Anti-asialo GM1 Research Articles

Suppressive Effect of Juzen‐Taiho‐To on Lung Metastasis of B16 Melanoma Cells In Vivo

Juzen-Taiho-To (JTT) is well known to be one of Kampo (Japanese herbal) medicine consisted of 10 component herbs and used for the supplemental therapy of cancer patients with remarkably success. However, the precise mechanisms by which JTT could favorably modify the clinical conditions of cancer patients are not well defined. The present study, therefore, was undertaken to examine the possible mechanisms of JTT on prevention of cancer metastasis using experimental mouse model. JTT was well mixed with rodent chow at concentrations of either 0.2 or 1.0%, and administered orally ad libitum, which was started 1 week before tumor cell injection and continue throughout the experiment. Oral administration of JTT at concentration 0.2 and 1.0% into C57BL/6 male mice significantly inhibited tumor metastasis in lungs, which was induced by the intravenous injection of 2 × 105 B16 melanoma cell. JTT at a concentration of 1.0% also significantly suppressed lung metastasis of B16 melanoma cell from hind footpad in C57BL/6 mice. In the second part of experiments, the influence of the depression of natural killer (NK) cell, natural killer T (NKT) cell and several types of cytokines on JTT-mediated inhibition of tumor cell metastasis. Intraperitoneal injection of anti asialo-GM1 antibody against NK cells and anti NK-1.1 monoclonal antibody (mAb) to NKT cells abrogated the inhibitory action of JTT on lung metastasis of B16 melanoma cells. Although intraperitoneal administration of anti-IFN-γ mAb scarcely affected the inhibitory action of JTT on tumor cell metastasis, injection of amrinone, which used for IL-12 suppression, significantly decreased the ability of JTT to prevent tumor cell metastasis. These results strongly suggest that oral administration of JTT caused increase in the production of IL-12, which is responsible for the activation of both NK cell and NKT cell, in the lungs and results in inhibition of B16 melanoma cell metastasis in the lungs.

Human Liver-Specific Nanocarrier in a Novel Mouse Xenograft Model Bearing Noncancerous Human Liver Tissue

For the targeted delivery of genes and drugs to the human liver, hepatitis B virus (HBV) envelope L particles, which form hollow nanoparticles and display a peptide that is indispensable for liver-specific infection by HBV in humans, should be a useful tool. To test the efficacy of these particles in vivo, in the present study we generated a small animal model harboring a functional human liver tissue xenograft. An anti-asialo GM1 antibody was administered to SCID mice to induce the depletion of natural-killer-cell-dependent immune responsibility and then the mice underwent transplantation of a noncancerous liver tissue originating in humans into the kidney capsule. Interestingly, human liver tissues were engrafted in 58% of the animals at least for 14 days shown by a human hepatocyte-specific antibody. The engineered HBV nanoparticles which contained fluorescent chemicals could selectively bind to the xenograft in these immune-deficient mice when they were administered systemically. These results suggested that the model animal was usable to demonstrate the efficacy of the nanoparticles that could deliver chemicals specific to the normal human liver tissue by systemic administration, which will facilitate the study of human liver cell biology, drug metabolism and infections with hepatotropic viruses.

Characteristics of Immune Reconstitution by Mixed Umbilical Cord Blood Transplantation In Mice: Predominant Engraftment of Major Histocompatibility Complex (MHC)-Matched Umbilical Cord Blood Stem Cell In Recipients' Bone Marrow.

AbstractAbstract 3721 Background:Currently, umbilical cord blood cells (UCBCs) are used as the primary source of hematopoietic stem cells (HSCs) for transplantation instead of bone marrow cells (BMCs). UCBC transplantation has several advantages over BMC transplantation, including the much larger size of the available donor pool, the rich proportion of hematopoietic progenitor cells, and the low content of mature T cells that might cause a graft-versus-host reaction. However, the limited quantity of cord blood samples that can be obtained from a pregnant woman is considered to be a disadvantage of UCBC transplantation. To overcome quantity limitations, the use of mixed cord blood might be necessary. Due to the lack of appropriate animal models, the analyses of the differentiation capacity of mixed UCBCs in recipients have been limited to in vivo xenogeneic experiments and clinical observations. In this study, we evaluated the reconstitution and functioning of immune systems induced by mixed UCBC transplantation in mice. Method:To deplete NK cells, female C57BL/6 (B6) [H-2b] recipient mice were intraperitoneally administered rabbit anti-asialo GM1 antibody 1 day before transplantation. On the following day, the B6 recipients irradiated with a radiation dose of 9 Gray were transplanted with 3 different combinations of mixed UCBC {[1] GFP-Tg B6 (H-2b) and C3H (H-2k); [2] GFP-Tg B6 (H-2b) and BALB/c (H-2d); and [3] C3H (H-2k) and BALB/c (H-2d)}, with each combination containing equal number of cells. Engraftment of cells in the recipients' peripheral blood was detected by flow cytometric analysis using a specific antibody against lineage markers such as CD3e (T cells), CD45R/B220 (B cells), CD11b (macrophages), or Ly-6G (granulocytes) at intervals of 4 weeks for 16 weeks. In addition, the donor origin of each lineage population was determined by H-2Kk and/or H-2Kd antibody staining. GFP+ lineage cells were found to have B6 donor origin. The dermis samples harvested from the B6, C3H, and BALB/c mice were simultaneously skingrafted on the shaved backs of the recipients at over 16 weeks after transplantation. After the recipients rejected the skin graft, alloreactive T cell and antibody responses were examined to evaluate the functional maturity of the reconstituted immune system in the recipients. Result:The survival rates of the recipient mice at 16 weeks after transplantation were as follows: (1), 73%, 8/11; (2), 92%, 12/13; and (3), 100%, 1/1. Flow cytometric analysis showed that cells of all lineages were reconstituted by only GFP+ cells in almost all the B6 recipients transplanted with mixed UCBCs: (1), 50%, 4/8 and (2), 100%, 12/12. This findings indicated predominant bone marrow engraftment of UCBC-HSCs from MHC-matched B6 donors. Furthermore, the allogeneic UCBCs in the recipients transplanted with combination [3] were eliminated from the recipients, and their lineage cells (GFP− H-2Kk− H-2Kd−) in peripheral blood were derived from the B6 recipient's own X-ray-resistant HSC in the bone marrow. The recipient mice in which the immune system was reconstituted by UCBC-HSCs from the B6 donors accepted the skin graft from the B6 donors, but completely rejected the skin grafts from the C3H and BALB/c donors. This finding indicates that both CD8+ killer and CD4+ helper T cells were functionally mature in the recipients. Furthermore, the functional competence of both cellular and humoral immunity in the recipients that rejected the skin grafts from the C3H and BALB/c donors was determined by evaluating alloreactive T cell and antibody responses against H-2k and H-2d in an in vitro experiment. Conclusion:This study showed the promising potential of transplantation of mixed UCBCs for achieving high survival rates and functional immune reconstitution. We found that only MHC-matched HSCs were involved in hematopoiesis in the recipients' bone marrow. This finding suggests the presence of a surveillance system in bone marrow with the ability to distinguish self from non-self with different MHC antigens. Disclosures:No relevant conflicts of interest to declare.

Interleukin-6 expression in response to innate immune regulatory factor stimulation

Innate immune regulatory factor (IIRF) is a serum-derived biological response modifier, or agent that modifies/bolsters the pathogenic immune response. IIRF has been shown to provide protection (e.g., increase host survival) against a variety of pathogenic challenges including Salmonella typhimurium and Pasteurella multocida. In this report, we analyzed cytokine production in mice, whole blood and cultured cells in response to IIRF challenge. When IIRF was administered to mice, it stimulated the release of pro-inflammatory cytokines. Both IL-6 and IL-10 were temporally stimulated when IIRF was introduced. Serum IL-6 peaked at 3 h (3957 pg/ml) before returning to baseline by 8 h, while IL-10 began to appear at 3 h, peaked at 8 h (734.5 pg/ml), and returned to baseline by 36 h. IIRF challenge also increased the expression of two positive acute phase proteins: haptoglobin increased 61-fold, while serum amyloid A levels increased ∼3500-fold in mice. A whole blood immunoassay indicated the effect of IIRF on production of IL-6 was dose- and time-dependent. Anti-asialo GM 1.1 provided to partially (> 95%) eliminate functional natural killer cells elevated IL-6 in whole blood. IIRF was also able to induce the production of IL-6 in cultured human monocytic THP-1 cells. Based upon this study and previously reported data, we propose that IIRF activates the innate immune response via induction of IL-6 production.

Cholesterol Lipids of Borrelia burgdorferi Form Lipid Rafts and Are Required for the Bactericidal Activity of a Complement-Independent Antibody

Borrelia burgdorferi, the agent of Lyme disease, is unusual as it contains free cholesterol and cholesterol glycolipids. It is also susceptible to complement-independent bactericidal antibodies, such as CB2, a monoclonal IgG1 against outer surface protein B (OspB). We find that the bactericidal actionof CB2 requires the presence of cholesterol glycolipids and cholesterol. Ultrastructural, biochemical, and biophysical analysis revealed that the bacterialcholesterol glycolipids exist as lipid raft-like microdomains in the outer membrane of cultured and mouse-derived B. burgdorferi and in model membranes from B. burgdorferi lipids. The order and size of the microdomains are temperature sensitive and correlate with the bactericidal activity of CB2. This study demonstrates the existence of cholesterol-containing lipid raft-like microdomains in a prokaryote, and we suggest that the temperature dependence of B. burgdorferi lipid raft organization may have significant implications in the transmission cycle of the spirochetes which are exposed to a range of temperatures.

Liver Xeno-Repopulation with Human Hepatocytes in Fah−/−Rag2−/− Mice after Pharmacological Immunosuppression

Functional human hepatocytes xeno-engrafted in mouse liver can be used as a model system to study hepatitis virus infection and vaccine efficacy. Significant liver xeno-repopulation has been reported in two kinds of genetically modified mice that have both immune deficiency and liver injury-induced donor hepatocyte selection: the uPA/SCID mice and Fah(-/-) Rag2(-/-)Il2rg(-/-) mice. The lack of hardy breeding and the overly elaborated technique in these two models may hinder the potential future application of these models to hepatitis virus infection and vaccination studies. Improving the transplantation protocol for liver xeno-repopulation from human hepatocytes will increase the model efficiency and application. In this study, we successfully apply immunosuppressive drug treatments of anti-asialo GM1 and FK506 in Fah(-/-)Rag2(-/-) mice, resulting in significant liver xeno-repopulation from human hepatocytes and human fetal liver cells. This methodology decreases the risk of animal mortality during breeding and surgery. When infected with hepatitis B virus (HBV) sera, Fah(-/-)Rag2(-/-) mice with liver xeno-repopulation from human hepatocytes accumulate significant levels of HBV DNA and HBV proteins. Our new protocol for humanized liver could be applied in the study of human hepatitis virus infection in vivo, as well as the pharmacokinetics and efficacy of potential vaccines.

An unexpected twist in alopecia areata pathogenesis: are NK cells protective and CD49b+ T cells pathogenic?

Natural killer (NK) cells have become a recent focus of interest in alopecia areata (AA) research. To further investigate their role in an established mouse model of AA, lesional skin from older C3H/HeJ mice with AA was grafted to young C3H/HeJ female mice, and NK cells were depleted by continuous administration of rabbit anti-asialo GM1. As expected, this significantly reduced the number of pure NK cells in murine skin, as assessed by NKp46 quantitative immunohistochemistry. Quite unexpectedly, however, the onset of hair loss in C3H/HeJ mice was accelerated, rather than retarded. NK cell depletion was accompanied by a significant increase in the number of perifollicular CD49b+T cells in the alopecic skin of anti-asialo GM1-treated mice. These findings underscore the need to carefully distinguish in future AA research between pure NK cells and defined subsets of CD49b+ lymphocytes, as they may exert diametrically opposed functions in hair follicle immunology and immunopathology.

125 Beta-blockers inhibit tumour cell migration: a potential use as anti-metastatic drugs

Liver metastases are a major cause of colorectal cancer death, and the perioperative period is believed to critically affect the metastatic process. Here we tested whether blocking excess release of catecholamines and prostaglandins during surgical procedures of different extent can reduce experimental liver metastasis of the syngeneic CT26 colon cancer in female and male BALB/c mice. Animals were either treated with the beta-blocker, propranolol, the COX-2 inhibitor, etodolac, both drugs, or vehicle. The role of NK cells in controlling CT26 hepatic metastasis and in mediating the effect of the drugs was assessed by in vivo depletion or stimulation of NK cells, using anti-asialo GM1 or CpG-C, respectively. Surgical extent was manipulated by adding laparotomy to small incision, extending surgical duration, and enabling hypothermia. The results indicated that combined administration of propranolol and etodolac, but neither drug alone, significantly improved host resistance to metastasis. These beneficial effects occurred in both minor and extensive surgeries, in both sexes, and in two tumor inoculation approaches. NK cell-mediated anti-CT26 activity is involved in mediating the beneficial effects of the drugs. Specifically, CpG-C treatment, known to profoundly activate mice marginating-hepatic NK cytotoxicity, reduced CT26 hepatic metastases; and NK-depletion increased metastases and prevented the beneficial effects of the drugs. Overall, given prevalent perioperative psychological and physiological stress responses in patients, and ample prostaglandin release by colorectal tumors and injured tissue, propranolol and etodolac could be tested clinically in laparoscopic and open colorectal surgeries, attempting to reduce patients’ metastatic disease.

NK Cells Influence Both Innate and Adaptive Immune Responses after Mucosal Immunization with Antigen and Mucosal Adjuvant

NK cells were found to be recruited in a temporally controlled manner to the nasal-associated lymphoid tissue and the cervical lymph nodes of mice after intranasal immunization with Ag85B-early secreted antigenic target 6 kDa from Mycobacterium tuberculosis mixed with Escherichia coli heat-labile toxin as adjuvant. These NK cells were activated and secreted a diverse range of cytokines and other immunomodulators. Using Ab depletion targeting anti-asialo GM1, we found evidence for altered trafficking, impaired activation, and cytokine secretion of dendritic cells, macrophages, and neutrophils in immunized NK cell-depleted mice compared with control animals. Analysis of Ag-specific immune responses revealed an attenuated Ab and cytokine response in immunized NK cell-depleted animals. Systemic administration of rIL-6 but not rIFN-gamma significantly restored immune responses in mice depleted of NK cells. In conclusion, cytokine production, particularly IL-6, via NK cells and NK cell-activated immune populations plays an important role in the establishment of local innate immune responses and the consequent development of adaptive immunity after mucosal immunization.

NK cells are required for defense against pulmonary K. pneumoniae infection in mice and mediate optimal production of protective cytokines, including IL-22. (37.41)

Abstract Among the products of Th17 cells are IL-17A and IL-22, which are required for protection against extracellular pathogens, such as K. pneumoniae. Although published data suggest that T cells are the source of these cytokines during pulmonary infection with K. pneumoniae, we found that Rag2-/- mice did not differ from wild-type mice in survival, bacterial counts or levels of IL-22 in the lungs after intratracheal infection with K. pneumoniae. By contrast, K. pneumoniae-infected Rag2-/-γc-/- mice failed to produce IL-22 and had higher bacterial counts as compared with wild-type controls. Together, these data suggested a possible role for NK cells in host defense, and in the production of IL-22, during K. pneumoniae infection. Lung NK cells showed a conventional surface phenotype and lacked NKp46 and CCR6, markers for IL-22-producing (“NK22”) NK cells. Moreover, we found no increase in lung NK cells during infection. However, as compared with control mice, mice depleted of NK-cells using anti-Asialo GM1 showed decreased survival and higher bacterial counts, as well as increased dissemination of K. pneumoniae to blood and liver. In addition, NK cell depletion led to decreased production of IL-22 and other cytokines important for protection, including IL-12, TNF-α, and IL-6. Together, our data show that NK cells are required for defense against pulmonary K. pneumoniae infection and optimal production of protective cytokines, including IL-22. Supported by the IRP, NIH, NIAID.

Acute myelogenous leukemia blasts contribute to a bone marrow-stroma in in vivo NOD/SCID mouse system

Abstract Abstract 1621 Poster Board I-647 Aims: Cancer stem cell theory has been developed, and whose precise characteristics have been reported. However, there have been no reports on the differentiation of cancer stem cells into the environmental stromal cells. We reported previously that non-adherent acute myelogenous leukemia (AML) cells were differentiated into myofibroblasts to create a microenvironment for proliferation of AML blasts in vitro. In this report we demonstrate that with severe combined immunodeficiency (SCID) mouse system AML blast cells also convert to myofibroblasts to form stroma in vivo. Materials and Methods Bone marrow cells were collected from informed AML (M2) patients who had chromosomal translocation of RUNX1 and ETO, from which mononuclear cells were separated with density-gradient sedimentation method. After discarded an adherent cell-fraction, the non-adherent mononuclear cells were injected to the 3.0 Gray-irradiated non-obese diabetes (NOD)/SCID mouse intravenously. For the inactivation of NK cells, anti-Asialo GM1 antibody was injected intra-peritoneally prior to the transplantation, and on each 11th day thereafter. Blood was collected to monitor Runx1 and ETO fusion transcript, and mice were sacrificed after chimeric mRNA was observed. Bone marrow cells were obtained, and sorted with anti-human CD133 antibody and -CD106 to select AML-derived human stromal myofibroblasts referred to the in vitro data. The isolated positive fraction was further cultured, and the biological and the molecular characteristics were analyzed. Results and Discussion When non-adherent AML (M2) blast cells were transplanted to NOD/SCID mice, cells were engrafted after 10 weeks. In murine bone marrow cells human stromal cells were identified, in which RUNX1 and ETO gene was fused with FISH analysis. When the parental AML blast cells were cultured on the expanded AML-derived myofibroblasts, AML cells grew extensively. These results indicate that AML cells can create their own microenvironment for proliferation in vivo. Disclosures No relevant conflicts of interest to declare.

Natural killer T cells are important in the pathogenesis of asthma: The many pathways to asthma

The pathogenesis of bronchial asthma, a complex trait associated with a number of environmental factors (eg, allergens, infection, air pollution, exercise, and obesity), involves multiple cell types and several distinct cellular and molecular pathways. These pathways include adaptive and innate immunity and involve T(H)2 cells, mast cells, basophils, eosinophils, neutrophils, airway epithelial cells, and subsets of a newly described cell type called natural killer T (NKT) cells. A role for subsets of NKT cells in asthma has been suggested by extensive studies in animal models of asthma induced with allergen, viral infection, ozone exposure, or bacterial components, suggesting that NKT cells function in concert with T(H)2 cells or independently of adaptive immunity in causing airway hyperreactivity. The clinical relevance of NKT cells in human asthma is supported by the observation that NKT cells are present in the lungs of some patients with asthma, particularly patients with severe, poorly controlled asthma, although additional research is required to more precisely define the specific role of NKT cells in human asthma. These studies of NKT cells greatly expand our understanding of possible mechanisms that drive the development of asthma, particularly in the case of asthma associated with neutrophils, viral infection, and air pollution.

Adoptive Transfer of Primed T Cells Mediates a Graft-Versus-Leukemia Effect against Pediatric ALL.

Abstract 1340Poster Board I-362 BackgroundPediatric Acute Lymphoblastic Leukemia (ALL) is the most common childhood malignancy. While current upfront therapy will cure over 80% of patients, treating relapse remains a major challenge. Blood or marrow transplantation (BMT) offers a therapeutic option but approximately 50% of patients undergoing BMT will not survive with relapse being the most common cause of death. Donor lymphocyte infusions (DLIs) have demonstrated efficacy in myeloid leukemia but are much less effective for ALL. Thus, we explored strategies to improve on the potency of the graft versus leukemia response against pediatric ALL using a murine model developed from E2a-PBX1 transgenic mice that express a recurring translocation t(1;19) present in approximately 5% of all pediatric ALL. MethodsE2a-PBX1 was administered intravenously (IV). Irradiated E2a-PBX1 was administered intraperitoneally for immunization both in the vaccine protection model and to prime DLI donors in the BMT models. Transplanted mice were lethally irradiated and given T-cell-depleted bone marrow cells followed by purified T cells as a DLI. In the syngeneic model, C57Bl/6 mice were used as both recipients and donors so that both donor cells were syngeneic to the tumor. In the allogeneic model, C57Bl/6 recipients received bone marrow and T cells from MHC-matched C3H.SW donors that are minor antigen-mismatched to both tumor and recipient, analogous to the clinical BMT setting. ResultsWe confirmed that E2a-PBX1 is a pre-B cell ALL that expresses B220, BP-1, CD43 and the alpha chain of the IL-7 receptor. The distribution of leukemia is similar to that observed in humans with bone marrow, lymph node, liver, spleen and central nervous system involvement confirmed by necropsy and imaging following injection of luciferase-expressing cells. Amazingly, E2a-PBX1 is lethal with as few as with as 1×102 cells (5/5 mice) in sublethally irradiated mice (250 cGy) and 1×104 cells in unirradiated mice (4/5 mice). Vaccination prior to tumor challenge protected 90-100% of mice from leukemia development. Mice that successfully rejected E2a-PBX1 were protected against rechallenge with a higher cell dose. Antibody depletion of either CD4+ or CD8+ T cells did not affect vaccine-mediated protection. However, depletion of both CD4+ and CD8+ T cells significantly diminished vaccine efficacy (n=7/group, p=0.0063). Interestingly, although data from killer inhibitory receptor (KIR) mismatched BMT would suggest that NK cells are less potent in ALL than in AML, antibody depletion of NK cells with either anti-asialo GM1 or anti-NK1.1 also had a significant impact on survival in our model. We next tested the therapeutic effect of purified T cells injected IV after E2aPBX1 injection. Administration of naive T cells (6×106) following syngeneic BMT was not effective at treating E2a-PBX1. The presence of minor antigen differences between donor T cells and tumor following allogeneic BMT did not increase the efficacy of naïve T cells consistent with the poor response rate to DLI in patients with ALL. However, primed T cell DLI from a donor immunized by irradiated E2a-PBX1 resulted in a statistically significant prolongation of survival in both syngeneic (p=.0067) and allogeneic (p=.008) BMT models. Conclusion: When administered in our allogeneic transplant model, E2a-PBX1 can be used to model relapse of pediatric ALL post-BMT. Naïve T cells (analogous to DLI given post-relapse in the clinic) were ineffective at treating ALL our model. However, priming of the DLI significantly prolonged survival demonstrating that the poor efficacy of DLI in ALL is not due to an inherent resistance of ALL to a T cell response. Our data supports the use of T cells to treat pediatric ALL relapse post-BMT but suggests manipulation of the T cell response will be required. DisclosuresNo relevant conflicts of interest to declare.

Acute Myelogenous Leukemia Blasts Contribute to a Bone Marrow-Stroma in In Vivo NOD/SCID Mouse System..

Abstract 1621Poster Board I-647Aims: Cancer stem cell theory has been developed, and whose precise characteristics have been reported. However, there have been no reports on the differentiation of cancer stem cells into the environmental stromal cells. We reported previously that non-adherent acute myelogenous leukemia (AML) cells were differentiated into myofibroblasts to create a microenvironment for proliferation of AML blasts in vitro. In this report we demonstrate that with severe combined immunodeficiency (SCID) mouse system AML blast cells also convert to myofibroblasts to form stroma in vivo. Materials and MethodsBone marrow cells were collected from informed AML (M2) patients who had chromosomal translocation of RUNX1 and ETO, from which mononuclear cells were separated with density-gradient sedimentation method. After discarded an adherent cell-fraction, the non-adherent mononuclear cells were injected to the 3.0 Gray-irradiated non-obese diabetes (NOD)/SCID mouse intravenously. For the inactivation of NK cells, anti-Asialo GM1 antibody was injected intra-peritoneally prior to the transplantation, and on each 11th day thereafter. Blood was collected to monitor Runx1 and ETO fusion transcript, and mice were sacrificed after chimeric mRNA was observed. Bone marrow cells were obtained, and sorted with anti-human CD133 antibody and -CD106 to select AML-derived human stromal myofibroblasts referred to the in vitro data. The isolated positive fraction was further cultured, and the biological and the molecular characteristics were analyzed. Results and DiscussionWhen non-adherent AML (M2) blast cells were transplanted to NOD/SCID mice, cells were engrafted after 10 weeks. In murine bone marrow cells human stromal cells were identified, in which RUNX1 and ETO gene was fused with FISH analysis. When the parental AML blast cells were cultured on the expanded AML-derived myofibroblasts, AML cells grew extensively. These results indicate that AML cells can create their own microenvironment for proliferation in vivo. DisclosuresNo relevant conflicts of interest to declare.

Chronic Myelogenous Leukemia Cells Contribute to a Bone Marrow-Stroma in In Vivo NOD/SCID Mouse System.

Abstract 2191Poster Board II-168Aims: The stroma-forming cells in a bone marrow are derived from hematopoietic stem cells. We reported previously that non-adherent leukemia blast cells converted into myofibroblasts to create a microenvironment for proliferation of leukemia blasts in vitro. In this report we demonstrate that with severe combined immunodeficiency (SCID) mouse system chronic myelogenous leukemia (CML) cells are also differentiated into myofibroblasts to contribute to a bone marrow-stroma in vivo. Materials and Methods: Bone marrow cells were collected from informed CML patients, from which mononuclear cells were separated with density-gradient sedimentation method. After discarded an adherent cell-fraction, non-adherent mononuclear cells were injected to the priory 2.5 Gray-irradiated non-obese diabetes (NOD)/SCID mice intravenously. For the inactivation of NK cells, anti-Asialo GM1 antibody was injected intra-peritoneally prior to the transplantation, and on each 11th day thereafter. Blood was collected to monitor Bcr-Abl transcript, and mice were sacrificed after chimeric mRNA was demonstrated. Bone marrow cells were obtained, and sorted with anti-human CD133 antibody and -CD106 to select CML-derived human stromal myofibroblasts referred to the in vitro data. The isolated positive fraction was further cultured, and the biological and the molecular characteristics were analyzed. Results and Discussion: When non-adherent CML cells were transplanted to NOD/SCID mice, CML cells were engrafted after 2 months. In the murine bone marrow human stromal cells were identified, in which BCR and ABL gene was fused with FISH analysis. When the parental CML cells were cultured on the CML-derived myofibroblasts, CML cells grew extensively in a vascular endothelial growth factor-A-dependent fashion. These results indicate that CML cells can create their own microenvironment for proliferation in vivo. Disclosures:No relevant conflicts of interest to declare.

Mechanisms of Antigen Dependent and Independent Rejection of High Grade B-Cell Lymphoma in a Murine Model.

Abstract 4091Poster Board III-1026The rejection of Non Hodgkin Lymphomas expressing foreign, for example viral, antigens is compromised despite the presence of specific T-cells. The underlying immunosuppressive mechanisms are poorly understood. Using a transgenic mouse lymphoma model, where the proto-oncogene c-myc is driven by parts of the immunoglobulin lambda locus representing a t(8;22) translocation as found in Burkitt's lymphoma, we investigated the anti-lymphoma activity of specific T-cells. By retroviral transduction of a specific foreign antigen (chicken Ovalbumin-IRES-GFP, OVA) into cell lines from primary c-myc transgenic lymphomas we established a syngeneic model that would allow us to address the role of interferon gamma signalling in rejection of high grade lymphomas. All primary lymphoma cells displayed normal MHC class I and II levels on the surface when compared to wildtype splenic B-cells. This expression could be enhanced by treatment with interferon gamma (IFNg,100U/ml) up to 10 fold. When retrovirally OVA-transduced lymphoma cells were injected into either wildtype or GFP transgenic recipients, animals displayed a significant delay in lymphoma growth compared to non transduced or IRES-GFP transduced control cell lines. 80% of the recipient mice rejected OVA expressing lymphomas. By contrast, we observed 100% mortality when GFP expressing control lymphomas were injected in GFP transgenic recipients, which are tolerant for GFP. Developing OVA expressing lymphomas (20%) displayed a loss of GFP expression indicating a selection for antigen negative cells (p=0.001). In spleens from mice rejecting OVA-expressing lymphomas we found up to 1.8% SIINFEKL specific T-cells. To gain more mechanistic insights, we transferred OVA expressing lymphoma cells into IFNg, Stat1-/- or IFNg-/-receptor deficient recipients. Lack of STAT1-/- or IFNg-receptor on the recipient side or inability to secrete IFNg was associated with fast lymphoma progression and growth was not different when compared to non transduced, antigen negative cell lines. When IFNg-receptor or STAT1 deficient OVA expressing cell lines were transferred into wildytpe mice, rejection was not influenced. Outgrowing OVA expressing lymphomas in wildtype mice displayed a high MHC class I and II expression compared to the cell line prior to injection. MHC induction was absent in lymphomas transferred to Stat1 or IFNg deficient recipients. Depletion of NK cells by anti AsialoGM antibody in wildtype recipients resulted in a significant reduction of disease free survival (80% vs. 50%, p=0.002) and animals developed larger tumors which were eventually rejected resulting in a comparable overall survival. In peripheral blood of NK depleted mice significantly more OVA specific T-cells were detectable through pentamer staining. When lymphoma cell lines were injected into Rag1-/- mice, NK cell mediated rejection was also significantly impaired upon depletion. Our results suggest that T-cell mediated rejection of high grade B-cell lymphomas is strongly dependent on host IFNg secretion and that NK cells substantially contribute to T-cell mediated rejection. Disclosures:No relevant conflicts of interest to declare.

Circulating blood monocytes traffic to and participate in the periprosthetic tissue inflammation

To examine the trafficking of human circulating blood monocytes and their influence on the inflammation of periprosthetic tissues using a novel mouse-human chimera model. Periprosthetic tissue and bone chips from patients with aseptic prosthetic loosening were implanted into the muscles of immune-deficient SCID mice depleted of host macrophages by periodic intraperitoneal injection of anti-asialo GM1 rabbit sera (ASGM1). Autologous patient peripheral blood monocytes (PBMCs) were labeled with PKH2 fluorescent dye and injected intraperitoneally into the implanted animals. Mice were sacrificed 14 days after PBMC transfusion for molecular and histological analyses. Patient periprosthetic tissues were well tolerated in SCID mice and preserved a high level of viability. Cell trafficking studies revealed the accumulation of fluorescent PBMC within the xenografts, with total cell counts in the xenografts significantly increased following the systemic PBMC infusion. PBMC infusion also promoted the expression of IL-1, IL-6, TNFalpha, and RANK within the periprosthetic tissue. Systemic PBMC migrated to the implanted periprosthetic tissues and contributed to the local inflammation. The data provide evidence that circulating blood monocytes may play a role in pathologic process during aseptic loosening of total joint replacement.

Changes in Mucosal Homeostasis Predispose NHE3 Knockout Mice to Increased Susceptibility to DSS-Induced Epithelial Injury

NHE3 is a target of inhibition by proinflammatory cytokines and pathogenic bacteria, an event contributing to diarrhea in infectious and idiopathic colitis. In mice, NHE3 deficiency leads to mild diarrhea, increased intestinal expression of interferon (IFN)-gamma, and distal colitis, suggesting its role in epithelial barrier homeostasis. Our aim was to investigate the role of NHE3 in maintaining mucosal integrity. Control or dextran sulfate sodium (DSS)-treated, 6- to 8-week-old wild-type (WT) and NHE3(-/-) mice were used for the experiments. Small intestines were dissected for further analysis. NHE3(-/-) mice have elevated numbers of CD8alpha(+) T and natural killer cells in the intraepithelial lymphocytes and lamina propria lymphocytes compartments, representing the source of IFN-gamma. NHE3(-/-) mice display alterations in epithelial gene and protein expression patterns that predispose them to a high susceptibility to DSS, with accelerated mortality resulting from intestinal bleeding, hypovolemic shock, and sepsis, even at a very low DSS concentration. Microarray analysis and intestinal hemorrhage indicate that NHE3 deficiency predisposes mice to DSS-induced small intestinal injury, a segment never reported as affected by DSS, and demonstrate major differences in the colonic response to DSS challenge in WT and NHE3(-/-) mice. In NHE3(-/-) mice, broad-spectrum oral antibiotics or anti-asialo GM1 antibodies reduce the expression of IFN-gamma and iNOS to basal levels and delay but do not prevent severe mortality in response to DSS treatment. These results suggest that NHE3 participates in mucosal responses to epithelial damage, acting as a modifier gene determining the extent of the gut inflammatory responses in the face of intestinal injury.

Natural killer cells suppress autoimmune myocarditis in mice (48.23)

Abstract Autoimmune myocarditis is induced by the adoptive immune response; however, there is evidence that the innate immune system plays a major role in modulating disease. Natural killer (NK) cells can function in early innate responses, as well affecting the later adaptive immune response. Depletion of NK cells has been shown to exacerbate the development of virus-induced myocarditis. We hypothesized that NK cells also modulate the virus-free model of myosin-induced experimental autoimmune myocarditis (EAM). To study the action of NK cells, we immunized BALB/c mice on days 0 and 7 with myocarditogenic peptide MyHC (614-629) in complete Freund adjuvant. The mice were treated with PBS, rabbit IgG or anti-asialo GM1 antibody every day for 6 days prior to the first immunization and every 2 days following day 0 until day 21. We found that depleting NK cells significantly increased the severity of disease, and serum antibodies specific to the myocarditogenic peptide. Splenic CD4+ T cells were significantly more activated in NK depleted mice compared to controls. Cytokine analysis of heart homogenates revealed a significant increase in eotaxin and IL-5 in the hearts of mice that were depleted of NK cells. Our data suggest that NK cells suppress the adoptive immune response and modulate eosinophil migration and T cell activation during EAM in mice.

NK cells in the CD19 − B220 + bone marrow fraction are increased in senescence and reduce E2A and surrogate light chain proteins in B cell precursors

E2A encoded proteins, key transcriptional regulators in B lineage specification and commitment, have been shown to decrease in B cell precursors in old age. E2A regulates genes encoding the surrogate light chain proteins λ5 and VpreB. In old age, B cell precursors express less surrogate light chain and this results in compromised pre-B cell receptor function and diminished expansion of new pre-B cells in senescence. Herein, we show that aged bone marrow has increased Hardy Fraction A (CD19 − B220 +) cells, including NK cells, that can inhibit both E47 (E2A) protein and surrogate light chain protein expression in B cell precursors. In vitro, NK-associated inhibition of E47 protein is contact-independent and partially reversed by neutralization of TNFα. In vivo, depletion of NK cells in aged mice by treatment with anti-asialo GM1 antibody led to restoration of surrogate light chain protein levels to that typical of young B cell precursors. These studies suggest that NK cells, within the CD19 − B220 + bone marrow cell fraction, may contribute to a bone marrow microenvironment that has the potential to negatively regulate E47 (E2A) as well as surrogate light chain levels in B cell precursors in old age.