Background characterization of an ultra-low background liquid scintillation counter.

The Ultra-Low Background Liquid Scintillation Counter developed by Pacific Northwest National Laboratory will expand the application of liquid scintillation counting by enabling lower detection limits and smaller sample volumes. By reducing the overall count rate of the background environment approximately 2 orders of magnitude below that of commercially available systems, backgrounds on the order of tens of counts per day over an energy range of ~3-3600keV can be realized. Initial test results of the ULB LSC show promising results for ultra-low background detection with liquid scintillation counting.

Osteocyte differentiation and the formation of an interconnected cellular network in vitro.

Extracellular matrix (ECM) stiffness and cell density can regulate osteoblast differentiation in two dimensional environments. However, it is not yet known how osteoblast-osteocyte differentiation is regulated within a 3D ECM environment, akin to that existing in vivo. In this study we test the hypothesis that osteocyte differentiation is regulated by a 3D cell environment, ECM stiffness and cell density. We encapsulated MC3T3-E1 pre-osteoblastic cells at varied cell densities (0.25, 1 and 2 × 106 cells/mL) within microbial transglutaminase (mtgase) gelatin hydrogels of low (0.58 kPa) and high (1.47 kPa) matrix stiffnesses. Cellular morphology was characterised from phalloidin-FITC and 4',6-diamidino-2-phenylindole (DAPI) dilactate staining. In particular, the expression of cell dendrites, which are phenotypic of osteocyte differentiation, were identified. Immunofluorescent staining for the osteocytes specific protein DMP-1 was conducted. Biochemical analyses were performed to determine cell number, alkaline phosphatase activity and mineralisation at 2.5 hours, 3, 21 and 56 days. We found that osteocyte differentiation and the formation of an interconnected network between dendritic cells was significantly increased within low stiffness 3D matrices, compared to cells within high stiffness matrices, at high cell densities. Moreover we saw that this network was interconnected, expressed DMP-1 and also connected with osteoblast-like cells at the matrix surface. This study shows for the first time the role of the 3D physical nature of the ECM and cell density for regulating osteocyte differentiation and the formation of the osteocyte network in vitro. Future studies could apply this method to develop 3D tissue engineered constructs with an osteocyte network in place.

Development of a low background liquid scintillation counter for a shallow underground laboratory.

Pacific Northwest National Laboratory has recently opened a shallow underground laboratory intended for measurement of low-concentration levels of radioactive isotopes in samples collected from the environment. The development of a low-background liquid scintillation counter is currently underway to further augment the measurement capabilities within this underground laboratory. Liquid scintillation counting is especially useful for measuring charged particle (e.g., ß and α) emitting isotopes with no (or very weak) gamma-ray yields. The combination of high-efficiency detection of charged particle emission in a liquid scintillation cocktail coupled with the low-background environment of an appropriately designed shield located in a clean underground laboratory provides the opportunity for increased-sensitivity measurements of a range of isotopes. To take advantage of the 35m-water-equivalent overburden of the underground laboratory, a series of simulations have evaluated the scintillation counter's shield design requirements to assess the possible background rate achievable. This report presents the design and background evaluation for a shallow underground, low background liquid scintillation counter design for sample measurements.

Bone cell mechanosensation of fluid flow stimulation: a fluid-structure interaction model characterising the role integrin attachments and primary cilia.

Load-induced fluid flow acts as an important biophysical signal for bone cell mechanotransduction in vivo, where the mechanical environment is thought to be monitored by integrin and primary cilia mechanoreceptors on the cell body. However, precisely how integrin- and primary cilia-based mechanosensors interact with the surrounding fluid flow stimulus and ultimately contribute to the biochemical response of bone cells within either the in vitro or in vivo environment remains poorly understood. In this study, we developed fluid-structure interaction models to characterise the deformation of integrin- and primary cilia-based mechanosensors in bone cells under fluid flow stimulation. Under in vitro fluid flow stimulation, these models predicted that integrin attachments on the cell-substrate interface were highly stimulated ε(eq) > 200,000 µÎµ, while the presence of a primary cilium on the cell also resulted in significant strain amplifications, arising at the ciliary base. As such, these mechanosensors likely play a role in mediating bone mechanotransduction in vitro. Under in vivo fluid flow stimulation, integrin attachments along the canalicular wall were highly stimulated and likely play a role in mediating cellular responses in vivo. The role of the primary cilium as a flow sensor in vivo depended upon its configuration within the lacunar cavity. Specifically, our results showed that a short free-standing primary cilium could not effectively fulfil a flow sensing role in vivo. However, a primary cilium that discretely attaches the lacunar wall can be highly stimulated, due to hydrodynamic pressure in the lacunocanalicular system and, as such, could play a role in mediating bone mechanotransduction in vivo.

Cell morphology and focal adhesion location alters internal cell stress.

Extracellular mechanical cues have been shown to have a profound effect on osteogenic cell behaviour. However, it is not known precisely how these cues alter intracellular mechanics to initiate changes in cell behaviour. In this study, a combination of in vitro culture of MC3T3-E1 cells and finite-element modelling was used to investigate the effects of passive differences in substrate stiffness on intracellular mechanics. Cells on collagen-based substrates were classified based on the presence of cell processes and the dimensions of various cellular features were quantified. Focal adhesion (FA) density was quantified from immunohistochemical staining, while cell and substrate stiffnesses were measured using a live-cell atomic force microscope. Computational models of cell morphologies were developed using an applied contraction of the cell body to simulate active cell contraction. The results showed that FA density is directly related to cell morphology, while the effect of substrate stiffness on internal cell tension was modulated by both cell morphology and FA density, as investigated by varying the number of adhesion sites present in each morphological model. We propose that the cells desire to achieve a homeostatic stress state may play a role in osteogenic cell differentiation in response to extracellular mechanical cues.

Osteocyte differentiation is regulated by extracellular matrix stiffness and intercellular separation.

Osteocytes are terminally differentiated bone cells, derived from osteoblasts, which are vital for the regulation of bone formation and resorption. ECM stiffness and cell seeding density have been shown to regulate osteoblast differentiation, but the precise cues that initiate osteoblast-osteocyte differentiation are not yet understood. In this study, we cultured MC3T3-E1 cells on (A) substrates of different chemical compositions and stiffnesses, as well as, (B) substrates of identical chemical composition but different stiffnesses. The effect of cell separation was investigated by seeding cells at different densities on each substrate. Cells were evaluated for morphology, alkaline phosphatase (ALP), matrix mineralisation, osteoblast specific genes (Type 1 collagen, Osteoblast specific factor (OSF-2)), and osteocyte specific proteins (dentin matrix protein 1 (DMP-1), sclerostin (Sost)). We found that osteocyte differentiation (confirmed by dendritic morphology, mineralisation, reduced ALP, Col type 1 and OSF-2 and increased DMP-1 and Sost expression) was significantly increased on soft collagen based substrates, at low seeding densities compared to cells on stiffer substrates or those plated at high seeding density. We propose that the physical nature of the ECM and the necessity for cells to establish a communication network contribute substantially to a concerted shift toward an osteocyte-like phenotype by osteoblasts in vitro.

Interferon-alpha after autologous stem cell transplantation in pediatric patients with advanced Hodgkin's lymphoma.

Thirteen children with refractory or recurrent Hodgkin's lymphoma (HL) received high-dose chemotherapy and autologous hematopoietic stem cell transplant (ASCT). After hematologic recovery, 10 patients were given interferon-alpha (IFN-alpha) as adjuvant therapy, starting at a dose of 0.5 x 10(6) U/m2 subcutaneously, three times a week. The dose was escalated as tolerated. Patients were treated for a median of 12 (4-24) months. Transient myelosuppression was the most common toxicity and led to temporary treatment interruption in five patients. The IFN-alpha dose was increased in nine patients, to a median final dose of 3.5 x 10(6) U/m2/week. With a median follow-up of 67 (range 25-114) months, nine of the 10 patients are alive and in continuous remission. One patient relapsed. Three patients were not treated with IFN-alpha initially, two because of rapidly progressive disease. One patient received IFN-alpha for treatment of relapse after transplant, and is alive in remission 10 years later. IFN-alpha has activity in children with advanced HL, and prolonged, low-dose treatment given after ASCT can be tolerated. Its therapeutic effect as a post-transplant adjuvant warrants further investigation.

Total body irradiation, fludarabine, melphalan, and allogeneic hematopoietic stem cell transplantation for advanced pediatric hematologic malignancies.

We evaluated the efficacy and toxicity of adding 9 Gy of total body irradiation (TBI), in three single daily fractions of 3 Gy, to the reduced intensity regimen of fludarabine 30 mg/m2 i.v. x 4 days and melphalan 140 mg/m2 i.v. x 1 day in advanced pediatric hematologic malignancies. Twenty-two acute lymphoblastic leukemia (ALL), six acute myeloid leukemia (AML), and one non-Hodgkin lymphoma patients were transplanted. Of these, 13 were beyond second remission, and five had prior hematopoietic stem cell transplant (HSCT). Twenty-one donors were unrelated, of which 19 were from cord blood (CB) units. Three of the eight related donors were genotypically disparate. Oral mucositis and diarrhea were the most common toxicities. Twenty-seven patients achieved neutrophil engraftment (median 16 days), and 23 had platelet engraftment (median 42 days). One patient had primary graft failure. Seven patients died of non-relapse causes in the first 100 days. With a median follow-up of 52 months, seven of 22 ALL, five of six AML, and one of one lymphoma patients are alive and in remission. The regimen of TBI, fludarabine, and melphalan allows the engraftment of allogeneic hematopoietic stem cells (including mismatched CB). It was fairly well tolerated in pediatric patients, even for second transplants. Its efficacy requires further evaluation.

Augmentation of post transplant immunity: antigen encounter at the time of hematopoietic stem cell transplantation enhances antigen-specific donor T-cell responses in the post transplant repertoire.

After transplant, the immune system is reconstituted by cells derived from both hematopoietic stem cells and peripheral expansion from differentiated donor T cells. After transplant, immune function is poor despite transplantation of mature lymphocytes from immune-competent donors. We tested the hypothesis that early antigen encounter at the time of cell transplant would improve the desired donor T-cell responses. Two independent models of peptide-specific T-cell responses were studied. The model for CD4 cells employed T cells from transgenic (Tg) DO11.11 mice that constitutively express the T-cell receptor for the class II-restricted ovalbumin peptide 323-339. The model for CD8 cells employed non-Tg H2-Db-restricted T-cell responses to the influenza nucleoprotein peptide 366-374. As measured both functionally and by direct imaging of T cells using clonotypic reagents, encounter with specific antigen at the time of T-cell transplantation led to clonal expansion of donor T cells and preservation of donor T-cell function in the post transplant immune environment. Antigen-specific donor T-cell function was poor if antigen encounter was delayed or omitted. Severe parent>F1 graft-versus-host reactions blocked the effect of early antigen exposure. Vaccination of transplant recipients against microbial or leukemia antigens may be worthy of study.

Analysis of immunodominance among minor histocompatibility antigens in allogeneic hematopoietic stem cell transplantation.

In major histocompatibility complex (MHC)-matched allogeneic hematopoietic stem cell transplantation (HSCT), donor responses are directed against multiple host minor histocompatibility antigens (mHAgs), producing graft-versus-host disease (GVHD) and graft-versus-tumor (GVT) effects. We studied MHC-matched, mHAg-mismatched C3H.SW>C57BL/6 HSCT in which three mHAg are molecularly defined (B6dom1, H3, H13) to determine if there is a hierarchy of immunodominance among the mHAgs and to learn the contribution of each to GVHD. We found that B6dom1 was the immunodominant mHAg. B6dom1 did not block responses to the subdominant mHAgs H3 and H13. The mechanism of immunodominance was not mHAg avidity or affinity for class I. B6dom1 elicited a broader variety of Vbeta clonotypes than either H3 or H13. Severe GVHD could occur in the absence of a strong B6dom1 response. Alloreactivity to isolated B6dom1, H3 or H13 differences did not produce severe GVHD. We concluded that immunodominance is explained by both mHAg density on host cells and the repertoire of donor T cells capable of responding to the mHAgs. Clinically significant GVHD requires donor responses to multiple mHAgs. Modulation of responses to a single immunodominant mHAg is insufficient for the prevention of GVHD, while immunotherapies directed against isolated mHAgs may not provoke severe GVHD.

Non-albicans Candida is the most common cause of candidemia in pediatric cancer patients.

GOALS: Candidemia is a serious infection that can severely complicate the care of children with cancer. We sought to determine the spectrum of Candida species in children with cancer, since effective therapy may depend on the species involved. PATIENTS AND METHODS: A retrospective review of candidemia episodes in our pediatric oncology patients over a 9-year period was conducted. During this period azole prophylaxis was not routine in this group. RESULTS: 38 episodes of candidemia were identified: C. albicans 29%, C. tropicalis 26%, C. parapsilosis 24%, C. krusei 8%, C. glabrata 8%, and C. lusitaniae 5%. Non-albicans Candida was common in patients not receiving azole prophylaxis. Species typically susceptible to azoles were common among patients not using azoles. Death attributed to the fungal infection occurred in 21% of episodes, with nearly all the deaths occurring in patients with C. albicans and C. tropicalis. CONCLUSIONS: C. albicans is not the predominant species in pediatric oncology patients experiencing candidemia, even in azole-naive patients.

Lorazepam for seizure prophylaxis during high-dose busulfan administration.

Seizure is a recognized complication of high-dose busulfan (BU) therapy and phenytoin (DPH) is widely used as prophylaxis. A number of adverse effects have been associated with DPH and it may also interfere with BU metabolism. We used lorazepam (median dose 0.022 mg/kg) i.v. or p.o. before each dose and for 24 h after the last dose of BU as seizure prophylaxis to 29 children undergoing hematopoietic stem cell transplantation. The regimen was well tolerated and drowsiness was the only significant side-effect. Twelve patients were able to receive the entire prophylaxis by mouth. No seizure developed during and within 48 h of BU. Concomitant pharmacokinetic studies showed no alternation of the absorption and clearance of BU during lorazepam administration. Lorazepam can be used as an alternative for seizure prophylaxis during high-dose BU treatment.

Clinical parameters associated with low bacteremia risk in 1100 pediatric oncology patients with fever and neutropenia.

BACKGROUND: Traditionally, children with malignant disease who present with fever and neutropenia are hospitalized for parenteral antibiotics. More recently, outpatient strategies have been proposed for lower risk cohorts of such patients. The authors sought to identify clinical and laboratory parameters that are associated with a low risk of bacteremia in children with malignant disease who presented with febrile neutropenia. METHODS: A multicenter, retrospective cohort of children with malignant disease and fever with neutropenia was established in three pediatric oncology centers over a 5-year period. A total of 1171 episodes of febrile neutropenia (absolute neutrophil count [ANC] < 500 cells per mm(3)) were identified in children with malignant disease age > 1 year. The endpoints examined were 1) bacteremia and 2) intensive care unit admission or death related to bacteremia. The odds ratio was used to determine which of the following admission parameters and cut-off values were associated with the lowest risk for bacteremia: ANC, absolute phagocyte count (APC), absolute monocyte count (AMC), platelet count, and admission temperature. RESULTS: A total of 189 episodes of bacteremia were identified among the 1171 episodes of febrile neutropenia (14% bacteremia). Only 11 of 1171 episodes (0.9%) resulted in intensive care unit admission, and 3 of these patients died. All 11 patients had an AMC < 30 cells per mm(3). The lowest frequency of bacteremia (6.1%) occurred in the children with an admission AMC of > or = 155 cells per mm(3). None of the patients identified as low risk by AMC required an intensive care unit admission or died. No level of ANC, APC, temperature, or platelet count was associated with a statistically significant decrease in the risk for bacteremia in the patient population. CONCLUSIONS: Adverse outcomes due to bacteremia are infrequent in pediatric oncology patients who present with fever and neutropenia are treated with parental antibiotics. Patients with fever and neutropenia and an AMC value of > or = 155 cells per mm(3) have the lowest risk for bacteremia and may be potential candidates for outpatient management.

Bone marrow transplant conditioning intensified with liposomal clodronate to eliminate residual host antigen presenting cells fails to ameliorate GVHD and increases PERI-BMT mortality.

BACKGROUND: Graft versus host disease (GVHD) mediated by allogeneic donor T cells may be initiated and/or exacerbated by residual host antigen presenting cells (APC) which survive the transplant conditioning regimen. We examined whether the depletion of hepatic and splenic APC could reduce the severity of hepatic GVHD after bone marrow transplantation (BMT). METHODS: Recipient mice were depleted for hepatic and splenic phagocytic APCs by i.v. injection of clodronate- (dichloromethylene diphosphonate) containing liposomes before fully allogeneic or MHC-matched, minor Ag-mismatched BMT. Severity of hepatic GVHD was scored on histological sections 2, 3, 4, or 9 weeks after BMT. RESULTS: No differences in the severity of GVHD were observed between APC-depleted mice and control mice. APC-depleted mice had increased peritransplant mortality due to sepsis. Bacterial clearance assays showed that APC-depleted mice were unable to efficiently clear bacteria, although nondepleted, transplanted mice were able to clear bacteria as quickly as naive control mice. CONCLUSIONS: Residual host phagocytic APC do not appear to play a role in the induction of GVHD after BMT. They are, however, essential for prevention of sepsis in the transplant host.

Pretransplant tumor antigen-specific immunization of allogeneic bone marrow transplant donors enhances graft-versus-tumor activity without exacerbation of graft-versus-host disease.

Allogeneic bone marrow transplantation (BMT) causes a beneficial graft-versus-tumor (GVT) immune response that is often associated with graft-versus-host disease (GVHD). There is substantial interest in developing therapeutic strategies that augment GVT without GVHD. We have demonstrated recently that immunization of BMT donors with cellular tumor vaccines leads to curative GVT but induces unacceptable GVHD because of the presence of recipient minor histocompatibility antigens (mHAgs) in whole-cell tumor vaccines. This study tested the hypothesis that immunization of BMT donors against a defined tumor-specific antigen with a vaccine not containing recipient mHAgs would help to separate the two responses by enhancing GVT activity without exacerbating GVHD, even when cellular vaccines were used after BMT. Recipient strain C57BL/6 fibrosarcoma cells engineered to express the well-characterized model tumor antigen, influenza nucleoprotein (NP), were used in these studies. C3H.SW donors were immunized against NP prior to BMT, and cytolytic T cells were transferred along with bone marrow into irradiated H-2-matched, mHAg-mismatched C57BL/6 recipients with established micrometastatic 205-NP tumors. Donor immunization led to a significant increase in GVT activity, as measured by reduction in tumor growth and enhanced survival. However, deaths in recipients of tumor antigen-specific immune BMT ultimately occurred because of the growth of antigen-loss variants; such tumor growth did not occur in animals receiving BMT from donors treated with whole-cell vaccines. Donor immunization did not lead to an exacerbation of GVHD, even when BMT recipients received additional immunization after BMT with a 205-NP "whole" tumor cell vaccine (which was shown to induce fatal GVHD when used for donor immunization). In conclusion, immunization of allogeneic BMT donors against a tumor-specific antigen significantly enhances GVT activity without an associated exacerbation of GVHD.

Unrelated umbilical cord blood transplantation in infancy for mucopolysaccharidosis type IIB (Hunter syndrome) complicated by autoimmune hemolytic anemia.

This report describes unrelated umbilical cord blood transplantation for a 10-month-old infant boy with mucopolysaccharidosis IIB (Hunter syndrome), an X-linked metabolic storage disorder due to deficiency of iduronate sulfatase. Two years after transplant approximately 55% normal plasma enzyme activity has been restored and abnormal urinary excretion of glycosaminoglycans has nearly completely resolved. The boy has exhibited normal growth and development after transplant. Nine months after transplant he developed severe autoimmune hemolytic anemia and required 14 months of corticosteroid treatment to prevent clinically significant anemia. Bone marrow transplantation for Hunter syndrome and post-transplant hemolytic anemia are reviewed. Bone Marrow Transplantation (2000).

Use of a herpes thymidine kinase/neomycin phosphotransferase chimeric gene for metabolic suicide gene transfer.

Metabolic suicide gene transfer is widely applied for gene therapy of cancer, and retroviral vectors expressing the herpes simplex virus thymidine kinase (HSV-tk) gene are commonly used in clinical trials. Most of these vectors contain positive selectable markers that undoubtedly facilitate the determination of viral titer and the identification of high-titer producer clones. However, the presence of additional transcriptional units may result in reduced expression of the gene of interest. The use of fusion genes expressing bifunctional proteins may help to overcome this problem. We have constructed a retroviral vector carrying the TNFUS69 chimeric gene, which originates from the fusion of the HSV-tk and neomycin phosphotransferase II genes, and evaluated the functional expression of the encoded fusion protein. In vitro, expression of the fusion gene conferred to target cells both resistance to neomycin and selective sensitivity to the antiherpetic drugs ganciclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine. Cells transduced with the fusion gene, however, showed reduced ability to phosphorylate ganciclovir compared with cells expressing the native HSV-tk. Therefore, although the fusion gene may be used as a constituent of retroviral cassettes for positive and negative selection in vitro, its usefulness for suicide gene transfer applications in vivo may depend upon the possibility of using (E)-5-(2-bromovinyl)-2'-deoxyuridine in a clinical context.

Immunization of allogeneic bone marrow transplant recipients with tumor cell vaccines enhances graft-versus-tumor activity without exacerbating graft-versus-host disease.

Allogeneic bone marrow transplantation (BMT) induces 2 closely associated immune responses: graft-versus-tumor (GVT) activity and graft-versus-host disease (GVHD). We have previously shown that pretransplant immunization of allogeneic BMT donors with a recipient-derived tumor cell vaccine increases both GVT activity and lethal GVHD because of the priming of donor T cells against putative minor histocompatibility antigens (mHAgs) on the tumor vaccine cells. The work reported here tested the hypothesis that tumor cell vaccination after BMT would produce an increase in GVT activity without exacerbating GVHD. C3H.SW donor bone marrow and splenocytes were transplanted into major histocompatibility complex-matched, mHAg-mismatched C57BL/6 recipients. One month after BMT, recipients were immunized against either a C57BL/6 myeloid leukemia (C1498) or fibrosarcoma (205). Immunized recipients had a significant increase in survival and protection against tumor growth in both tumor models, and significant tumor protection was seen even in recipients with preexisting micrometastatic cancer before immunization. Alloreactivity appeared to contribute to the in vitro anti-tumor cytolytic activity, but in vivo immunity was tumor specific, and no exacerbation of GVHD was observed. Although the immunodominant mHAg B6(dom1) was shown to be expressed by all B6 tumors tested and was largely responsible for the alloreactivity resulting from tumor immunization of donors, the in vitro alloreactivity of immune recipients was more restricted and was not mediated by recognition of B6(dom1). In conclusion, post-transplant tumor immunization of allogeneic BMT recipients against either a leukemia or a solid tumor can increase GVT activity and survival without exacerbating GVHD.