Objective. Recent evidence suggests that hematopoietic stem cells (HSCs) in the bone marrow (BM) are located in areas where the environment is hypoxic. Although previous studies have demonstrated positive effects by hypoxia, its role in HSC maintenance has not been fully elucidated, neither has the molecular mechanisms been delineated. Here, we have investigated the consequence of in vitro incubation of HSCs in hypoxia prior to transplantation and analyzed the role of hypoxia-inducible factor (HIF)-1 alpha. Materials and Methods. HSC and progenitor populations isolated from mouse BM were cultured in 20% or 1% O-2, and analyzed for effects on cell cycle, expression of cyclin-dependent kinase inhibitors genes, and reconstituting ability to lethally irradiated mice. The involvement of HIF-1 alpha was studied using methods of protein stabilization and gene silencing. Results. When long-term FLT3(-)CD34(-)Lin(-)Sca-1(+)c-Kit(+) (LSK) cells were cultured in hypoxia, cell numbers were significantly reduced in comparison to normoxia. This was due to a decrease in proliferation and more cells accumulating in G(0). Moreover, the proportion of HSCs with long-term engraftment potential was increased. Whereas expression of the cyclin-dependent kinase inhibitor genes p21(cip1), p27(Kip1), and p57(Kip2) increased in LSK cells by hypoxia, only p21(cip1) was upregulated in FLT3(-)CD34(-)LSK cells. We could demonstrate that expression of p27(KiP1) and p57(Kip2) was dependent of HIF-1 alpha. Surprisingly, overexpression of constitutively active HIF-1 alpha or treatment with the HIF stabilizer agent FG-4497 led to a reduction in HSC reconstituting ability. Conclusions. Our results imply that hypoxia, in part via HIF-1 alpha, maintains HSCs by decreasing proliferation and favoring quiescence.

Immune-induced prostaglandin E2 (PGE2) synthesis is critical for fever and other centrally elicited disease symptoms. The production of PGE2 depends on cyclooxygenase-2 and microsomal prostaglandin E synthase-1 (mPGES-1), but the identity of the cells involved has been a matter of controversy. We generated mice expressing mPGES-1 either in cells of hematopoietic or nonhematopoietic origin. Mice lacking mPGES-1 in hematopoietic cells displayed an intact febrile response to lipopolysaccharide, associated with elevated levels of PGE2 in the cerebrospinal fluid. In contrast, mice that expressed mPGES-1 only in hematopoietic cells, although displaying elevated PGE2 levels in plasma but not in the cerebrospinal fluid, showed no febrile response to lipopolysaccharide, thus pointing to the critical role of brain-derived PGE2 for fever. Immunohistochemical stainings showed that induced cyclooxygenase-2 expression in the brain exclusively occurred in endothelial cells, and quantitative PCR analysis on brain cells isolated by flow cytometry demonstrated that mPGES-1 is induced in endothelial cells and not in vascular wall macrophages. Similar analysis on liver cells showed induced expression in macrophages and not in endothelial cells, pointing at the distinct role for brain endothelial cells in PGE2 synthesis. These results identify the brain endothelial cells as the PGE2-producing cells critical for immune-induced fever.

The air kerma area product P_KA is an important quantity used by hospital physicists in quality assurance and optimization processes in diagnostic radiology and is recommended by national authorities for setting of diagnostic reference levels. P_KA can be measured using a transmission ionization chamber (kerma area product (KAP) meter) mounted on the collimator housing. Its signal Q_KAP must be calibrated to give values of PKA. The objective of this thesis is to analyze the factors influencing the accuracy of the calibration coefficients k= P_KA/Q_KAP and of reported PKA-values.

Due to attenuation and scatter in the KAP-meter and presence of extra-focal radiation, values of P_KA depend on the choice of integration area A and the distance of the reference plane from the focal spot yielding values of PKA that may differ by as much as 23% depending on this choice. The two extremes correspond to (1) P_KA=P_KA,o integrated over the exit surface of the KAP-meter resulting in geometry independent calibration coefficients and (2) P_KA=P_KA,Anom integrated over the nominal beam area in the patient entrance plane resulting in geometry dependent calibration coefficients.

Three calibration methods are analysed. Method 1 aims at determine P_KA,Anom, for clinical use at the patient entrance plane. At standard laboratories, the method is used to calibrate with respect to radiation incident on the KAP-meter. Problems with extra-focal and scattered radiation are then avoided resulting in calibration coefficients with low standard uncertainty (±1.5 %, coverage factor 2). Method 2 was designed in this work to approach determination of P_KA,o using thermoluminescent detectors to monitor contributions from extra-focal radiation and account for the heel effect. The uncertainty in derived calibration coefficients was ± 3% (coverage factor 2). Method 3 uses a Master KAP-meter calibrated at a standard laboratory for incident radiation to calibrate clinical KAP-meters. It has potential to become the standard method in the future replacing the tedious method 2 for calibrations aiming at determination of P_KA,o.

Commercially available KAP-meters use conducting layers of indium oxide causing a strong energy dependence of their calibration coefficients. This dependence is investigated using Monte Carlo simulations and measurements. It may introduce substantial uncertainties in reported P_KA– values since calibration coefficients as obtained from standard laboratories are often available only at one filtration (2.5 mm Al) as function of tube voltage or HVL. This is not sufficient since higher filtrations are commonly used in practice, including filters of Cu. In extreme cases, calibration coefficients for the same value of HVL but using different tube voltages and filtrations can deviate by as much as 30%. If standardised calibration methods are not used and choice of calibration coefficients not carefully chosen with respect to beam quality, the total uncertainty in reported P_KA–values may be as large as 40-45%. Conversion of PKA-values to risk related quantities is briefly discussed. The large energy dependence of the conversion coefficients, ε/P_KA, for determination of energy imparted,ε, to the patient reduces to a lower energy dependence of calibration coefficients CQ,ε = ε/Q_KAP for determination of ε from the KAP-meter signal.

This report was prepared in order to give more details to the uncertainty evaluation of the Kerma area product meter calibration procedure described in the paper:

Larsson J P Persliden J Sandborg S and Alm Carlsson G 1996 Transmission ionization chambers for measurements of air collision kerma integrated over beam area. Factors limiting the accuracy of calibration. Phys. Med. Biol. 41 2381-2398.

Figures and equations referred to in this report will be found in the paperabove.

For convenience, however, the equations in the paper that are used in the uncertainty analysis are retyped on the next two sides, see text in section 2.7. in the paper for further details. The numbering of the equations are kept as in the paper.

Calibrations of kerma-area product meters (KAP meters) are often performed using simplified methods. The accuracy thus obtained can be insufficient, especially when the KAP meters are used for optimizing radiological procedures. The deviations between the best available calibration factor (k) and the simplified calibration factor were measured at different clinical x-ray installations. Depending on the type of x-ray installation and calibration method, the quotient ranged from 0.83 to 1.19, reflecting the error made in practice using these methods. A simple alternative calibration method based on comparison with a KAP meter calibrated by the best available method is described. Depending on tube potential and the stability of the electrometers, the uncertainty in the calibration factor derived with this method was between 3.8% and 5.6% (at 95% confidence level).

Kerma - area product meters (KAP meters) are frequently used in diagnostic radiology to measure the integral of air-collision kerma over an area perpendicular to the x-ray beam. In this work, a precise method for calibrating a KAP meter to measure is described and calibration factors determined for a broad range of tube potentials (40 - 200 kV). The integral is determined using a large number of TL dosimeters spread over and outside the nominal field area defined as the area within 50% of maximum . The method is compared to a simplified calibration method which approximates the integral by multiplying the kerma in the centre of the field by the nominal field area . While the calibration factor using the precise method is independent of field area and distance from the source, that using the simplified method depends on both. This can be accounted for by field inhomogeneities caused by the heel effect, extrafocal radiation and scattered radiation from the KAP meter. The deviations between the calibration factors were as large as for collimator apertures of and distances from the source of 50 - 160 cm. The uncertainty in the calibration factor using the precise method was carefully evaluated and the expanded relative uncertainty estimated to be with a confidence level of 95%.

The Monte Carlo method was used to study the dependence of the calibration coefficient on the tube voltage, beam aperture and reference plane in simplified over-couch geometries modelling VacuTec's type 70157 KAP-meter both with and without an additional filter. The MCNP5 code was used to calculate (i) energy imparted to air cavities of the KAP-meter and (ii) spatial distribution of air collision kerma at entrance and exit planes of the KAP-meter and at a plane close to the patient. From these data, the air kerma area product and calibration coefficient were calculated and their dependence on the tube voltage and beam aperture was analysed. It was found that the variation of the calibration coefficient as a function of tube voltage was up to 40% when the additional filter was used. The additional filter placed closely in front of the KAP-meter decreased the calibration coefficient for the patient plane by about 10% compared to the ideal additional filter. The effect of the beam aperture was small at the patient plane and negligible for the exit plane.