flow cytometry analysis using anti-human CD19 and CD45 (hCD19-APC, hCD45-FITC) versus anti-murine CD45 (mCD45-PerCP-Cy5.5). Flow cytometric data was analyzed using FACS/AriaIII. Results: Screening over one hundred BCP-ALL samples identified that approximately ten percent of these samples show hypersensitivity to dasatinib. TCF3-rearranged ALL and BCR-ABL1 ALL had a majority of samples with IC50’s less than 10nM. Throughout all known subsets of ALL except ETV6-RUNX1, there also appeared to be individual samples that have IC50 values less than 30nM, suggesting significant sensitivity to this drug. Of these, three individual TCF3-rearranged ALL samples were identified and xenografted into NSG mice, expanded and injected into secondary recipients. All dasatinib treated cohorts showed significantly less leukemic peripheral blood chimerisms as compared to their vehicle control counterparts. Further, in vitro treatment of xenografted cells with dasatinib indicated inhibition of the pre-BCR by decrease in pan-phospho-SRC. Intriguingly, dasatinib did not completely abolish disease in all TCF3-rearranged ALL, suggesting other important mechanisms for cell viability. Conclusions: These studies show in vivo therapeutic benefits of dasatinib as treatment for TCF3-rearranged ALL, and open the possibility of adding this drug to their treatment. Further studies are underway to address the mechanisms of dasatinib sensitivity of other subsets of ALL identified in our screen in hopes of adding targeted therapies to their treatment. [Display omitted] DisclosuresDruker:Molecular MD: Consultancy, Equity Ownership, Scientific Founder. Some clinical trials on which I participate as PI or co-investigator utilize MolecularMD for molecular testing. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU. Other; Bristol-Myers Squibb: Clinical trial funding: PI and co-investigator on ARIAD clinical trials. OHSU has contracts with ARIAD to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. Clinical trial funding: PI and co-investigator on ARIAD clinical trials. OHSU has contracts with ARIAD to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. Other. Off Label Use: Dasatinib use as potential therapy in ALL.

Arhgap21 Expression in Bone Marrow Niche Is Crucial for Hematopoietic Progenitor Homing and Short Term Reconstitution after Transplantation

The microenvironment of the bone marrow (BM) is essential for retention and migration of hematopoietic progenitor cells. ARHGAP21 is a negative regulator of RhoGTPAses, involved in cellular migration and adhesion, however the role of ARHGAP21 in hematopoiesis is unknown.In order to investigate whether downregulation of Arhgap21 in microenvironment modulates bone marrow homing and reconstitution, we generated Arhgap21+/- mice using Embryonic Stem cell containing a vector insertion in Arhgap21 gene obtained from GeneTrap consortium and we then performed homing and bone marrow reconstitution assays.Subletally irradiated (9.5Gy) Arhgap21+/- and wild type (WT) mice received 1 x 106 BM GFP+cells by IV injection. For homing assay, 19 hours after the transplant, Lin-GFP+ cells were analyzed by flow cytometry. In reconstitution and self-renew assays, the GFP+ cell percentage in peripheral blood were analyzed 4, 8, 12 and 16 weeks after transplantation. Hematopoietic stem cells [GFP+Lin-Sca+c-Kit+ (LSK)] were counted after 8 and 16 weeks in bone marrow after primary transplant and 16 weeks after secondary transplant.The percentage of Lin-GFP+ hematopoietic progenitor cells that homed to Arhgap21+/- recipient (mean± SD) (2.07 ± 0.85) bone marrow was lower than those that homed to the WT recipient (4.76 ± 2.60); p=0.03. In addition, we observed a reduction (WT: 4.22 ±1.39; Arhgap21+/-: 2.17 ± 0.69; p=0.001) of Lin- GFP+ cells in Arhgap21+/-receptor spleen together with an increase of Lin- GFP+ population in Arhgap21+/-receptor peripheral blood (WT: 8.07 ± 3.85; Arhgap21+/-: 14.07 ±5.20; p=0.01), suggesting that hematopoietic progenitor cells which inefficiently homed to Arhgap21+/- bone marrow and spleen were retained in the blood stream.In bone marrow reconstitution assay, Arhgap21+/-receptor presented reduced LSK GFP+ cells after 8 weeks (WT: 0.19 ±0.03; Arhgap21+/-0.12±0.05; p=0.02) though not after 16 weeks from primary and secondary transplantation. The reduced LSK percentage after short term reconstitution was reflected in the lower GFP+ cells in peripheral blood 12 weeks after transplantation (WT: 96.2 ±1.1; Arhgap21+/-94.3±1.6; p=0.008). No difference was observed in secondary transplantation, indicating that Arhgap21reduction in microenvironment does not affect normal hematopoietic stem cell self-renewal.The knowledge of the niche process in regulation of hematopoiesis and their components helps to better understand the disordered niche function and gives rise to the prospect of improving regeneration after injury or hematopoietic stem and progenitor cell transplantation.In previous studies, the majority of vascular niche cells were affected after sublethal irradiation, however osteoblasts and mesenchymal stem cells were maintained (Massimo Dominici et al.; Blood; 2009.). RhoGTPase RhoA, which is inactivated by ARHGAP21 (Lazarini et al.; Biochim Biophys acta; 2013), has been described to be crucial for osteoblasts and mesenchymal stem cell support of hematopoiesis (Raman et al.; Leukemia; 2013). Taken together, these results suggest that Arhgap21 expression in bone marrow niche is essential for homing and short term reconstitution support. Moreover, this is the first study to investigate the role of Arhgap21 in bone marrow niche. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.