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Cardiac exposure to ionizing radiation can damage both the microvasculature and coronary arteries, as well as increase the long-term risk of heart disease, myocardial fibrosis, and conduction abnormalities. Therapeutic agents capable of promoting recovery from radiation injury to the heart are limited. Growth hormone is linked to improved cardiac function following injury. Here, we leveraged a cynomolgus macaque model to determine the long-term outcomes of recombinant human growth hormone (rhGH) therapy on the heart following low-dose ionizing radiation. Macaques were exposed to 2 Gy radiation, treated with rhGH for one month, and assessed after 2 years. Overall, plasma lipid profile, cardiac function, and coronary artery disease were similar between rhGH and placebo treated animals. However, a subgroup of rhGH-treated animals exhibited more extensive atherosclerotic plaques in the coronary arteries. Together, these findings indicate that transient human growth hormone therapy subsequent to a single low dose of ionizing radiation involving the heart does not result in long-term changes to plasma cholesterol but may promote exacerbated coronary artery disease in a subset of individuals.
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OBJECTIVES: In the United States, cannabis is legal for adult recreational use in 24 states and Washington, DC. Unintentional pediatric cannabis exposures have increased in many states following legalization.We evaluated the relationship between recreational cannabis legalization and the rates of unintentional pediatric exposures in a neighboring state that had not undergone legalization. METHODS: We obtained cannabis exposure cases for children 0-12 years from the New Mexico Poison and Drug Information Center electronic database. Only deidentified patient data from closed-case exposure encounters were abstracted. Data were grouped as precommercial and postcommercial availability in neighboring Colorado, demarcated by January 2014. We coded cannabis products as edible, not edible, or unknown. For bivariable comparisons, we used odds ratios, risk ratio, χ2 test, and Wilcoxon rank sum test. We chose a type 1 error rate of 0.05 to determine significance. RESULTS: There were 269 exposures over 24 years of calls. Following neighboring legalization, the median number of exposures per year increased from 4 (interquartile range 2, 5) to 24.5 (16.5, 34), the median age increased from 1.9 to 3.0 (P = 0.007), and the relative risk of the exposure involving edible products was double (relative risk = 2.0, 95% confidence interval = 1.6, 2.6). The severity of the exposures' medical effects also increased (P = 0.008). CONCLUSIONS: The number, severity, and type of pediatric cannabis exposures in New Mexico changed after neighboring recreational cannabis legalization. States neighboring those undergoing cannabis legalization should be prepared to respond to increased acute exposures in children.
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Extracellular vesicles (EVs) have been recognized as a novel way of cell-to-cell communication in the last several decades. It is believed that EVs exert their functions on nearby or distant cells through transfer of the cargo that they carry. In this review, we focus on EVs produced by endothelial cells, with emphasis on their role in hematopoiesis. We first describe how endothelial cells interact with hematopoietic stem/progenitor cells during development and in disease conditions. We then discuss EVs, ranging from their subtypes to isolation methods and analysis of EVs. With the above background information, we next review the literature related to endothelial cell derived EVs (ECEVs), including physiological functions and their clinical uses. In the last sections, we summarize the current results about the effect of ECEVs on hematopoiesis under physiological and stress conditions.
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Células Endoteliales , Vesículas Extracelulares , Hematopoyesis , Humanos , Vesículas Extracelulares/fisiología , Vesículas Extracelulares/metabolismo , Células Endoteliales/citología , Células Endoteliales/fisiología , Animales , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Células Madre Hematopoyéticas/metabolismo , Comunicación CelularRESUMEN
In this study, we examine the adiabat-to-diabat (ATD) angles for trajectories in 2-dimensional vibrational subspace of the seam space of two degenerate states. In circulating around the tangential touching degeneracy center, the ATD angle is changed by 2 π ${2\pi }$ or 0, similar to the Renner-Teller problem and the pseudo-Jahn-Teller problem, respectively. These ATD angle profiles may be indistinguishable from those of circulating multiple conical intersections or a pseudo-Jahn-Teller center. Methods to discern those seemingly indistinguishable cases are proposed. A sharp zigzag variation of the ATD angle is seen as a feature for trajectories that graze a pseudo-Jahn-Teller-type tangential touching center, in contrast to the monotonic steep variation for grazing a conical intersection or a Renner-Teller-type tangential touching center.
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De novo immune responses to myeloid and other blood-borne tumors are notably limited and ineffective, making our ability to promote immune responses with vaccines a major challenge. While focus has been largely on cytotoxic cell-mediated tumor eradication, B-cells and the antibodies they produce also have roles in anti-tumor responses. Indeed, therapeutic antibody-mediated tumor cell killing is routinely employed in patients with hematolymphoid cancers, but whether endogenous antibody responses can be incited to blood-born tumors remains poorly studied. A major limitation of immunoglobulin therapies is that cell surface expression of tumor-associated antigen (TAA) targets is dynamic and varied, making promotion of polyclonal, endogenous B cell responses appealing. Since many TAAs are self-antigens, developing tumor vaccines that enable production of antibodies to non-polymorphic antigen targets remains a challenge. As B cell responses to RNA vaccines are known to occur, we employed the Viral Replicon Particles (VRP) which was constructed to encode mouse FLT3. The VRP-FLT3 vaccine provoked a rapid IgG B-cell response to this self-antigen in leukemia and lymphoma mouse models. In addition, IgGs to other TAAs were also produced. Our data suggest that vaccination with RNA viral particle vectors incites a loss of B-cell tolerance that enables production of anti-tumor antibodies. This proof of principle work provides impetus to employ such strategies that lead to a break in B-cell tolerance and enable production of broadly reactive anti-TAA antibodies as potential future therapeutic agents for patients with hematolymphoid cancers.
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Alphavirus , Vacunas contra el Cáncer , Neoplasias , Vacunas Virales , Animales , Antígenos de Neoplasias , Humanos , Inmunoglobulina G , Ratones , Neoplasias/genética , ReplicónRESUMEN
Alloreactive donor T cells undergo extensive metabolic reprogramming to become activated and induce graft-versus-host disease (GVHD) upon alloantigen encounter. It is generally thought that glycolysis, which promotes T cell growth and clonal expansion, is employed in this process. However, conflicting data have been reported regarding the requirement of glycolysis to induce T cell-mediated GVHD due to the lack of T cell-specific treatments using glycolysis inhibitors. Importantly, previous studies have not evaluated whether graft-versus-leukemia (GVL) activity is preserved in donor T cells deficient for glycolysis. As a critical component affecting the clinical outcome, it is necessary to assess the anti-tumor activity following treatment with metabolic modulators in preclinical models. In the present study, we utilized T cells selectively deficient for glucose transporter 1 (Glut1T-KO), to examine the role of glycolysis exclusively in alloreactive T cells without off-targeting effects from antigen presenting cells and other cell types that are dependent on glycolysis. We demonstrated that transfer of Glut1T-KO T cells significantly improved acute GVHD outcomes through increased apoptotic rates, impaired expansion, and decreased proinflammatory cytokine production. In addition to impaired GVHD development, donor Glut1T-KO T cells mediated sufficient GVL activity to protect recipients from tumor development. A clinically relevant approach using donor T cells treated with a small molecule inhibitor of glycolysis, 2-Deoxy-D-glucose ex vivo, further demonstrated protection from tumor development. These findings indicate that treatment with glycolysis inhibitors prior to transplantation selectively eliminates alloreactive T cells, but spares non-alloreactive T cells including those that protect against tumor growth. The present study has established a definitive role for glycolysis in acute GVHD and demonstrated that acute GVHD can be selectively prevented through targeting glycolysis.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Leucemia , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Glucólisis , Humanos , Linfocitos TRESUMEN
Patients with chronic graft-versus-host disease (cGVHD) have increased B cell-activating factor (BAFF) levels, but whether BAFF promotes disease after allogeneic bone marrow transplantation (allo-BMT) remains unknown. In a major histocompatibility complex-mismatched model with cGVHD-like manifestations, we first examined B-lymphopenic µMT allo-BMT recipients and found that increased BAFF levels in cGVHD mice were not merely a reflection of B-cell number. Mice that later developed cGVHD had significantly increased numbers of recipient fibroblastic reticular cells with higher BAFF transcript levels. Increased BAFF production by donor cells also likely contributed to cGVHD, because BAFF transcript in CD4+ T cells from diseased mice and patients was increased. cGVHD manifestations in mice were associated with high BAFF/B-cell ratios and persistence of B-cell receptor (BCR)-activated B cells in peripheral blood and lesional tissue. By employing BAFF transgenic (Tg) mice donor cells, we addressed whether high BAFF contributed to BCR activation in cGVHD. BAFF increased NOTCH2 expression on B cells, augmenting BCR responsiveness to surrogate antigen and NOTCH ligand. BAFF Tg B cells had significantly increased protein levels of the proximal BCR signaling molecule SYK, and high SYK protein was maintained by BAFF after in vitro BCR activation or when alloantigen was present in vivo. Using T cell-depleted (BM only) BAFF Tg donors, we found that BAFF promoted cGVHD manifestations, circulating GL7+ B cells, and alloantibody production. We demonstrate that pathologic production of BAFF promotes an altered B-cell compartment and augments BCR responsiveness. Our findings compel studies of therapeutic targeting of BAFF and BCR pathways in patients with cGVHD.
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Factor Activador de Células B/metabolismo , Trasplante de Médula Ósea/efectos adversos , Enfermedad Injerto contra Huésped/patología , Proteínas Proto-Oncogénicas c-bcr/metabolismo , Receptor Notch2/metabolismo , Quinasa Syk/metabolismo , Linfocitos T/inmunología , Animales , Factor Activador de Células B/genética , Femenino , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/metabolismo , Isoanticuerpos/inmunología , Isoantígenos/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-bcr/genética , Receptor Notch2/genética , Quinasa Syk/genética , Trasplante HomólogoRESUMEN
Prophylactic donor lymphocyte infusions (DLI) are used to augment post-transplant immune recovery to reduce both infectious complications and disease recurrence. Preclinical studies implicate the naive T-cell subset as the primary driver of graft-versus-host disease (GvHD). In this phase I dose escalation study, we assessed the safety of a DLI that was depleted of CD45RA+ naive T cells. Sixteen adult patients received a prophylactic DLI at a median of 113 days (range 76-280 days) following an HLA-identical, non-myeloablative allogeneic hematopoietic stem cell transplantation. Three patients each received the naive T-cell depleted DLI with a CD3+ dose of 1 × 105/kg, 1 × 106/kg, and 5 × 106/kg. The maximum dose of 1 × 107/kg was expanded to 7 patients. No dose-limiting grade III/IV acute GvHD or adverse events attributable to the DLI were observed at any dose level. One patient developed grade 2 acute GvHD of skin and upper intestines, and another developed moderate chronic GvHD of the lungs following the DLI. With a median follow-up of 2.8 years, 2-year progression-free and overall survival is 50.0% and 68.8%, respectively. In conclusion, these data suggest that a DLI that has been depleted of CD45RA+ naive T cells is feasible and carries a low risk of acute or chronic GvHD.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Adulto , Humanos , Transfusión de Linfocitos , Recurrencia Local de Neoplasia , Linfocitos TRESUMEN
Thrombocytopenia (TCP) may cause severe and life-threatening bleeding. While this may be prevented by platelet transfusions, transfusions are associated with potential complications, do not always work (platelet refractory) and are not always available. There is an urgent need for a synthetic alternative. We evaluated the ability of fibrinogen-coated nanospheres (FCNs) to prevent TCP-related bleeding. FCNs are made of human albumin polymerized into a 100-nm sphere and coated with fibrinogen. We hypothesized that FCNs would bind to platelets through fibrinogen-GPIIb/IIIa interactions, contributing to hemostasis in the setting of TCP. We used two murine models to test these effects: in the first model, BALB/c mice received 7.25 Gy total-body irradiation (TBI); in the second model, lower dose TBI (7.0 Gy) was combined with an anti-platelet antibody (anti-CD41) to induce severe TCP. Deaths in both models were due to gastrointestinal or intracranial bleeding. Addition of antiplatelet antibody to 7.0 Gy TBI significantly worsened TCP and increased mortality compared to 7.0 Gy TBI alone. FCNs significantly improved survival compared to saline control in both models, suggesting it ameliorated TCP-related bleeding. Additionally, in a saphenous vein bleeding model of antibody-induced TCP, FCNs shortened bleeding times. There were no clinical or histological findings of thrombosis or laboratory findings of disseminated intravascular coagulation after FCN treatment. In support of safety, fluorescence microscopy suggests that FCNs bind to platelets only upon platelet activation with collagen, limiting activity to areas of endothelial damage. To our knowledge, this is the first biosynthetic agent to demonstrate a survival advantage in TCP-related bleeding.
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Albúminas/química , Fibrinógeno/química , Fibrinógeno/farmacología , Hemorragia/complicaciones , Hemorragia/prevención & control , Nanosferas , Trombocitopenia/complicaciones , Animales , Endotelio/metabolismo , Fibrinógeno/metabolismo , Hemorragia/metabolismo , Hemorragia/fisiopatología , Ratones , Agregación Plaquetaria/efectos de los fármacos , Análisis de SupervivenciaRESUMEN
Graft-versus-host disease (GVHD) is a major source of morbidity and mortality following allogeneic hematopoietic stem cell transplant (allo-HSCT), one of the most effective approaches to treat hematopoietic malignancies.1 However, current prophylaxis regimens and treatments that reduce the detrimental effect of acute GVHD can be offset by increased incidence in opportunistic infections and relapse of the primary malignancy.2 In addition, the majority of the approaches that inhibit T cell responses are non-specific, resulting in the inhibition of both alloreactive T cells and protective T cells from the donor. Therefore, there is an increase in the demand to develop novel approaches that selectively target alloreactive T cells. One potential means to address this issue is to take advantage of the unique metabolic profile of activated T cells.
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Cellular heterogeneity is frequently observed in cancer, but the biological significance of heterogeneous tumor clones is not well defined. Using multicolor reporters and CRISPR-Cas9 barcoding, we trace clonal dynamics in a mouse model of sarcoma. We show that primary tumor growth is associated with a reduction in clonal heterogeneity. Local recurrence of tumors following surgery or radiation therapy is driven by multiple clones. In contrast, advanced metastasis to the lungs is driven by clonal selection of a single metastatic clone (MC). Using RNA sequencing (RNA-seq) and in vivo assays, we identify candidate suppressors of metastasis, namely, Rasd1, Reck, and Aldh1a2. These genes are downregulated in MCs of the primary tumors prior to the formation of metastases. Overexpression of these suppressors of metastasis impair the ability of sarcoma cells to colonize the lungs. Overall, this study reveals clonal dynamics during each step of tumor progression, from initiation to growth, recurrence, and distant metastasis.
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Evolución Clonal/genética , Células Clonales/metabolismo , Recurrencia Local de Neoplasia/metabolismo , Sarcoma/metabolismo , Sarcoma/secundario , Familia de Aldehído Deshidrogenasa 1/genética , Familia de Aldehído Deshidrogenasa 1/metabolismo , Animales , Linaje de la Célula , Células Clonales/citología , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Proteínas Luminiscentes , Ratones , Ratones Desnudos , Recurrencia Local de Neoplasia/genética , RNA-Seq , Retinal-Deshidrogenasa/genética , Retinal-Deshidrogenasa/metabolismo , Sarcoma/genética , Sarcoma/patología , Transcriptoma/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Aim: To quantitatively and sensitively investigate the biodistribution of immune cells after systemic administration. Methods: Immune cells were loaded with plasmonic gold nanostars (GNS) tracking probes. Inductively coupled plasma mass spectrometry (ICP-MS) was used for quantitative gold mass measurement and two-photon photoluminescence (TPL) was used for high-resolution sensitive optical imaging. Results: GNS nanoparticles were loaded successfully into immune cells without negative effect on cellular vitality. Liver and spleen were identified to be the major organs for macrophage cells uptake after systematic administration. A small amount of macrophage cells were detected in the tumor site in our murine lymphoma animal model. Conclusion: GNS has great potential as a biocompatible marker for quantitative tracking and high-resolution imaging of immune cells at the cellular level.
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Oro/química , Linfocitos/metabolismo , Nanopartículas del Metal/química , Animales , Línea Celular Tumoral , Supervivencia Celular , Macrófagos/metabolismo , Nanopartículas del Metal/administración & dosificación , Nanopartículas del Metal/ultraestructura , Ratones Endogámicos C57BL , Distribución TisularRESUMEN
Data from both animal models and humans have demonstrated that effector memory T cells (TEM) and central memory T cells (TCM) from unprimed donors have decreased ability to induce graft-vs-host disease (GVHD). Allospecific TEM from primed donors do not mediate GVHD. However, the potential of alloreactive TCM to induce GVHD is not clear. In this study, we sought to answer this question using a novel GVHD model induced by T cell receptor (TCR) transgenic OT-II T cells. Separated from OT-II mice immunized with OVA protein 8 weeks earlier, the allospecific CD44high TCM were able to mediate skin graft rejection after transfer to naive mice, yet had dramatically decreased ability to induce GVHD. We also found that these allospecific CD44high TCM persisted in GVHD target organs for more than 30 days post-transplantation, while the expansion of these cells was dramatically decreased during GVHD, suggesting an anergic or exhausted state. These observations provide insights into how allospecific CD4+ TCM respond to alloantigen during GVHD and underscore the fundamental difference of alloresponses mediated by allospecific TCM in graft rejection and GVHD settings.
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Linfocitos T CD4-Positivos/inmunología , Enfermedad Injerto contra Huésped/inmunología , Receptores de Hialuranos/inmunología , Memoria Inmunológica , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Linfocitos T CD4-Positivos/patología , Modelos Animales de Enfermedad , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/patología , Receptores de Hialuranos/genética , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
PURPOSE: Extracellular vesicles (EVs) are shed vesicles that bear a combination of nucleic acids and proteins. EVs are becoming recognized as a mode of cell-to-cell communication. Because hematopoietic stem cells reside in proximity to endothelial cells (ECs), we investigated whether EC-derived EVs could regulate hematopoietic stem cell regeneration after ionizing radiation. METHODS AND MATERIALS: We generated EVs derived from primary murine marrow ECs. We sought to determine the response of irradiated hematopoietic stem and progenitor cells to syngeneic or allogeneic EVs in culture assays. Starting 24 hours after either sublethal or lethal irradiation, mice were treated with EVs or saline or cultured primary marrow endothelial cells to determine the hematopoietic response in vivo. RESULTS: We demonstrate that EVs bear nuclear material and express EC-specific markers. Treatment with EVs promoted cell expansion and increased the number of colony-forming units compared to irradiated, hematopoietic cell cultures treated with cytokines alone. After total body irradiation, EV-treated mice displayed preserved marrow cellularity, marrow vessel integrity, and prolonged overall survival compared with controls treated with saline. Treatment of irradiated hematopoietic stem/progenitor cells (HSPCs) with EVs from different genetic strains showed results similar to treatment of HSPCs from syngeneic EVs. Mechanistically, treatment of irradiated HSPCs with EVs resulted in decreased levels of annexin V+ apoptotic cell death, which is mediated in part by tissue inhibitor of metalloproteinase-1. CONCLUSIONS: Our findings show that syngeneic or allogeneic EVs could serve as cell-derived therapy to deliver physiologic doses of nucleic acids and growth factors to hematopoietic cells to accelerate hematopoietic regeneration.
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Células Endoteliales , Vesículas Extracelulares , Células Madre Hematopoyéticas/fisiología , Células Madre Hematopoyéticas/efectos de la radiación , Traumatismos por Radiación/terapia , Regeneración , Animales , Anexina A5/metabolismo , Apoptosis , Comunicación Celular , Proliferación Celular , Supervivencia Celular , Vesículas Extracelulares/fisiología , Trasplante de Células Madre Mesenquimatosas , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Inhibidor Tisular de Metaloproteinasa-1/análisis , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Irradiación Corporal TotalRESUMEN
Graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HCT). The tyrosine kinase SYK contributes to both acute and chronic GVHD development, making it an attractive target for GVHD prevention. Entospletinib (ENTO) is a second-generation highly selective SYK inhibitor with a high safety profile. Potential utility of ENTO as GVHD prophylaxis in patients was examined using a preclinical mouse model of eye and skin GVHD and ENTO-compounded chow. We found that early SYK inhibition improved blood immune cell reconstitution in GVHD mice and prolonged survival, with 60% of mice surviving to day +120 compared with 10% of mice treated with placebo. Compared with mice receiving placebo, mice receiving ENTO had dramatic improvements in clinical eye scores, alopecia scores, and skin scores. Infiltrating SYK+ cells expressing B220 or F4/80, resembling SYK+ cells found in lichenoid skin lesions of chronic GVHD patients, were abundant in the skin of placebo mice but were rare in ENTO-treated mice. Thus, ENTO given early after HCT safely prevented GVHD.
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Enfermedad Injerto contra Huésped/prevención & control , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Indazoles/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirazinas/administración & dosificación , Quinasa Syk/antagonistas & inhibidores , Administración Oral , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Ojo/efectos de los fármacos , Ojo/inmunología , Ojo/patología , Femenino , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/mortalidad , Humanos , Ratones , Piel/efectos de los fármacos , Piel/inmunología , Piel/patología , Análisis de Supervivencia , Quinasa Syk/inmunología , Quinasa Syk/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Resultado del TratamientoRESUMEN
Hematopoietic regeneration following chemotherapy may be distinct from regeneration following radiation. While we have shown that epidermal growth factor (EGF) accelerates regeneration following radiation, its role following chemotherapy is currently unknown. We sought to identify EGF as a hematopoietic growth factor for chemotherapy-induced myelosuppression. Following 5-fluorouracil (5-FU), EGF accelerated hematopoietic stem cell regeneration and prolonged survival compared with saline-treated mice. To mitigate chemotherapy-induced injury to endothelial cells in vivo, we deleted Bax in VEcadherin+ cells (VEcadherinCre;BaxFL/FL mice). Following 5-FU, VEcadherinCre;BaxFL/FL mice displayed preserved hematopoietic stem/progenitor content compared with littermate controls. 5-FU and EGF treatment resulted in increased cellular proliferation, decreased apoptosis, and increased DNA double-strand break repair by non-homologous end-joining recombination compared with saline-treated control mice. When granulocyte colony stimulating factor (G-CSF) is given with EGF, this combination was synergistic for regeneration compared with either G-CSF or EGF alone. EGF increased G-CSF receptor (G-CSFR) expression following 5-FU. Conversely, G-CSF treatment increased both EGF receptor (EGFR) and phosphorylation of EGFR in hematopoietic stem/progenitor cells. In humans, the expression of EGFR is increased in patients with colorectal cancer treated with 5-FU compared with cancer patients not on 5-FU. Similarly, EGFR signaling is responsive to G-CSF in humans in vivo with both increased EGFR and phospho-EGFR in healthy human donors following G-CSF treatment compared with donors who did not receive G-CSF. These data identify EGF as a hematopoietic growth factor following myelosuppressive chemotherapy and that dual therapy with EGF and G-CSF may be an effective method to accelerate hematopoietic regeneration. Stem Cells 2018;36:252-264.
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Factor de Crecimiento Epidérmico/farmacología , Factor Estimulante de Colonias de Granulocitos/farmacología , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Receptores ErbB/metabolismo , Fluorouracilo/farmacología , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Radiation exposure poses a significant threat to public health. Hematopoietic injury is one of the major manifestations of acute radiation sickness. Protection and/or mitigation of hematopoietic stem cells (HSCs) from radiation injury is an important goal in the development of medical countermeasure agents (MCM). We recently identified thioredoxin (TXN) as a novel molecule that has marked protective and proliferative effects on HSCs. In the current study, we investigated the effectiveness of TXN in rescuing mice from a lethal dose of total body radiation (TBI) and in enhancing hematopoietic reconstitution following a lethal dose of irradiation. METHODS: We used in-vivo and in-vitro methods to understand the biological and molecular mechanisms of TXN on radiation mitigation. BABL/c mice were used for the survival study and a flow cytometer was used to quantify the HSC population and cell senescence. A hematology analyzer was used for the peripheral blood cell count, including white blood cells (WBCs), red blood cells (RBCs), hemoglobin, and platelets. Colony forming unit (CFU) assay was used to study the colongenic function of HSCs. Hematoxylin and eosin staining was used to determine the bone marrow cellularity. Senescence-associated ß-galactosidase assay was used for cell senescence. Western blot analysis was used to evaluate the DNA damage and senescence protein expression. Immunofluorescence staining was used to measure the expression of γ-H2AX foci for DNA damage. RESULTS: We found that administration of TXN 24 h following irradiation significantly mitigates BALB/c mice from TBI-induced death: 70% of TXN-treated mice survived, whereas only 25% of saline-treated mice survived. TXN administration led to enhanced recovery of peripheral blood cell counts, bone marrow cellularity, and HSC population as measured by c-Kit+Sca-1+Lin- (KSL) cells, SLAM + KSL cells and CFUs. TXN treatment reduced cell senescence and radiation-induced double-strand DNA breaks in both murine bone marrow lineage-negative (Lin-) cells and primary fibroblasts. Furthermore, TXN decreased the expression of p16 and phosphorylated p38. Our data suggest that TXN modulates diverse cellular processes of HSCs. CONCLUSIONS: Administration of TXN 24 h following irradiation mitigates radiation-induced lethality. To the best of our knowledge, this is the first report demonstrating that TXN reduces radiation-induced lethality. TXN shows potential utility in the mitigation of radiation-induced hematopoietic injury.
Asunto(s)
Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Traumatismos Experimentales por Radiación/prevención & control , Protectores contra Radiación/farmacología , Tiorredoxinas/farmacología , Irradiación Corporal Total , Animales , Antígenos Ly/genética , Antígenos Ly/metabolismo , Biomarcadores/metabolismo , Recuento de Células Sanguíneas , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Médula Ósea/patología , Médula Ósea/efectos de la radiación , Línea Celular , Senescencia Celular/efectos de los fármacos , Senescencia Celular/efectos de la radiación , Ensayo de Unidades Formadoras de Colonias , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Expresión Génica , Hematócrito , Hematopoyesis/genética , Hematopoyesis/efectos de la radiación , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/efectos de la radiación , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Traumatismos Experimentales por Radiación/genética , Traumatismos Experimentales por Radiación/mortalidad , Traumatismos Experimentales por Radiación/patología , Proteínas Recombinantes/farmacología , Análisis de Supervivencia , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Autoantibodies mediate organ destruction in multiple autoimmune diseases, yet their origins in patients remain poorly understood. To probe the genetic origins and structure of disease-associated autoantibodies, we engrafted immunodeficient mice with human CD34+ hematopoietic stem cells and immunized with the non-collagenous-1 (NC1) domain of the alpha3 chain of type IV collagen. This antigen is expressed in lungs and kidneys and is targeted by autoantibodies in anti-glomerular basement membrane (GBM) nephritis and Goodpasture syndrome (GPS), prototypic human organ-specific autoimmune diseases. Using Epstein Barr virus transformation and cell fusion, six human anti-alpha3(IV)NC1 collagen monoclonal autoantibodies (mAb) were recovered, including subsets reactive with human kidney and with epitopes recognized by patients' IgG. Sequence analysis reveals a long to exceptionally long heavy chain complementarity determining region3 (HCDR3), the major site of antigen binding, in all six mAb. Mean HCDR3 length is 25.5 amino acids (range 20-36), generated from inherently long DH and JH genes and extended regions of non-templated N-nucleotides. Long HCDR3 are suited to forming noncontiguous antigen contacts and to binding recessed, immunologically silent epitopes hidden from conventional antibodies, as seen with self-antigen crossreactive broadly neutralizing anti-HIV Ig (bnAb). The anti-alpha3(IV)NC1 collagen mAb also show preferential use of unmutated variable region genes that are enriched among human chronic lymphocytic leukemia antibodies that share features with natural polyreactive Ig. Our findings suggest unexpected relationships between pathogenic anti-collagen Ig, bnAb, and autoreactive Ig associated with malignancy, all of which arise from B cells expressing unconventional structural elements that may require transient escape from tolerance for successful expansion.