Comparison of Cesium-137 and X-ray Irradiators by Using Bone Marrow Transplant Reconstitution in C57BL/6J Mice

Mice are used extensively in transplantation studies involving bone marrow ablation. Due to the increasing security issues and expenses involved with γ irradiators, self-contained X-ray irradiators have been increasing in popularity. We hypothesized that bone marrow ablation by irradiation of mice with a ¹³⁷Cs irradiator would be comparable to that from an X-ray source irradiator. A lethal-dose curve was obtained by irradiating C57BL/6J mice with 500, 700, 900, and 1100 cGy from either source. These data were used to determine the lethal radiation exposure range for a noncompetitive bone marrow engraftment curve for each source. At 90 d after reconstitution, the bone marrow engraftment curves revealed significant differences between the 2 sources in the establishment of B cell, myeloid, and T cell lineages. Murine B cell reconstitution after exposure to a ¹³⁷Cs source was greater than that after X-ray exposure at each dose level, whereas the converse was true for myeloid cell reconstitution. At the 1050- and 1100-cGy doses, mice irradiated by using the X-ray source demonstrated higher levels of T cell reconstitution but decreased survival compared with mice irradiated with the ¹³⁷Cs source. We concluded that although both sources ablated endogenous bone marrow sufficiently to enable stem cell engraftment, there are distinct physiologic responses that should be considered when choosing the optimal source for use in a study and that irradiation from the ¹³⁷Cs source was associated with lower overall morbidity due to opportunistic infection.Abbreviations: B3, barrier level; B4, barrier level 4; BMT, bone marrow transplantationStudies of hematopoietic stem cell transplantation have evolved over the past 60 y.^1,9 Many preclinical investigations involving cell and gene therapy and hematopoietic stem cell function are performed in mouse models, and techniques such as adoptive cell transfer and bone marrow transplant are commonly used in these studies. Such techniques often require a supralethal dose of irradiation to ensure adequate engraftment with donor cells and subsequent survival. Conventional γ-emitting irradiators (¹³⁷Cs and ⁶⁰Co sources) have been used to deliver myeloablative doses of radiation prior to bone marrow transplantation (BMT). After the terrorist attack of 11 September 11 2001, security measures regarding active radioactive source irradiators have been heightened. In 2005, the US Congress passed the Energy Policy Act, in which the US Nuclear Regulatory Commission was assigned to evaluate and prevent malicious misuse of radioactive materials. As a result, increased security controls were imposed on radioactive material sources and quantities of concern, including shielded active source irradiators.¹⁴ Mandated security measures now include fingerprinting and a criminal-history record check to allow persons unescorted access to various radioactive materials.¹⁵ Background checks and fingerprinting procedures can be time-consuming and present an additional expense that usually is passed on to individual investigators. These enhanced security measures have significantly increased the expense associated with use of these irradiators, and federal regulations as proposed in 10 CFR Part 37 are likely to become more stringent in coming years.¹⁶ This situation has correspondingly led to an increased interest in the use of X-ray irradiators as a substitute for γ-ray sources such as ¹³⁷Cs, and many animal facilities across the country have begun to purchase these units, even though there is no unbiased comparative information regarding the effectiveness of the instruments.In addition to decreased security requirements, X-ray irradiators are substantially less expensive to purchase than are active-source irradiators. After reviewing quotes, we estimate that the initial purchase price of an X-ray irradiator is about one sixth that of a cesium source. These figures do not include the costs of shipping, installation, or disposal of old active-source machines, and thus actual starts up costs are much higher. Annual maintenance as well as annual or semiannual dosimetry assessment costs are relatively comparable between the 2 sources. X-ray irradiators offer an additional financial advantage in that they do not require the strict security measures required for active γ-source irradiators. Given the number of disadvantages for the possession and use of γ-emitting irradiators, the use of X-ray irradiators in research likely will increase in the future.Extensive review of the literature did not reveal any studies in which bone marrow transplantation (BMT) efficiencies, kinetics, or overall responses in mice were compared between ¹³⁷Cs and X-ray irradiators. We hypothesized that both the ¹³⁷Cs and X-ray sources would ablate the bone marrow effectively and allow for comparable donor bone marrow reconstitution, and we sought to compare any differences in cell population engraftment after the use of each source. Recipient hematologic recovery after irradiation and reconstitution with bone marrow was assessed by determining the percentages of B and T lymphocytes and myeloid cells in the peripheral blood at 90 d after engraftment. In light of previously published work, we hypothesized that using the X-ray source before BMT would require a reduced dosage of radiation compared with that for the ¹³⁷Cs source.^4,6,9Historically, lethal-dose curves have been generated to calculate the dose which is lethal for 50% of the irradiated animals (mice, in this case) over a 30-d period (that is, the LD_50:30); this method allows approximation of the radiation sensitivity of a cohort of experimental mice.¹¹ A mouse in which 100% of the bone marrow has been ablated will be unable to recover hematopoietic function and will die. A priori, if the animal dies, one can assume that the minimal lethal dose has been reached or exceeded; conversely, if the mouse survives, the minimal lethal dose was not achieved. Because of the number of mice needed to calculate an accurate LD_50:30, we elected to perform a broad lethal-dose curve (1100 to 500 cGy in 200-cGy increments) to determine the point at which 100% death was reached for both sources. We then used this information as the lower radiation exposure limit for a bone marrow reconstitution curve (refined into 50-cGy increments), thereby allowing us to examine the bone marrow reconstitution response after differing radiation exposures.⁹ There is ample support in the literature for the broad lethal-dose test range chosen in this study.^4,9 Previous work with thymocyte reconstitution after bone marrow ablation has demonstrated that irradiation exposures of approximately 400 cGy are required to establish a population of donor-derived thymocytes in the recipient.⁵ Therefore, we used a dosage test range above 400cGy in the current study. The dose range for this study was 500 to 1100 cGy, and we expected to see morbidity and mortality primarily due to bone marrow failure between 8 and 20 d in the lethal-dose curve.¹²     

Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes

Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.     

Pentacycloundecane lactam vs lactone norstatine type protease HIV inhibitors: binding energy calculations and DFT study

Background

Novel pentacycloundecane (PCU)-lactone-CO-EAIS peptide inhibitors were designed, synthesized, and evaluated against wild-type C-South African (C-SA) HIV-1 protease. Three compounds are reported herein, two of which displayed IC₅₀ values of less than 1.00 μM. A comparative MM-PB(GB)SA binding free energy of solvation values of PCU-lactam and lactone models and their enantiomers as well as the PCU-lactam-NH-EAIS and lactone-CO-EAIS peptide inhibitors and their corresponding diastereomers complexed with South African HIV protease (C-SA) was performed. This will enable us to rationalize the considerable difference between inhibitory concentration (IC₅₀) of PCU-lactam-NH-EAIS and PCU-lactone-CO-EAIS peptides.

Results

The PCU-lactam model exhibited more negative calculated binding free energies of solvation than the PCU-lactone model. The same trend was observed for the PCU-peptide inhibitors, which correspond to the experimental activities for the PCU-lactam-NH-EAIS peptide (IC₅₀ = 0.076 μM) and the PCU-lactone-CO-EAIS peptide inhibitors (IC₅₀ = 0.850 μM). Furthermore, a density functional theory (DFT) study on the natural atomic charges of the nitrogen and oxygen atoms of the three PCU-lactam, PCU-lactim and PCU-lactone models were performed using natural bond orbital (NBO) analysis. Electrostatic potential maps were also used to visualize the electron density around electron-rich regions. The asymmetry parameter (η) and quadrupole coupling constant (χ) values of the nitrogen and oxygen nuclei of the model compounds were calculated at the same level of theory. Electronic molecular properties including polarizability and electric dipole moments were also calculated and compared. The Gibbs theoretical free solvation energies of solvation (∆G_solv) were also considered.

Conclusions

A general trend is observed that the lactam species appears to have a larger negative charge distribution around the heteroatoms, larger quadrupole constant, dipole moment and better solvation energy, in comparison to the PCU-lactone model. It can be argued that these characteristics will ensure better eletronic interaction between the lactam and the receptor, corresponding to the observed HIV protease activities in terms of experimental IC₅₀ data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0115-5) contains supplementary material, which is available to authorized users.     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

Evidence for Multidrug Resistance-1 P-Glycoprotein-dependent Regulation of Cellular ATP Permeability

The mechanisms responsible for regulating epithelial ATP permeability and purinergic signaling are not well defined. Based on the observations that members of the ATP-binding cassette (ABC)¹ family of proteins may contribute to ATP release, the purpose of these studies was to assess whether multidrug resistance-1 (MDR1) proteins are involved in ATP release from HTC hepatoma cells. Using a bioluminescence assay to detect extracellular ATP, increases in cell volume increased ATP release ∼3-fold. The MDR1 inhibitors cyclosporine A (10 μm) and verapramil (10 μm) inhibited ATP release by 69% and 62%, respectively (p < 0.001). Similarly, in whole-cell patch-clamp recordings, intracellular dialysis with C219 antibodies to inhibit MDR1 decreased ATP-dependent volume-sensitive Cl⁻ current density from −33.1 ± 12.5 pA/pF to −2.0 ± 0.3 pA/pF (−80 mV, p≤ 0.02). In contrast, overexpression of MDR1 in NIH 3T3 cells increased ATP release rates. Inhibition of ATP release by Gd³⁺ had no effect on transport of the MDR1 substrate rhodamine-123; and alteration of MDR1-substrate selectivity by mutation of G185 to V185 had no effect on ATP release. Since the effects of P-glycoproteins on ATP release can be dissociated from P-glycoprotein substrate transport, MDR1 is not likely to function as an ATP channel, but instead serves as a potent regulator of other cellular ATP transport pathways. Received: 20 November 2000/Revised: 25 May 2001     

Development of enzyme-immunoassays based on egg yolk antibodies for the detection of mycotoxins

Enzyme-linked immunosorbent assays (ELISA) proved to be a fast and simple method for the detection of mycotoxins and other undesired contaminants in food and feed. The present study is focused on the optimisation and exploitation of the egg yolk antibody technology in order to develop competitive ELISAs for the detection of mycotoxins in cereals. Due to its importance as one of the most relevant Fusarium mycotoxins, the trichothecene deoxynivalenol (DON) was selected as representative. Chickens were immunised with different protein conjugates performing varying booster intervals. The antibodies were isolated by the poly(ethylene glycol) precipitation method according to Polson. By use of these antibodies an indirect competitive ELISA was developed for the detection of DON. First investigations of naturally contaminated wheat samples showed a good correspondence with results obtained by GC-ECD when calibration in blank wheat extracts was performed.     

RETRACTED ARTICLE: Mast cells are the main interleukin 17-positive cells in anticitrullinated protein antibody-positive and -negative rheumatoid arthritis and osteoarthritis synovium

Introduction  

Mast cells have been implicated to play a functional role in arthritis, especially in autoantibody-positive disease. Among the cytokines involved in rheumatoid arthritis (RA), IL-17 is an important inflammatory mediator. Recent data suggest that the synovial mast cell is a main producer of IL-17, although T cells have also been implicated as prominent IL-17 producers as well. We aimed to identify IL-17 expression by mast cells and T cells in synovium of arthritis patients.