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Whole blood (WB) transfusion is routinely used to resuscitate severely injured military trauma patients. Blood can be stored refrigerated while still maintaining reasonable function but is susceptible to environmental influences, including radiation exposure. Immune-compromised patients are transfused with irradiated blood to inactivate donor lymphocyte function (25 Gy per Association for the Advancement of Blood and Biotherapies [AARB] standard 5.7.3.2). However, there is limited information on function of WB exposed to high radiation doses.This study aimed to determine if stored irradiated WB still retains function. This will be important if the stored blood supply is exposed to radiation in a combat situation or mass casualty incident when the need for blood will be high.Whole blood collected from healthy donors was irradiated at 0, 25, or 75 Gy and stored at 4°C. Blood cell count, blood gas chemistry, thromboelastometry, platelet aggregation, and reactive oxygen species were measured before irradiation and at 1, 7, and 14 days of storage. Irradiated WB was compared with nonirradiated WB controls.Irradiated WB stored for up to 14 days was not significantly different than nonirradiated WB in most of the parameters measured. Stored blood showed expected changes associated with functional decline at longer storage times, but irradiation did not hasten the decline. There was a significant change in potassium and sodium ion concentrations after irradiation, but the functional relevance is not clear.High-dose irradiation had little effect on stored WB. Although there were changes in plasma sodium and potassium levels, there was little to no effect on hemostasis and blood cell viability. This suggests that stored blood subjected to a radiation event generating at least a dose of 75 Gy is still suitable for transfusion, which could be particularly important in the event of a mass casualty event where a large amount of blood is needed.

Graphical abstract Traumatic injuries are a leading cause of death and disability in both military and civilian populations. Given the complexity and diversity of traumatic injuries, novel and individualized treatment strategies are required to optimize outcomes. Cellular therapies have potential benefit for the treatment of acute or chronic injuries, and various cell-based pharmaceuticals are currently being tested in preclinical studies or in clinical trials. Cellular therapeutics may have the ability to complement existing therapies, especially in restoring organ function lost due to tissue disruption, prolonged hypoxia or inflammatory damage. In this article we highlight the current status and discuss future directions of cellular therapies for the treatment of traumatic injury. Both published research and ongoing clinical trials are discussed here.

A perfluorocarbon (PFC) investigated for treatment of traumatic brain injury (TBI) delivers oxygen to support brain function, but causes transient thrombocytopenia. TBI can cause acute inflammation with resulting thrombocytopenia; an interaction between the PFC effects and TBI inflammation might exacerbate thrombocytopenia. Therefore, PFC effects on platelet (PLT) function and hemostasis in a lipopolysaccharide (LPS) model of inflammation in the baboon were studied. Animals were randomized to receive saline ±LPS, and ± one of two doses of PFC. PLT count, transmission electron microscopy, and microparticle populations were quantified at baseline (BL) and at 2, 24, 48, 72, and 96 hours; hemostatic parameters for aggregometry and for blood clotting were measured at baseline (BL) and days 3 and 4. Injection of vehicle and LPS caused thrombocytopenia within hours; PFCs caused delayed thrombocytopenia beginning 48 hours post-infusion. LPS+PFC produced a more prolonged PLT decline and decreased clot strength. LPS+PFC increased ADP-stimulated aggregation, but PFC alone did not. Microparticle abundance was greatest in the LPS+PFC groups. LPS+PFC caused diffuse microvascular hemorrhage and death in 2 of 5 baboons in the low dose LPS-PFC group and 2 of 2 in the high dose LPS-PFC group. Necropsy and histology suggested death was caused by shock associated with hemorrhage in multiple organs. Abnormal morphology of platelets and red blood cells were notable for PFC inclusions. In summary, PFC infusion caused clinically significant thrombocytopenia and exacerbated LPS-induced platelet activation. The interaction between these effects resulted in decreased hemostatic capacity, diffuse bleeding, shock and death.

Background Cellular therapeutic agents may benefit trauma patients by modulating the immune response to injury, and by reducing inflammation and vascular leakage. Administration of allogeneic mesenchymal stromal cells (MSCs) shows some benefit in preclinical and clinical trials, but less testing has been performed with other cell types. Human primary fibroblasts (FBs) were compared to MSCs in assays designed to evaluate MSCs to determine if these assays actually evaluate properties unique to MSCs or whether related cell types perform similarly. Study design and methods MSC-related surface marker expression, tissue factor, and human leukocyte antigen-D related were evaluated by flow cytometry, and in vitro adipogenic and osteogenic differentiation potential were determined. Procoagulant activity was determined by thromboelastography. Two potency assays correlated with immunomodulation potential were utilized: the mixed lymphocyte reaction and indoleamine 2,3-dioxygenase enzyme activity assays. Results Human primary FBs performed similarly to MSCs in assays designed to evaluate MSC characteristics and potency. Although similar for MSC-positive cell surface marker expression, FBs did not show robust adipose differentiation and expressed some level of markers not expected on MSCs. Conclusions Human primary FBs are very similar to human MSCs, at least in assays currently used to evaluate MSC potency. Preclinical and clinical testing are required to determine if FBs show similar activity to MSCs in vivo. If FBs show inferior activity in vivo, development of new MSC-specific potency assays will be necessary to evaluate properties relevant to their unique clinical benefits.

Hepatocellular carcinoma (HCC) remains a deadly cancer, underscoring the need for relevant preclinical models. Male C3HeB/FeJ mice model spontaneous HCC with some hepatocarcinogenesis susceptibility loci corresponding to syntenic regions of human chromosomes altered in HCC. We tested other properties of C3HeB/FeJ tumors for similarity to human HCC. C3HeB/FeJ tumors were grossly visible at 4 months of age, with prevalence and size increasing until about 11 months of age. Histologic features shared with human HCC include hepatosteatosis, tumor progression from dysplasia to poorly differentiated, vascular invasion, and trabecular, oncocytic, vacuolar, and clear cell variants. More tumor cells displayed cytoplasmic APE1 staining versus normal liver. Ultrasound effectively detected and monitored tumors, with 85.7% sensitivity. Over 5000 genes were differentially expressed based on the GSE62232 and GSE63898 human HCC datasets. Of these, 158 and 198 genes, respectively, were also differentially expressed in C3HeB/FeJ. Common cancer pathways, cell cycle, p53 signaling and other molecular aspects, were shared between human and mouse differentially expressed genes. We established eigengenes that distinguish HCC from normal liver in the C3HeB/FeJ model and a subset of human HCC. These features extend the relevance and improve the utility of the C3HeB/FeJ line for HCC studies.

Background There is broad interest in the use of cell therapies and cell products for treatment of a variety of diseases and problems. Of interest to the military, cellular therapies have the potential to confer tremendous benefit for treatment of both acute and chronic injuries. Although many different cell therapy products are currently under investigation, mesenchymal stromal cells (MSCs) are good candidates, based on their ability to respond to inflammation, limit vascular permeability, and modulate immune responses to injury. Although a large number of clinical trials utilize MSCs or their products, there is no firm consensus defining the characteristics and activities of a good MSC product. Here, we test multiple human MSCs in several assays designed to test potency, to determine if functionally relevant differences between MSCs can be defined using in vitro assays, allowing identification of superior MSC products for preclinical or clinical testing. Methods Human MSCs derived from several tissue sources (adipose, bone marrow, umbilical cord) were evaluated for their ability to respond to inflammatory signaling by upregulating indoleamine-2,3-dioxygenase and TSG6, suppress lymphocyte proliferation, alter the polarization of macrophages, and affect tube formation by endothelial cells. Results All MSCs tested displayed activity in the functional assays utilized, but differences in potency were observed in each assay. Conclusion The indoleamine-2,3-dioxygenase enzyme activity assay represents a simple way to screen multiple samples. The mixed lymphocyte reaction and monocyte assays used to test interactions between MSCs and immune cells are more involved but give direct information on immunomodulation potential. The endothelial cell tube formation assay is relatively simple to perform but a large number of images must be generated and analyzed. However, it tests a functional activity other than immunomodulation and, therefore, adds another facet to MSC evaluation.

Background This study evaluated blood components processed by the platelet rich plasma (PRP) method from fresh whole blood (FWB) treated with a pathogen reduction technology (PRT). The effects of storage temperature on PRT treated platelet concentrates (PCs) were also examined. Study design and methods PRT was performed using riboflavin and ultraviolet light on FWB in citrate phosphate dextrose anticoagulant. Following PRT, red blood cells (RBCs), PCs, and plasma for fresh frozen plasma (FFP), were isolated by sequential centrifugation. RBCs were stored at 4°C, FFP at -80°C, and PC at 22°C or at 4°C. Components were assayed throughout their storage times for blood gases, chemistry and CBC, hemostatic function as well as platelet (PLT) and RBC integrity. Results Component processing following PRT resulted in a significant drop in platelet recovery. Most PRT-PC bags fell below AABB guidelines for platelet count. PRT-PC also showed a decrease in clot strength and decreased aggregometry response. Platelet caspases were activated by PRT. Storage at 4°C improved platelet function. In PRT-FFP, prothrombin time and partial thromboplastin time (PT and aPTT) were prolonged; factors V, VII, VIII, and XI, protein C, and fibrinogen were significantly decreased. Free hemoglobin was elevated two-fold in PRT-RBC. Conclusion Blood components isolated by the PRP method from PRT-treated WB result in a high percentage of PC that fail to meet AABB guidelines. FFP also shows diminished coagulation capacity. However, PRT-RBC are comparable to control-RBC. PRT-WB retains acceptable hemostatic function but alternatives to the PRP method of component separation may be more suitable.

BACKGROUND Mesenchymal stem/stromal cell (MSC)-derived extracellular vesicles (EVs) are a possible cell-free alternative to MSCs because they retain the regenerative potential of MSCs, while still mitigating some of their limitations (such as the possible elicitation of host immune responses). The promotion and restoration of angiogenesis, however, is an important component in treating trauma-related injuries, and has not been fully explored with EVs. Herein, we describe the effects of monolayer adipose-derived EVs, spheroid adipose-derived EVs (SAd-EVs), monolayer bone marrow-derived EVs (MBM-EVs), and spheroid bone marrow-derived EVs (SBM-EVs) on human umbilical vein endothelial cell (HUVEC) tube formation and mitochondrial respiration. METHODS The successful isolation of EVs derived from adipose MSCs or bone marrow MSCs in monolayer or spheroid cultures was confirmed by NanoSight (particle size distribution) and Western blot (surface marker expression). The EV angiogenic potential was measured using a 24-hour HUVEC tube formation assay. The EV effects on HUVEC mitochondrial function were evaluated using the Seahorse respirometer machine. RESULTS The number of junctions, branches, and the average length of branches formed at 24 hours of tube formation were significantly affected by cell and culture type; overall adipose-derived EVs outperformed bone marrow-derived EVs, and spheroid-derived EVs outperformed monolayer-derived EVs. Additionally, adipose-derived EVs resulted in significantly increased HUVEC mitochondrial maximal respiration and adenosine triphosphate (ATP) production, while only MBM-EVs negatively impacted HUVEC proton leak. CONCLUSION Adipose-derived EVs promoted HUVEC tube formation significantly more than bone marrow-derived EVs, while also maximizing HUVEC mitochondria function. Results demonstrate that, as with MSC therapies, it is possible to tailor EV culture and production to optimize therapeutic potential. LEVEL OF EVIDENCE Basic or Foundational Research.

Background Immunomodulation by mesenchymal stromal cells (MSCs) is a potentially important therapeutic modality. MSCs suppress peripheral blood mononuclear cell (PBMC) proliferation in vitro, suggesting a mechanism for suppressing inflammatory responses in vivo. This study details the interactions of PBMCs and MSCs. Methods Pooled human PBMCs and MSCs were co-cultured at different MSC:PBMC ratios and harvested from 0 to 120 h, with and without phytohaemagglutin A (PHA) stimulation. Proliferation of adherent MSCs and non-adherent PBMCs was assessed by quantitation of ATP levels. PBMC surface marker expression was analyzed by flow cytometry. Indoleamine 2,3-dioxygenase (IDO) activity was determined by kynurenine assay and IDO mRNA by RT-PCR. Cytokine release was measured by ELISA. Immunofluorescent microscopy detected MSC, PBMC, monocyte (CD14+) and apoptotic events. Results PBMC proliferation in response to PHA gave a robust ATP signal by 72 h, which was suppressed by co-culture with densely plated MSCs. Very low level MSC seeding densities relative to PBMC number reproducibly stimulated PBMC proliferation. The CD4+/CD3+ population significantly decreased over time while the CD8+/CD3+ population significantly increased. No change in CD4+/CD8+ ratio is seen with high density MSC co-culture; very low density MSCs augment the changes seen in PHA stimulated PBMCs alone. IDO activity in MSCs co-cultured with PBMCs correlated with PBMC suppression. MSCs increased the secretion of IL-10 and IL-6 from stimulated co-cultures and decreased TNF-α secretion. In stimulated co-culture, low density MSCs decreased in number; fluorescence immunomicroscopy detected association of PBMC with MSC and phosphatidyl serine externalization in both cell populations. Conclusions A bidirectional interaction between MSCs and PBMCs occurs during co-culture. High numbers of MSCs inhibit PHA-stimulated PBMC proliferation and the PBMC response to stimulation; low numbers of MSCs augment these responses. Low density MSCs are susceptible to attrition, apparently by PBMC-induced apoptosis. These results may have direct application when considering therapeutic dosing of patients; low MSC doses may have unintended detrimental consequences.

Allogeneic mesenchymal stem cells (MSCs) show great potential for the treatment of military and civilian trauma based on their reduced immunogenicity and ability to modulate inflammation and immune function in the recipient. Although generally considered to be safe, MSCs express tissue factor (TF), a potent activator of coagulation. In the current study, we evaluated multiple MSC populations for tissue factor expression and procoagulant activity to characterize safety considerations for systemic use of MSCs in trauma patients who may have altered coagulation homeostasis.Multiple MSC populations derived from either human adipose tissue or bone marrow were expanded in the recommended stem cell media. Stem cell identity was confirmed using a well-characterized panel of positive and negative markers. Tissue factor expression on the cell surface was evaluated by flow cytometry with anti-CD142 antibody. Effects on blood coagulation were determined by thromboelastography and calibrated automated thrombogram assays using platelet-poor plasma or whole blood.Mesenchymal stem cells express tissue factor on their surfaces and are procoagulant in the presence of blood or plasma. The adipose-derived MSCs (Ad-MSC) evaluated were more procoagulant and expressed more tissue factor than bone marrow MSCs (BM-MSCs), which showed a greater variability in TF expression. Bone marrow MSCs were identified that exhibited low procoagulant activity, whereas all Ad-MSCs examined exhibited high procoagulant activity. The percentage of cells in a given population expressing surface tissue factor correlates roughly with functional procoagulant activity. Mesenchymal stem cell tissue factor expression and procoagulant activity change over time in culture.All MSC populations are not equivalent; care should be taken to select cells for clinical use that minimize potential safety problems and maximize chance of patient benefit. Adipose-derived MSCs seem more consistently procoagulant than BM-MSCs, presenting a potential safety concern for systemic administration in coagulopathic patients. Donor variation exists between different cell populations, and culture handling conditions may also determine coagulation activity. Cells must be routinely monitored during preparation to ensure that they retain the desired characteristics before patient administration.

Objectives Autotransfusion of shed blood from traumatic hemothorax is an attractive option for resuscitation of trauma patients in austere environments. However, previous analyses revealed that shed hemothorax (HX) blood is defibrinated, thrombocytopenic, and contains elevated levels of D-dimer. Mixing studies with normal pooled plasma demonstrated hypercoagulability, evoking concern for potentiation of acute traumatic coagulopathy. We hypothesized that induction of coagulopathic changes by shed HX blood may be due to increases in cellular microparticles (MP) and that these may also affect recipient platelet function. Methods Shed HX blood was obtained from 17 adult trauma patients under an Institutional Review Board approved prospective observational protocol. Blood samples were collected every hour up to 4 h after thoracostomy tube placement. The corresponding plasma was isolated and frozen for analysis. The effects of shed HX frozen plasma (HFP) and isolated HX microparticles (HMP) on coagulation and platelet function were assessed through mixing studies with platelet-rich plasma at various dilutions followed by analysis with thromboelastometry (ROTEM), platelet aggregometry (Multiplate), enzyme-linked immunosorbent assays, and flow cytometry. Furthermore, HFP was assessed for von Willebrand factor antigen levels and multimer content, and plasma-free hemoglobin. Results ROTEM analysis demonstrated that diluted HFP and isolated HMP samples decreased clotting time, clotting formation time, and increased α angle, irrespective of sample concentrations, when compared with diluted control plasma. Isolated HMP inhibited platelet aggregation in response to adenosine diphosphate, arachidonic acid, and collagen. HFP contained elevated levels of fibrin-degradation products and tissue factor compared with control fresh frozen plasma samples. MP concentrations in HFP were significantly increased and enriched in events positive for phosphatidylserine, tissue factor, CD235, CD45, CD41a, and CD14. von Willebrand factor (vWF) multimer analysis revealed significant loss of high molecular weight multimers in HFP samples. Plasma-free hemoglobin levels were 8-fold higher in HFP compared with fresh frozen plasma. Conclusion HFP induces plasma hypercoagulability that is likely related to increased tissue factor and phosphatidylserine expression originating from cell-derived MP. In contrast, platelet dysfunction is induced by HMP, potentially aggravated by depletion of high molecular weight multimers of vWF. Thus, autologous transfusion of shed traumatic hemothorax blood may induce a range of undesirable effects in patients with acute traumatic coagulopathy.

Acute traumatic coagulopathy (ATC) is the failure of coagulation homeostasis that can rapidly arise following traumatic injury, hemorrhage, and shock; it is associated with higher injury severity, coagulation abnormalities, and increased blood transfusions. Acute traumatic coagulopathy has historically been defined by a prolonged prothrombin time, although newer, more informative measurements of hemostatic function have been used to improve diagnosis and support clinical decision making. The underlying biochemical mechanisms of and best practice therapeutics for ATC remain under active investigation because of its significant correlation to poor outcomes. The wide range of hypothesized mechanisms for ATC results from the large number of symptoms, phenotypes, and altered states in these patients as observed by multiple research groups. Much like the ancient fable of blind men describing an elephant from their limited perspectives, the limited nature of clinical and laboratory tools used to diagnose coagulopathy or evaluate hemostatic function has made finding causation difficult. The prolonged prothrombin time, degree of fibrinolysis, depletion of coagulation factors and inhibitors, and general failure of the blood have all been identified as being primary indicators for ATC. Therapeutic interventions including recombinant coagulation factors, antifibrinolytics, and blood products have been used with varying degrees of success as they are used to address specific symptoms. To truly understand the causes of ATC, research efforts must recognize the complexity of the hemostatic system and get to the heart of the matter by answering the question: "Is ATC a pathological condition that develops from the observed deficiencies in coagulation, fibrinolysis, and autoregulation, or is ATC an adaptive response generated as the body attempts to restore perfusion and avoid massive organ failure?" Because patient management must proceed without definitive answers regarding the entire causative chain, the current therapeutic focus should be on using what knowledge has been gained to the patient's advantage: control hemorrhage, maintain appropriate homeostatic balances of coagulation proteins, and restore oxygen perfusion.

BACKGROUND Ordinarily, whole blood (WB) is separated into components before storage. We assessed the posttransfusion viability and function of platelets (PLTs) if they were stored within WB at 4°C. STUDY DESIGN AND METHODS Whole blood was obtained from 30 normal subjects and stored at 4°C without agitation for 12 days and for 10, 15, or 22 days with agitation. After WB storage, a PLT concentrate was prepared, and a fresh PLT sample was obtained from each donor. The stored PLTs were labeled with 111 In and the fresh with 51 Cr, and both were simultaneously transfused into their donor. Blood samples were obtained after transfusion to determine PLT recoveries and survivals. PLT samples from WB before and after storage were also assayed for PLT function and biochemistry. RESULTS After storage for 12 days without WB rotation, poststorage PLT counts averaged only 49 ± 12% of baseline values. After storage for 10, 15, or 22 days with end-over-end WB rotation, PLT counts averaged 76 ± 14% of baseline values. Fifteen-day poststorage radiolabeled PLT recoveries averaged 27 ± 11% (49 ± 16% of fresh), and survivals averaged 1.2 ± 0.4 days (16 ± 6% of fresh). in vitro assays demonstrated marked PLT activation after any storage time, and although PLT function decreased over time, stored PLTs were still considered acceptable. CONCLUSION These data suggest that, during rotated WB storage at 4°C for up to 15 days, PLT yields, poststorage PLT recoveries and survivals, and PLT function should be sufficient to support the short-term hemostatic needs of traumatized patients.

Background Mesenchymal stromal cells (MSCs) have been proposed for treatment of acute respiratory distress syndrome (ARDS), graft versus host disease (GVHD), wound healing and trauma. A consensus is building that immunomodulation by MSCs is important for therapeutic potential. MSCs suppress peripheral blood mononuclear cell (PBMC) proliferation in vitro, potentially reflecting an ability to suppress PBMC inflammatory responses in vivo. Current mixed lymphocyte reaction (MLR) assays commonly used to evaluate MSC potency generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated PBMCs in the presence of mitotically inactive MSCs. Proliferation of PBMCs is monitored by several methods, including incorporation of radiolabeled nucleotides, BRDU labeling and ELISA assay or flow cytometry of carboxyfluorescein labeled PBMCs. Here we present a streamlined assay using MSCs in a direct co-culture system with unmodified MSCs using a luminescent ATP assay to evaluate both PBMC and MSC proliferation/survival. Methods PBMCs were isolated from fresh anti-coagulated whole blood by centrifugation over Ficoll-Paque in LeucoSep tubes. Isolated PBMCs from 8 to 10 donors were pooled and cryopreserved at 1 × 107/ml in 50% RPMI medium,10% DMSO, 40% human AB serum. MSCs derived from bone marrow, adipose tissue or umbilical cord (BM-MSC, Ad-MSC, UC-MSC, respectively) were serially diluted starting at 50–60,000 cells/well and cultured in 96 well plates for 4–48 h in their respective medium. On Day 0, MSCs were washed, resuspended in PBMC media (RPMI with 10% FBS, 2 mM Glutamine, 10 mM HEPES, pH 7.4) and incubated with or without 150,000 freshly thawed pooled PBMCs/well, in the presence or absence of phytohemagglutinin A (PHA, 0–5 μg/ml). Proliferation of both MSCs (adherent) and PBMCs (non-adherent) was assessed by quantitation of ATP levels using the bioluminescent reagent Cell Titer-Glo (Promega). Culture supernatant contained PBMC, while washed adherent cells were primarily MSCs. Both cell types were incubated for 30 min with an equal volume of Cell Titer-Glo reagent and then assayed in white plates on a luminescence plate reader. Results PBMC proliferation in response to PHA stimulation resulted in a robust increase in ATP by 72 h, with >6 fold increase over unstimulated PBMCs, which showed no increase. MSC proliferation was decreased Conclusions Direct co-culture of live unmodified MSCs with freshly thawed pooled PBMCs gives a robust determination of immunosuppression by MSCs with unparalleled ease. Graded responses can be determined, allowing comparison of potency between MSC preparations as in comparisons between freshly thawed and cultured MSCs as well as interferon-γ licensed MSCs. With the 96 well plate assay, far fewer PBMCs are generally required than in a typical flow cytometry determination. This streamlined assay can be performed within 72 h, without irradiating cells and without specialized equipment.

Hepatocellular carcinoma is increasingly important in the United States as the incidence rate rose over the last 30 years. C3HeB/FeJ mice serve as a unique model to study hepatocellular carcinoma tumorigenesis because they mimic human hepatocellular carcinoma with delayed onset, male gender bias, approximately 50% incidence, and susceptibility to tumorigenesis is mediated through multiple genetic loci. Because a human O6-methylguanine-DNA methyltransferase (hMGMT) transgene reduces spontaneous tumorigenesis in this model, we hypothesized that hMGMT would also protect from methylation-induced hepatocarcinogenesis. To test this hypothesis, wild-type and hMGMT transgenic C3HeB/FeJ male mice were treated with two monofunctional alkylating agents: diethylnitrosamine (DEN; 0.025 μmol/g body weight) on day 12 of life with evaluation for glucose-6-phosphatase-deficient (G6PD) foci at 16, 24, and 32 weeks or N-methyl-N-nitrosurea (MNU; 25 mg MNU/kg body weight) once monthly for 7 months starting at 3 months of age with evaluation for liver tumors at 12 to 15 months of age. No difference in abundance or size of G6PD foci was measured with DEN treatment. In contrast, it was unexpectedly found that MNU reduces liver tumor prevalence in wild-type and hMGMT transgenic mice despite increased tumor prevalence in other tissues. hMGMT and MNU protections were additive, suggesting that MNU protects through a different mechanism, perhaps through the cytotoxic N7-alkylguanine and N3-alkyladenine lesions which have low mutagenic potential compared with O6-alkylguanine lesions. Together, these results suggest that targeting the repair of cytotoxic lesions may be a good preventative for patients at high risk of developing hepatocellular carcinoma. Cancer Prev Res; 9(3); 245–52. ©2015 AACR.

BackgroundTo study central hypovolemia in humans, lower body negative pressure (LBNP) is a recognized alternative to blood removal (HEM). While LBNP mimics the cardiovascular responses of HEM in baboons, similarities in hemostatic responses to LBNP and HEM remain unknown in this species.MethodsThirteen anesthetized baboons were exposed to progressive hypovolemia by HEM and, four weeks later, by LBNP. Hemostatic activity was evaluated by plasma markers, thromboelastography (TEG), flow cytometry, and platelet aggregometry at baseline (BL), during and after hypovolemia.ResultsBL values were indistinguishable for most parameters although platelet count, maximal clot strength (MA), protein C, thrombin anti-thrombin complex (TAT), thrombin activatable fibrinolysis inhibitor (TAFI) activity significantly differed between HEM and LBNP. Central hypovolemia induced by either method activated coagulation; TEG R-time decreased and MA increased during and after hypovolemia compared to BL. Platelets displayed activation by flow cytometry; platelet count and functional aggregometry were unchanged. TAFI activity and protein, Factors V and VIII, vWF, Proteins C and S all demonstrated hemodilution during HEM and hemoconcentration during LBNP, whereas tissue plasminogen activator (tPA), plasmin/anti-plasmin complex, and plasminogen activator inhibitor-1 did not. Fibrinolysis (TEG LY30) was unchanged by either method; however, at BL, fibrinolysis varied greatly. Post-hoc analysis separated baboons into low-lysis (LY30 2%) whose fibrinolytic state matched at both HEM and LBNP BL. In high-lysis, BL tPA and LY30 correlated strongly (r = 0.95; PConclusionsCentral hypovolemia induced by either LBNP or HEM resulted in activation of coagulation; thus, LBNP is an adjunct to study hemorrhage-induced pro-coagulation in baboons. Furthermore, this study revealed a subset of baboons with baseline hyperfibrinolysis, which was strongly coupled to tPA and uncoupled from TAFI activity.

Background: Lower body negative pressure (LBNP) has been established in human studies as an alternative method to blood removal (HEM) to study central hypovolemia. In a baboon model, LBNP also mimics the cardiovascular response of HEM. Hemostatic responses, including platelet activation, coagulation and fibrinolysis in response to LBNP and HEM in this baboon model are reported here. A previous meeting report from this study focused on hemostatic interactions; this report expands the assay of fibrinolytic proteins and includes detailed post-hoc analysis of interactions. Methods: Thirteen anesthetized baboons were exposed to progressive central hypovolemia by HEM and, four weeks later, by LBNP. Whole blood cell counts were determined; pro-coagulant and fibrinolytic activity was evaluated by plasma markers, thrombelastography, flow cytometry and platelet aggregometry at baseline (BL), intermediate hypovolemia (MID), presyncope or maximal hypovolemia (MAX) and after recovery (REC). Results: Baseline values for HEM (day 0) and LBNP (day 28) were indistinguishable for most analyzed parameters; however platelet number, maximal clotting (MA), Protein C, TAT, TAFI-activity, plasma renin, angiotensin and epinephrine were all significantly different (p2) whose fibrinolytic state matched at both HEM and LBNP BL. High lysis samples (n=8) showed a strong correlation (r=0.95; p Conclusions: Central hypovolemia resulted in activation of coagulation by either LBNP or HEM; thus LBNP is a powerful model for studying hemorrhage. This study also revealed a subset of baboons with baseline activation of fibrinolysis which was strongly coupled to tPA and uncoupled from TAFI activity. The methods demonstrated hemodilution with blood removal and hemoconcentration with LBNP, as has been observed in humans, but these modest changes had no significant effect on coagulation parameters. Unexpectedly, this study demonstrated that the effects of blood removal were sustained 28 days later on baseline parameters. Overall, these results in large primates suggest that baseline differences between apparently healthy and superficially homogeneous individuals may determine outcomes in studies of hemostasis. Disclosures No relevant conflicts of interest to declare.

Background Formulation of a medical preparedness plan for treating severely bleeding casualties during naval deployment is a significant challenge because of territory covered during most missions. The aim of this study was to evaluate the concept of "walking blood bank" as a supportable plan for supplying safe blood and blood products. Methods In 2013, the Royal Norwegian Navy conducted antipiracy operations from a frigate, beginning in the Gulf of Aden and ending in the Indian Ocean. Crews were on 24-hour emergency alert in preparation for an enemy assault on the frigate. Under an approved command protocol, a "walking blood bank," using crew blood donations, was established for use on board and on missions conducted in rigid-hulled inflatable boats, during which freeze-dried plasma and leukoreduced, group O low anti-A/anti-B titer, cold-stored whole blood were stored in Golden Hour Boxes. Data demonstrating the ability to collect, store, and provide whole blood were collected to establish feasibility of implementing a whole blood-focused remote damage-control resuscitation program aboard a naval vessel. In addition, ROTEM data were collected to demonstrate feasibility of performing this analysis on a large naval vessel and to also measure hemostatic efficacy of cold-stored leukoreduced whole blood (CWB) stored during a period of 14 days. ROTEM data on CWB was compared with reconstituted whole blood. Results Drills simulating massive transfusion activation were conducted, in which 2 U of warm fresh whole blood with platelet sparing leukoreduction were produced in 40 minutes, followed by collection of two additional units at 15-minute increments. The ROTEM machine performed well during ship-rolling, as shown by the overlapping calculated and measured mechanical piston movements measured by the ROTEM device. Error messages were recorded in 4 (1.5%) of 267 tests. CWB yielded reproducible ROTEM results demonstrating preserved fibrinogen function and platelet function for at least 3.5 weeks and 2 weeks, respectively. The frequency of ROTEM tests were as follows: EXTEM (n = 88), INTEM (n = 85), FIBTEM (n = 82), and APTEM (n = 12). CWB results were grouped. Compared with Days 0 to 2, EXTEM maximum clot firmness was significantly reduced, beginning on Days 10 to 14; however, results through that date remained within reference ranges and were comparable with the EXTEM maximum clot firmness for the reconstituted whole blood samples containing Day 5 room temperature-stored platelets. Conclusion A "walking blood bank" can provide a balanced transfusion product to support damage-control resuscitation/remote damage-control resuscitation aboard a frigate in the absence of conventional blood bank products. ROTEM analysis is feasible to monitor damage-control resuscitation and blood product quality. ROTEM analysis was possible in challenging operational conditions. Level of evidence Therapeutic study, level V.

Smoke inhalation and burn injury remain a major source of morbidity and mortality. There is known dysregulation of hemostasis in burn patients, but either hypercoagulation or hypocoagulation states are reported. Sheep are an established animal model for studying burn pathology and provide robust data on hemostatic function at baseline and after injury.After an IACUC-approved protocol, 15 sheep were anesthetized and subjected to a 40% full thickness burn with smoke inhalation. Blood was sampled at baseline, 1 day postinjury (early effects) and days 2, 3, and 4 (late effects) after injury. Assays at each timepoint assessed: hemostatic function by thromboelastography (TEG), platelet counts and function by flow cytometry and aggregometry, coagulation protein levels, and free hemoglobin. Data were analyzed by the Wilcoxon paired test (nonparametric) with significance set at less than 0.05.By 24 hours postinjury, platelet counts had dropped, whereas the percent activated platelets increased. Absolute platelet functional response to the agonist adenosine diphosphate (ADP) decreased, whereas response to collagen showed no significant difference. On a per platelet basis, ADP response was unchanged, whereas the collagen response was elevated. Prothrombin time and activated partial thromboplastin time were prolonged. TEG parameters decreased significantly from baseline. Fibrinogen and factor V were trending up; coagulation proteins ATIII, factors IX and X were decreased.Late effects were followed in six animals. At day 4, platelet counts remained depressed compared with baseline with a nadir at day 2; responses to agonist on a per platelet basis remained the same for ADP and stayed elevated for collagen. Platelets continued to have elevated activation levels. Fibrinogen and factor V remained significantly elevated, whereas TEG parameters and prothrombin time, factors IX and X returned to near baseline levels.Coagulation parameters and hemostasis are dysregulated in sheep after smoke inhalation and burn. By 24 hours, sheep were hypocoagulable and subsequently became hypercoagulable by day 4. These results suggest a three-stage coagulopathy in burn injuries with a known early consumptive hypercoagulable state which is followed by a relatively hypocoagulable state with increased bleeding risk and then a return to a relatively unknown hypercoagulability with increased susceptibility to thrombotic disorders.

The prevalence of hepatocellular carcinoma (HCC) was diminished from 60% to 18% at 15 months of age in C3HeB/FeJ male transgenic mice expressing hMGMT in our previous studies. To directly test if the methyltransferase activity is required for diminished tumor prevalence, two separate lines of transgenic mice bearing an enzymatically inactive form of hMGMT were used. In these lines, cysteine 145 was substituted with alanine (C145A). Expression of the hMGMT C145A transgene in liver was demonstrated by Northern blots and Western blots. Immunohistochemistry revealed predominantly nuclear localization of the hMGMT C145A protein. hMGMT C145A transgenic mice were crossed with lacI transgenic mice to assess mutant frequencies in the presence of the mutant protein. Mutant frequencies were similar among livers of lacI × hMGMT C145A bi-transgenic mice and lacI × wild-type (WT) mice. DNA sequence analysis of recovered lacI mutants revealed similar mutation spectra for hMGMT C145A and WT mice. The prevalence of HCC was also similar for the two tested lines of hMGMT C145A mice, 45% and 48% prevalence with median tumor sizes of 11 and 8 mm, and WT mice, 40% prevalence and median tumor size of 10 mm. These results provide evidence that residue C145 in hMGMT is required to reduce the prevalence of HCC in C3HeB/FeJ mice transgenic for hMGMT. © 2011 Wiley Periodicals, Inc.

DNA damage and mutagenesis are suggested to contribute to aging through their ability to mediate cellular dysfunction. The base excision repair (BER) pathway ameliorates a large number of DNA lesions that arise spontaneously. Many of these lesions are reported to increase with age. Oxidized guanine, repaired largely via base excision repair, is particularly well studied and shown to increase with age. Spontaneous mutant frequencies also increase with age which suggests that mutagenesis may contribute to aging. It is widely accepted that genetic instability contributes to age-related occurrences of cancer and potentially other age-related pathologies. BER activity decreases with age in multiple tissues. The specific BER protein that appears to limit activity varies among tissues. DNA polymerase-beta is reduced in brain from aged mice and rats while AP endonuclease is reduced in spermatogenic cells obtained from old mice. The differences in proteins that appear to limit BER activity among tissues may represent true tissue-specific differences in activity or may be due to differences in techniques, environmental conditions or other unidentified differences among the experimental approaches. Much remains to be addressed concerning the potential role of BER in aging and age-related health span.

Background Many military and civilian centers have shifted to a damage-control resuscitation approach, focused on providing oxygen-carrying capacity while simultaneously mitigating coagulopathy with a balanced ratio of platelets and plasma to red blood cells. It is unclear to what degree this strategy is used during burn or soft tissue excision. Here, we characterized blood product transfusion during burn and soft tissue surgery and reviewed the published literature regarding intraoperative coagulation changes. We hypothesized that blood product resuscitation during burn and soft tissue excision is not hemostatic and would be insufficient to address hemorrhage-induced coagulopathy. Methods Consented adult patients were enrolled into an institutional review board-approved prospective observational study. Number, component type, volume, and age of the blood products transfused were recorded during burn excision/grafting or soft tissue debridement. Component bags (packed red blood cells, fresh frozen plasma, platelets, and cryoprecipitate) were collected, and the remaining sample was harvested from the bag and tubing. Aliquots of 1/1,000th the original volume of each blood product were obtained and combined, producing an amalgam sample containing the same ratio of product transfused. Platelet count, rotational thromboelastometry, and impedance aggregometry were measured. Significance was set at p Results Amalgamated transfusate samples produced abnormally weak clots (p ≤ 0.001) particularly if they did not contain platelets. Clot strength (48.8 [2.6] mm; reference range, 49-71 mm) for platelet-containing amalgams was below the lower limit of the reference range despite platelet-red blood cell ratios greater than 1:1. Platelet aggregation was abnormally low; transfused platelets were functionally inferior to native platelets. Conclusion Our study and focused review demonstrate that further work is needed to fully understand the needs of patients undergoing tissue excision. The three studies reviewed and the results of our observational work suggest that coagulopathy and thrombocytopenia may contribute to intraoperative hemorrhage. Blood product resuscitation during burn and soft tissue excision is not hemostatic. Level of evidence Epidemiologic study, level V.

AT-rich minisatellites (AT islands) are sites of genomic instability in cancer cells and targets for extremely lethal AT-specific drugs, such as bizelesin. Here we investigated the AT islands in the FRA16B fragile site region for their possible roles in the organization of DNA on the nuclear matrix. The FRA16B AT island nominally spans approximately 3 kb of mostly >90% A/T DNA. In silico analysis indicates that this domain exhibits characteristics of nuclear matrix attachment regions (MARs): an exceptionally intense computed 'MAR potential' and profound duplex destabilization and flexibility. FRA16B repeats specifically bind to isolated nuclear matrices, which indicates their in vitro MAR function. This binding is several-fold greater than that of a known MAR in the c-myc gene. AT islands in fragile sites FRA16B and FRA16D are significantly more abundant in CEM cells that are hypersensitive to bizelesin compared to normal WI-38 cells. FRA16B overabundance in CEM is due to an approximately 10-fold expansion of FRA16B repeats. The expanded FRA16B minisatellites in CEM cells preferentially localize to the nuclear matrix-associated DNA indicating their in vivo MAR function. The unexpanded repeats in WI-38 cells localize to the loop DNA. The c-myc MAR is also matrix-associated in CEM cells while localizing to loop DNA in WI-38 cells. These results are the first to demonstrate that AT islands in fragile sites can function as MARs both in vitro and in vivo. The ability of FRA16B-mediated MAR sites to rearrange depending on the repeat expansion status could be relevant to both genomic instability of cancer cells and their sensitivity to AT-island targeting drugs.

Damage to cellular DNA is believed to determine the antiproliferative properties of platinum (Pt) drugs. This study characterized DNA damage by oxaliplatin, a diaminocyclohexane Pt drug with clinical antitumor activity. Compared with cisplatin, oxaliplatin formed significantly fewer Pt-DNA adducts (e.g., 0.86+/-0.04 versus 1.36+/- 0.01 adducts/10(6) base pairs/10 microM drug/1 h, respectively, in CEM cells, P

Hydroxymethylacylfulvene (HMAF, MGI 114) is a novel antitumor drug and a potent pro-apoptotic agent that has the potential to alkylate cellular nucleophiles. The objective of these studies was to characterize drug uptake and cellular targets for drug binding in human leukemia CEM cells. The uptake of [14C]HMAF had two components: a rapid phase (0-10 min) and a slow phase. At 10 microM drug (37 degrees), the rapid and slower phase amounted to 0.86 and 0.13 pmol/min/10(6)cells, respectively. HMAF uptake was inhibited 82% by low temperature (4 degrees) at 4 hr. Cell-associated HMAF localized to nuclear (50%), cytoplasmic (37%), and membrane fractions (10%). Continued drug uptake appeared to be driven by covalent binding to cellular macromolecules. Approximately 1/4 and 2/3 of cell-associated HMAF formed covalent adducts after 10 min and 4 hr, respectively, as found by perchloric acid precipitation. Drug adducts were not readily reversible; 77% of the covalently bound radiolabel was retained by the cells 20 hr after drug treatment. Combinations of DNase, RNase, and proteinase K with perchloric acid precipitation showed that approximately 60, 30, and 10% of the covalently bound drug was associated with the protein, DNA, and RNA fractions, respectively. Incubation of 100 microM [14C]HMAF (24 hr) with purified DNA, serum albumin, thioredoxin, and thioredoxin reductase resulted in 6, 22, 14, and 11 pmol [14C]HMAF/microg DNA or protein, respectively. Results indicate that multiple targets for HMAF binding may contribute to the pro-apoptotic and antiproliferative action of the drug.

The role of transforming growth factor-beta (TGF-β) signaling in hepatocarcinogenesis remains controversial. We aimed to reveal TGF-β signaling status in human and murine tissues of hepatocellular carcinoma (HCC) and the mechanisms that mediate TGF-β's role in regulating HCC malignancy. Here, TGF-β pathway component expression and activation in human and murine HCC tissues were measured with quantitative RT-PCR and Western blotting assays. The role of TGF-β receptor and Smad signaling in the growth and survival of several HCC cell lines was determined with several in vitro and in vivo approaches. We found that TGF-β receptor II (TβRII) expression was downregulated in two different HCC patient cohorts. Consistently, Smad3 phosphorylation was also downregulated in HCC tissues in comparison to that in adjacent normal tissues. Interestingly, many HCC cell lines were sensitive to TGF-β and growth-inhibited by exogenous TGF-β. However, stable knockdown of TβRII inhibited cell growth on plastic and in soft agar, and induced apoptosis resulting in suppressed subcutaneous tumor growth and metastatic potential in vivo. Furthermore, knockdown of Smad4 also led to a significant inhibition of growth on plastic and in soft agar with concomitant increase of apoptosis, PTEN expression, and reduced nuclear accumulation of linker region-phosphorylated Smad3. Taken together, TGF-β signaling pathway plays a dichotomous role in hepatocellular carcinogenesis. It appears to suppress HCC development, but is retained for HCC cell survival and malignancy. Furthermore, Smad4 can mediate both growth inhibitory activity induced by exogenous TGF-β and the survival activity induced by autocrine TGF-β revealing a delicate selection of the two opposing activities of TGF-β during HCC evolution.

Chemical cross-linking was used to analyze the binding sites for the agonist bradykinin (BK) and the antagonists NPC17731 and HOE140 on the bovine B2 bradykinin receptor. [3H]BK and [3H]NPC17731 bound with high affinity to the same B2 receptor in bovine myometrial membranes as determined by the total number of specific binding sites and pharmacological specificity of the binding of these two radioligands. Cross-linking experiments were done using a series of bifunctional reagents reactive either primarily to amines (homobifunctional) or reactive to amines in one end and to sulfhydryls in the opposite end (heterobifunctional). All the heterobifunctional reagents plus the homobifunctional arylhalide 1,5-difluoro-2,4-dinitrobenzene were effective in cross-linking the [3H]BK N terminus specifically to a sulfhydryl in the receptor, and this cross-linking occurred at 5-100 microM reagent. In contrast, the homobifunctional N-hydroxysuccinimide ester reagents, ator = 1 mM, were only able to cross-link [3H]BK to membrane proteins nonspecifically. The sulfhydryl reagents N-ethylmaleimide, iodoacetamide, and phenylarsine oxide blocked cross-linking, whereas these reagents did not inhibit reversible specific [3H]BK binding. Immunoblotting with anti-BK antiserum revealed that low concentrations of BK (5-50 nM) were cross-linked to a receptor-specific species of 65 kDa. All cross-linking of [3H]NPC17731 was nonspecific with both homobifunctional and heterobifunctional reagents. The 65-kDa receptor-specific species was observed on anti-HOE140 immunoblots, but only when proteins were cross-linked with very high concentrations of HOE140 (or = 500 nM). Our results provide direct biochemical evidence that the binding site for the agonist BK in the bovine B2 receptor is adjacent to a cysteine and is differentiated from the binding site(s) for the antagonists NPC17731 and HOE140.

Introduction Hemostatic resuscitation in combat casualty care presents unique challenges not typically encountered in metropolitan areas. Fresh whole blood and blood components are considered preferable to crystalloid or colloid solutions, but availability may be limited in austere wartime environments. Autologous shed blood from a traumatic hemothorax or hemoperitoneum, in theory, is an attractive alternative. This position is supported by the American Academy of Blood Banking, Eastern Association for the Surgery of Trauma, and Society of Critical Care Medicine, despite the fact that the safety and efficacy of shed blood has never been evaluated in randomized trials. Recent reports suggest that shed blood has poor hemostatic properties. This study was performed to evaluate the effects of shed hemothorax plasma and microparticles on platelet function and global tests of coagulation. Methods Shed traumatic hemothorax blood was obtained from 17 adult trauma patients requiring tube thoracostomy, every hour up to 4 hours after placement, according to IRB approved protocol. The hemothorax plasma was then isolated and frozen for later analysis. The effects of shed frozen plasma at 1 and 4 hours (HFP1; HFP4) was examined by performing mixing studies with fresh platelet rich plasma from normal donors at pre-specified serial dilutions of 1:8, 1:4, and 1:2 to mimic intravascular resuscitation . This was then analyzed by several assays. Thromboelastometry (ROTEM) of HFP was done to evaluate clotting time (CT), clot formation time (CFT), alpha angle, and lysis index at 45 minutes. Platelet aggregometry was done to assess the effect of shed HFP on platelet function in response to adenosine diphosphate (ADP), arachidonic acid (ASPI) and collagen. Fibrin degradation products (FDP), von Willebrand factor (vWF) and tissue factor (TF) levels in HFP were determined by enzyme-linked immunosorbent assays (ELISA ). vWF multimers in HFP were further separated by size using gel electrophoresis and detected by immunostaining. Microparticle analysis was performed using isolated microparticles from ultra-centrifuged HFP1 and HFP4 (HMP1 and HMP4) and compared to control isolated microparticles from fresh frozen plasma (FMP). HMP and FMP were then diluted with platelet rich plasma to a predetermined platelet count after which, ROTEM and platelet aggregometry were performed as described above. Non-diluted HFP and resuspended HMP1, HMP4 and FMP were then analyzed by flow cytometry for a variety of microparticle (MP) derived antigens including APC-H7-CD41a (GPIIb/IIIa derived MP), and APC-CD235a (Glycophorin A from red cell derived MP), and FITC-Lactadherin phosphatidylserine (platelet derived MP). Results ROTEM of HFP demonstrated a CT of 55.8% and 60.6% at the 1:8 dilution and 69.3% and 71.4% at the 1:2 dilution, for HFP1 and HFP4 respectively, compared to platelet rich plasma (PRP) and FFP (p HMP1 and HMP4 inhibited platelet aggregation in response to ADP, TXA2, and collagen with data expressed as area under the curve (U), when compared to FMP and PRP, all statistically significant (p700ng/mL vs 0ng/mL p500 pg/mL vs Flow cytometry showed increased microparticle concentrations in HFP1, HFP4, HMP1, and HMP4 samples with greater than 10 fold increase (MP/uL) in several markers (p Conclusion These results demonstrate that autologous shed hemothorax blood promotes a prothrombotic state due to increased tissue factor and microparticle content. Furthermore, clot localization may be impaired through inhibition of platelet aggregation by microparticles and degradation of vWF. These data suggest that randomized trials are indicated to establish the safety and efficacy of autologous shed traumatic hemothorax blood for trauma resuscitation. Disclosures No relevant conflicts of interest to declare.

Several B2 bradykinin (BK) receptor-specific antagonists including HOE140, NPC17731, and NPC567 exhibited negative intrinsic activity, which was observed as a decrease in basal phosphoinositide hydrolysis in primary cultures of rat myometrial cells, and this response was opposite to that elicited by the agonist BK. The order of potency of the antagonists in attenuating basal activity was essentially the same as that in competing both [3H]BK and [3H]NPC17731 for binding to B2 receptors on both intact rat myometrial cells and bovine myometrial membranes. We previously proposed a three-state model for the binding of agonists to G-protein-coupled B2 receptors in bovine myometrial membranes (Leeb-Lundberg, L. M. F. and Mathis, S. A. (1990) J. Biol. Chem. 265, 9621-9627). This model was based on the ability of BK to promote the sequential formation of three receptor binding states where formation of the third, equilibrium state was blocked by Gpp(NH)p (guanyl-5'-yl imidodiphosphate) identifying it as the G-protein-coupled state of the receptor. Here, we show that, in contrast to BK, these antagonists bound preferentially to a G-protein-uncoupled state of the receptor. These results indicate that B2 receptor antagonists that stabilize a G-protein-uncoupled state of the receptor act as inverse agonists. Furthermore, these results provide strong evidence that endogenous G-protein-coupled receptors exhibit spontaneous activity in their natural environment in the absence of agonist occupancy.

The overexpression of Bcl-2 is implicated in the resistance of cancer cells to apoptosis. This study explored the potential of irofulven (hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863), a novel DNA- and protein-reactive anticancer drug, to overcome the anti-apoptotic properties of Bcl-2 in HeLa cells with controlled Bcl-2 overexpression. Irofulven treatment resulted in rapid (12 hr) dissipation of the mitochondrial membrane potential, phosphatidylserine externalization, and apoptotic DNA fragmentation, with progressive changes after 24 hr. Bcl-2 overexpression caused marginal or partial inhibition of these effects after treatment times ranging from 12 to 48 hr. Both Bcl-2-dependent and -independent responses to irofulven were abrogated by a broad-spectrum caspase inhibitor. Despite the somewhat decreased apoptotic indices, cell growth inhibition by irofulven was unaffected by Bcl-2 status. In comparison, Bcl-2 overexpression drastically reduced apoptotic DNA fragmentation by etoposide, acting via topoisomerase II-mediated DNA damage, but had no effect on apoptotic DNA fragmentation by helenalin A, which reacts with proteins but not DNA. Irofulven retains its pro-apoptotic and growth inhibitory potential in cell lines that have naturally high Bcl-2 expression. Collectively, the results implicate multiple mechanisms of apoptosis induction by irofulven, which may differ in time course and Bcl-2 dependence. It is possible that the sustained ability of irofulven to induce profound apoptosis and to block cell growth despite Bcl-2 overexpression may be related to its dual reactivity with both DNA and proteins.

Caspase-3 deficiency can limit the efficiency of pro-apoptotic anticancer treatments. Irofulven (hydroxymethylacyl-fulvene, HMAF. MGI 114, NSC 683863) is an antitumor drug, currently in a Phase III and multiple Phase II trials, which can differentiate between tumor and normal cells in apoptosis induction. This study investigated whether apoptosis induced by irofulven requires caspase-3. Irofulven action was compared in breast cancer cells differing in caspase-3 status: deficient MCF-7 cells and proficient MDA-MB-231 cells and in normal human mammary epithelial cells, HMEC. Irofulven induces significant, concentration and time-dependent apoptotic DNA fragmentation in breast cancer cell lines, regardless of caspase-3 status. After 12, 24 and 48 h incubation at 1 microM irofulven (approximately 3 x GI50), fragmented DNA comprised 3.7, 14.1 and 34.6% and 8.4, 12.6 and 20.3% of total DNA in MCF-7 and MDA-MB-231 cells, respectively. Cell viability (trypan blue exclusion) remained largely unaffected during the first 24 h but decreased markedly after 48 h, indicating secondary necrosis. Net losses in cell numbers were apparent at 48 h. Normal HMEC cells were refractory to 1 microM drug with only approximately 3-9% fragmented DNA after 12-48 h, although apoptosis was observed at drug levels >3 microM. The broad-spectrum caspase inhibitor Z-VAD-fmk inhibited irofulven-induced apoptosis of all cell lines at 20 microM with nearly complete abrogation of apoptosis at 100 microM. Irofulven treatment resulted in marginal caspase-3 processing in MDA-MB-231 and HMEC cells. These results indicate that whereas the caspase cascade mediates irofulven- induced apoptosis, caspase-3 is dispensable (supported by NIH CA70091 and CA78706).

A 3rd generation perfluorocarbon (PFC) is under development for the treatment of traumatic brain injury (TBI). PFCs have high gas solubility and can deliver oxygen to support ischemic brain tissue. This drug increased brain tissue oxygen consumption, decreased infarct size, and improved functional deficits in animal models of brain injury, but it also caused transient thrombocytopenia of unknown etiology. Acute inflammation also causes platelet deficits and is common in the setting of severe TBI. The interaction between the two could exacerbate thrombocytopenia. Here we studied PFC effects on platelet function, platelet morphology and global hemostasis in a lipopolysaccharide (LPS) baboon model of inflammation. Perfluorocarbon (Oxycyte”, Oxygen Biotherapeutics Inc, Morrisville, NC) infusion was performed with and without administration of LPS. The groups studied included: 1. Saline control (20 ml saline + 12 ml/kg saline: SALINE) 2. Saline and LPS control (0.3 mg/kg LPS in 20 ml saline + 12 ml/kg saline: SAL-LPS) 3. Medium dose PFC with saline vehicle (20 ml SALINE + 3 ml/kg PFC: SAL-PFC3) 4. High dose PFC with saline vehicle (20 ml SALINE + 12 ml/kg PFC: SAL-PFC12) 5. Medium dose PFC with LPS (LPS 0.3 mg/kg in 20 ml SALINE + 3 ml/kg PFC: LPS -PFC3) 6. High dose PFC with with LPS (LPS 0.3 mg/kg in 20 ml SALINE + 12 ml/kg PFC: LPS –PFC12) Platelet count, transmission electron microscopy, and flow cytometry were performed at baseline (BL) and at 2, 24, 48, 72, and 96 hours after perfluorocarbon infusion. Microparticles were quantified and characterized by flow cytometry and morphology examined via transmission electron microscopy. Impedance aggregometry and thromboelastography (TEG) were quantified at baseline (BL) and at T2, T72, and T96 (hours). Statistical analysis was by ANOVA, followed by pairwise comparisons and a Bonferroni adjustment. PFCs caused delayed thrombocytopenia which began 2 days post-infusion and persisted beyond the 5-day period of observation (nadirs≤50% of baseline at day 5, p PFC infusion caused clinically significant thrombocytopenia, particularly after LPS administration, and exacerbated LPS-induced platelet activation. The interaction between the two resulted in decreased hemostatic capacity, diffuse bleeding, and shock. Disclosures: Shade: Oxygen Biotherapeutics, Inc.: Research Funding. Anderson:Oxygen Biotherapeutics, Inc.: Employment.

This investigation compared the effects of hydroxymethylacylfulvene (HMAF), a novel antitumor drug with alkylating properties, in eight human tumor (prostate, colon, and leukemia) cell lines, and five human normal (prostate and renal proximal tubule epithelial, colon mucosa, fibroblasts, and endothelial) cell lines. Drug-induced growth inhibition paralleled the uptake of HMAF into both tumor and normal cells, although normal cells were 3- to 4-fold more tolerant to the accumulated drug. In both tumor and normal cells, approximately two-thirds of internalized [14C]HMAF-derived radioactivity was bound covalently to macromolecules. Trypan blue exclusion and cell counts indicated that HMAF was cytotoxic in tumor but cytostatic in normal cells. Correspondingly, profound apoptosis was detected in all tumor cell lines examined. A 4-hr treatment with HMAF followed by 20-hr post-incubation induced a potent DNA fragmentation in nearly all tumor lines. Apoptosis-resistant PC-3 and HT-29 cells underwent significant DNA fragmentation after 24 hr of continuous treatment with HMAF. In contrast to tumor cell lines, marginal or very low levels of apoptosis were detected in the normal cells even after prolonged treatments with HMAF at concentrations that exceeded 15- to 800-fold the gi 50 values in tumor cells. This resistance of normal cells to apoptosis could not be accounted for by differences in drug accumulation or drug covalent binding to macromolecules. The qualitatively different responses of the tumor and normal cells studied suggest a greater tolerance of normal cells to HMAF–macromolecular adducts. The demonstrated differential cytotoxic/cytostatic and apoptotic effects of HMAF can be of significance for the clinical use of this promising new agent.

Bizelesin is a bifunctional AT-specific DNA alkylating drug. Our study characterized the ability of bizelesin to induce interstrand crosslinks, a potential lethal lesion. In genomic DNA of BSC-1 cells, bizelesin formed from approx. 0.3 to 6.03±0.85 interstrand crosslinks per 10 6 base pairs, at 5–100 nM drug concentration, respectively, comparable to the number of total adducts previously determined in the same system (J.M. Woynarowski, M.M. McHugh, L.S. Gawron, T.A. Beerman, Biochemistry 34 (1995) 13042–13050). Bizelesin did not induce DNA-protein crosslinks or strand breaks. A model defined target, intracellular simian virus 40 (SV40) DNA, was employed to map at the nucleotide level sites of bizelesin adducts, including potential interstrand crosslinks. Preferential adduct formation was observed at AT tracts which are abundant in the SV40 matrix associated region and the origin of replication. Many sites, including each occurrence of 5′-T(A/T) 4 A-3′, co-mapped on both DNA strands suggesting interstrand crosslinks, although monoadducts were also formed. Bizelesin adducts in naked SV40 DNA were found at similar sites. The localization of bizelesin-induced crosslinks in AT-rich tracts of replication-related regions is consistent with the potent anti-replicative properties of bizelesin. Given the apparent lack of other types of lesions in genomic DNA, interstrand crosslinks localized in AT-rich tracts, and to some extent perhaps also monoadducts, are likely to be lethal effects of bizelesin.

Oxaliplatin is a clinical anticancer drug with a pharmacological profile distinct from that of cisplatin. Our studies compared site- and region-specificity of lesions induced by oxaliplatin and cisplatin in naked and intracellular DNA, respectively. Oxaliplatin adducts in naked Simian virus 40 (SV40 DNA) were mapped by repetitive primer extension. The sites of oxaliplatin adducts were nearly identical to the sites of cisplatin adducts and were focused in G clusters and GNG motifs probably reflecting intrastrand cross-links. Although alkaline agarose electrophoresis of specific SV40 fragments showed that oxaliplatin formed interstrand cross-links, the levels of this lesion type were low. Drug-induced lesions in discrete loci of cellular DNA were assessed by the polymerase chain reaction stop assay in human tumor A2780 cells. Oxaliplatin at 200 microM induced approximately 1300, approximately 1500, approximately 800, and approximately 300 lesions/10(6) bp in the human beta-globin, c-myc, and HPRT genes and in mitochondrial DNA, respectively. Cisplatin formed two to six times more lesions in the same regions. For both drugs, lesion frequencies seem to parallel the density of drug-binding motifs in the nuclear regions, whereas mitochondrial DNA was disproportionately less affected. Despite less potent induction of DNA lesions, oxaliplatin was more cytotoxic than cisplatin against A2780 cells. Because our findings clearly demonstrate that oxaliplatin forms covalent adducts with a similar sequence- and region-specificity to that of cisplatin, other properties of oxaliplatin adducts, factors other than DNA binding, or both determine the unique features of the mechanism of action of oxaliplatin.

Chemical cross-linking combined with site-directed mutagenesis was used to evaluate the role of extracellular cysteines and their positions relative to the binding site for the agonist bradykinin (BK) in the human BK B2 receptor. All extracellular cysteines, Cys20, Cys103, Cys184, and Cys277, in the receptor were mutated to serines, and single and double mutants were transfected into COS-7 cells. The Ser20 and Ser277 single mutants and the Ser20/Ser277 double mutant bound [3H]BK and the antagonist [3H]NPC17731 with pharmacological profiles identical to the wild-type B2 receptor. In contrast, the Ser103 and Ser184 single mutants were unable to bind either of the two radioligands. However, these mutants were still expressed as determined by immunoblotting with anti-B2 receptor antibodies. Previous studies on the bovine B2 receptor showed that bifunctional reagents, which are reactive to amines at one end and to free sulfhydryls in the opposite end, cross-link the N terminus of receptor-bound BK to a sulfhydryl in the receptor (Herzig, M. C. S., and Leeb-Lundberg, L. M. F. (1995) J. Biol. Chem. 270, 20591-20598). Here, we show that m-maleimidobenzoyl-N-hydroxysuccinimide ester and 1,5-difluoro-2, 4-dinitrobenzene cross-linked BK to the wild-type human B2 receptor and the Ser20 and Ser277 single mutant receptors, whereas these reagents were unable to cross-link BK to the Ser20/Ser277 double mutant. These results show that Cys103 and Cys184 are both required for expression of high affinity agonist and antagonist binding sites in the human B2 receptor, while Cys20 and Cys277 are not required. Furthermore, the results provide direct biochemical evidence that the N terminus of BK, when bound to the B2 receptor, is adjacent to Cys277 in extracellular domain 4 and Cys20 in extracellular domain 1 of the receptor.

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. Its development and progression have been shown to be regulated by various cytokines including TGFβ. However, the role of TGFβ signaling in the progression of HCC remains controversial. In the current study, we examined the expression levels of TGFβ pathway components and the activation status of Smad2/3 proteins as reflected by phospho-Smad2/3 levels in human and mouse HCC tissues and human HCC cell lines. We also investigated whether abrogation of TGFβ signaling in HCC cell lines that are responsive to TGFβ affects the growth and metastasis of transplanted HCC cells in nude mice. Quantitative real-time RT-PCR and Western immune blotting analyses reveal widespread down-regulation of TGFβ signaling pathway components including Smad2/3 and TGFβ type I and II receptors in HCC tissues of patients and mice in comparison to adjacent normal tissues. Treatment of our HCC cell lines (SNU398, SNU423, HepG2, and Sk-Hep-1) with TGFβ1 revealed that the SNU398 cell line was refractory whereas the other three cell lines were sensitive to TGFβ as determined with a number of in vitro assays including Smad phosphorylation, gene transcription, and growth inhibition assays. Knockdown of TGFβ type II receptor with shRNA significantly reduced TGFβ signaling activity in the Sk-Hep-1 cell line resulting in suppressed growth of its subcutaneous tumors and reduced metastasis potential when the cells were inoculated through tail vein injection. Our results suggest that TGFβ signaling may play a suppressive role in early stage of HCC development, but a promoting role in advanced stage, which is represented by the Sk-Hep-1 cell model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3259. doi:1538-7445.AM2012-3259

Epidemiological studies indicated that aging is a significant risk factor for breast cancer. Yet, there is insufficient knowledge on the mechanisms affecting breast cancer initiation and promotion in older women. Recent studies indicated epithelial stem/progenitor cell population, responsible for lifelong tissue maintenance and regeneration, as a target of transformation. It has been reported earlier by serial transplantation of murine mammary tissues that functional mammary outgrowths indicating stem cell function is unaffected by the age of the donor. But it is unclear that whether spontaneous or induced mutagenesis in the aging stem/progenitor cell population has the potential to impair stem/progenitor functions and/or to initiate tumorigenesis. In this study, first we have isolated lineage negative (CD31−CD45−Ter119−) mammary epithelial cells (Lin−MEC) from the thoracic/inguinal mammary glands of young (6 months, n=7) and old (28 months, n=6) mice by serial enzymatic dissociation followed by magnetic nanoparticle separation method (StemCell Technology). These cells were utilized to form mammospheres, representing mammary stem/early progenitor cells. Although the frequency of mammosphere formation remained the same, the number of larger mammospheres (>60µm) from old mice were significantly lower than the young mice, probably indicating decreased stem cell activity in the older population. To study the effect of mutagen in vivo, we treated two young and two old mice with methyl nitrosourea (MNU, 25mg/kg body weight) once a week for three consecutive weeks. Animals were sacrificed one week after the last treatment. Both total and larger mammospheres (>60µm) from the isolated Lin−MEC of the treated older mice were observed in significantly higher number than the treated younger mice. In a recent clinical study larger mammospheres (>50µm) obtained from metastatic breast cancer cells has been correlated to their ability to induce tumors. These Lin−MEC cells were also analyzed and compared for DNA damage by alkaline comet assay and a higher percentage of cells (>10%) with irreversible DNA damage in the older population were observed compared to younger population ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3469.