Using a dose-area product for absolute measurements in small fields: a feasibility study.

To extend the dosimetric reference system to field sizes smaller than 2 cm × 2 cm, the LNE-LNHB laboratory is studying an approach based on a new dosimetric quantity named the dose-area product instead of the commonly used absorbed dose at a point. A graphite calorimeter and a plane parallel ion chamber with a sensitive surface of 3 cm diameter were designed and built for measurements in fields of 2, 1 and 0.75 cm diameter. The detector surface being larger than the beam section, most of the issues linked with absolute dose measurements at a point could be avoided. Calibration factors of the plane parallel ionization chamber were established in terms of dose-area product in water for small fields with an uncertainty smaller than 0.9%.

A new single crystal diamond dosimeter for small beam: comparison with different commercial active detectors.

Recent developments of new therapy techniques using small photon beams, such as stereotactic radiotherapy, require suitable detectors to determine the delivered dose with a high accuracy. The dosimeter has to be as close as possible to tissue equivalence and to exhibit a small detection volume compared to the size of the irradiation field, because of the lack of lateral electronic equilibrium in small beam. Characteristics of single crystal diamond (tissue equivalent material Z = 6, high density) make it an ideal candidate to fulfil most of small beam dosimetry requirements. A commercially available Element Six electronic grade synthetic diamond was used to develop a single crystal diamond dosimeter (SCDDo) with a small detection volume (0.165 mm(3)). Long term stability was studied by irradiating the SCDDo in a (60)Co beam over 14 h. A good stability (deviation less than ± 0.1%) was observed. Repeatability, dose linearity, dose rate dependence and energy dependence were studied in a 10 × 10 cm(2) beam produced by a Varian Clinac 2100 C linear accelerator. SCDDo lateral dose profile, depth dose curve and output factor (OF) measurements were performed for small photon beams with a micro multileaf collimator m3 (BrainLab) attached to the linac. This study is focused on the comparison of SCDDo measurements to those obtained with different commercially available active detectors: an unshielded silicon diode (PTW 60017), a shielded silicon diode (Sun Nuclear EDGE), a PinPoint ionization chamber (PTW 31014) and two natural diamond detectors (PTW 60003). SCDDo presents an excellent spatial resolution for dose profile measurements, due to its small detection volume. Low energy dependence (variation of 1.2% between 6 and 18 MV photon beam) and low dose rate dependence of the SCDDo (variation of 1% between 0.53 and 2.64 Gy min(-1)) are obtained, explaining the good agreement between the SCDDo and the efficient unshielded diode (PTW 60017) in depth dose curve measurements. For field sizes ranging from 0.6 × 0.6 to 10 × 10 cm(2), OFs obtained with the SCDDo are between the OFs measured with the PinPoint ionization chamber and the Sun Nuclear EDGE diode that are known to respectively underestimate and overestimate OF values in small beam, due to the large detection volume of the chamber and the non-water equivalence of both detectors.

Assessment of small volume ionization chambers as reference dosimeters in high-energy photon beams.

LNE-LNHB is involved in a European project aiming at establishing absorbed dose-to-water standards for photon-radiation fields down to 2 × 2 cm². This requires the calibration of reference ionization chambers of small volume. Twenty-four ionization chambers of eight different types with volume ranging from 0.007 to 0.057 cm³ were tested in a 6°Co beam. For each chamber, two major characteristics were investigated: (1) the stability of the measured current as a function of the irradiation time under continuous irradiation. At LNE-LNHB, the variation of the current should be less than ±0.1% in comparison with its first value (over a 16 h irradiation time); (2) the variation of the ionization current with the applied polarizing voltage and polarity. Leakage currents were also measured. Results show that (1) every tested PTW (31015, 31016 and 31014) and Exradin A1SL chambers demonstrate a satisfying stability under irradiation. Other types of chambers have a stability complying with the stability criterion for some or none of them. (2) IBA CC01, IBA CC04 and Exradin A1SL show a proper response as a function of applied voltage for both polarities. PTW, Exradin A14SL and Exradin A16 do not. Only three types of chambers were deemed suitable as reference chambers according to LNE-LNHB requirements and specifications from McEwen (2010 Med. Phys. 37 2179-93): Exradin A1SL chambers (3/3), IBA CC04 (2/3) and IBA CC01 (1/3). The Exradin A1SL type with an applied polarizing voltage of 150 V was chosen as an LNE-LNHB reference chamber type in 2 × 2 cm² radiation fields.

Vesicular transport of newly synthesized cytochromes P4501A to the outside of rat hepatocyte plasma membranes.

Anti-cytochrome P450 (CYP)1A2 autoantibodies are found in dihydralazine-induced hepatitis, and CYPs2B and 2C have been shown to follow vesicular flow to the plasma membrane (PM). However, it is unknown whether other CYPs follow this route, whether NADPH-CYP reductase is present on the hepatocyte surface, and whether autoimmune hepatitis-inducing drugs increase PM CYPs. In this study, we determined the transmembrane topology and transport of CYPs1A in rat hepatocytes. In cultured hepatocytes, colchicine and other vesicular transport inhibitors decreased PM CYPs1A assessed by flow cytometry. Colchicine administration also decreased PM CYPs1A in vivo. Pulse chase experiments with [(35)S]methionine showed that only the newly synthesized CYP molecules are transferred to the PM, whereas microsomal CYP1A2 was stably radiolabeled for several hours. In contrast, radiolabeled CYP1A2 reached the PM and disappeared from the PM with half-lives of less than 30 min. Confocal microscopy, biotinylation, and coimmunoprecipitation experiments showed that PM CYPs1A and CYP reductase are present on the cell surface, and that the reductase is closely associated with PM CYPs. Exposure of whole cells to an anti-CYP1A1/2 antibody at 4 degrees C, before five washes and PM preparation, abolished PM CYPs1A-supported monooxygenase activity, indicating that PM CYPs are mostly located on the external surface. Dihydralazine and other CYPs1A inducers increased PM CYPs1A. In conclusion, newly synthesized CYPs1A follow vesicular flow to the outside of the PM, and NADPH-CYP reductase also is located on the hepatocyte surface. Dihydralazine administration increases PM CYP1A2, its autoimmune target.

Antigenic targets in tienilic acid hepatitis. Both cytochrome P450 2C11 and 2C11-tienilic acid adducts are transported to the plasma membrane of rat hepatocytes and recognized by human sera.

Patients with tienilic acid hepatitis exhibit autoantibodies that recognize unalkylated cytochrome P450 2C9 in humans but recognize 2C11 in rats. Our aim was to determine whether the immune reaction is also directed against neoantigens. Rats were treated with tienilic acid and hepatocytes were isolated. Immunoprecipitation, immunoblotting, and flow cytometry experiments were performed with an anti-tienilic acid or an anti-cytochrome P450 2C11 antibody. Cytochrome P450 2C11 was the main microsomal or plasma membrane protein that was alkylated by tienilic acid. Inhibitors of vesicular transport decreased flow cytometric recognition of both unalkylated and tienilic acid-alkylated cytochrome P450 2C11 on the plasma membrane of cultured hepatocytes. Tienilic acid hepatitis sera that were preadsorbed on microsomes from untreated rats (to remove autoantibodies), poorly recognized untreated hepatocytes in flow cytometry experiments, but better recognized tienilic acid-treated hepatocytes. This recognition was decreased by adsorption with tienilic acid or by preexposure to the anti-tienilic acid or the anti-cytochrome P450 2C11 antibody. We conclude that cytochrome P450 2C11 is alkylated by tienilic acid and follows a vesicular route to the plasma membrane. Tienilic acid hepatitis sera contain antibodies against this tienilic acid adduct, in addition to the previously described anticytochrome P450 autoantibodies.

Characterization and distribution in human tissues of a glycoproteic antigen defined by monoclonal antibody 1BE12 raised against the human breast cancer cell line T47D.

Spleen cells from inbred Biozzi mice, immunized against the human breast cancer cell line T47D, were fused with murine myeloma SP2O cells to generate monoclonal antibodies. One of these, 1BE12, of IgM isotype, reacted with five of six human breast tumor cell lines, while no binding was detectable with normal lymphocytes, RBC, or fibroblasts. The antigen recognized by monoclonal antibody 1BE12 was localized on the surface of T47D and MCF7 cells and was detected in cell-free supernatants of cultures. The antigen was found also on the surface of milk secretory cells. Immunohistochemical staining of frozen and paraffin-embedded sections of human tissues showed apical polarized reactivity in normal breast glands, while in all breast cancers staining was either cytoplasmic or membranous and heterogeneously distributed. Immunostaining was also observed in some other normal epithelia, including salivary gland, gastroduodenal mucosa, exocrine pancreas, and cervix. The antigen was not detectable in secretory endometrium, whereas proliferative endometrium was strongly stained. Colon carcinoma, and cancers of the bladder and endometrium were strongly reactive. No staining was detected in melanoma, lymphoma, mesothelioma, non-small cell lung carcinoma, and thyroid, renal, and ovarian carcinomas. Lectin absorption of MCF7 membrane extracts reduced 1BE12 binding. A large reduction in 1BE12 reactivity was observed after digestion of T47D and MCF7 membrane extracts with proteases. Treatment with sodium periodate resulted in complete loss of antigenicity, while neuraminidase treatment did not affect 1BE12 binding. These findings suggest that the 1BE12 epitope is expressed on the carbohydrate moiety of a glycoprotein and does not contain sialic acid. Immunoblotting of the perchloric acid-soluble fraction of MCF7 membrane extracts after electrophoresis in 1% agarose detected the antigen as a high molecular weight species (Mr greater than 900,000). The antigen was purified by perchloric acid extraction of MCF7 membrane preparations followed by affinity chromatography on 1BE12 antibody coupled to Sepharose-4B and gel exclusion fast protein liquid chromatography. No reactivity of the purified material was found with monoclonal antibodies directed against human milk fat globule membrane-associated mucins HMFG1 and DF3.