Comparative dosimetry of diode and diamond detectors in electron beams for intraoperative radiation therapy

The aim of the present study is to examine the validity of using silicon semiconductor detectors in degraded electron beams with a broad energy spectrum and a wide angular distribution. A comparison is made with diamond detector measurements, which is the dosimeter considered to give the best results provided that dose rate effects are corrected for. Two-dimensional relative absorbed dose distributions in electron beams (6-20 MeV) for intraoperative radiation therapy (IORT) are measured in a water phantom. To quantify deviations between the detectors, a dose comparison tool that simultaneously examines the dose difference and distance to agreement (DTA) is used to evaluate the results in low- and high-dose gradient regions, respectively. Uncertainties of the experimental measurement setup (+/- 1% and +/- 0.5 mm) are taken into account by calculating a composite distribution that fails this dose-difference and DTA acceptance limit. Thus, the resulting area of disagreement should be related to differences in detector performance. The dose distributions obtained with the diode are generally in very good agreement with diamond detector measurements. The buildup region and the dose falloff region show good agreement with increasing electron energy, while the region outside the radiation field close to the water surface shows an increased difference with energy. The small discrepancies in the composite distributions are due to several factors: (a) variation of the silicon-to-water collision stopping-power ratio with electron energy, (b) a more pronounced directional dependence for diodes than for diamonds, and (c) variation of the electron fluence perturbation correction factor with depth. For all investigated treatment cones and energies, the deviation is within dose-difference and DTA acceptance criteria of +/- 3% and +/- 1 mm, respectively. Therefore, p-type silicon diodes are well suited, in the sense that they give results in close agreement with diamond detectors, for practical measurements of relative absorbed dose distributions in degraded electron beams used for IORT.     

Significant expression of IGFBP2 in breast cancer compared with benign lesions

BACKGROUND/AIM: Insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) play a role in the normal development of breast tissue, and possibly in breast cancer aetiology. IGFBP2, one of six members of the IGFBP superfamily, acts as regulator of the IGFs and has pleiotropic effects in normal and neoplastic tissues. Because IGFs have mitogenic effects on mammary epithelia, this study investigated IGFBP2 expression in mammary tissues of different benign and malignant entities. METHODS: Immunohistochemistry was used to study correlations between the presence and intensity of IGFBP2 staining and tumour type and grade, in addition to steroid hormone receptor status, in 120 breast specimens. Expression was measured by quantitative colour video image analysis and semiquantitative evaluation, and the measurements correlated well (r = 0.92; p<0.05). RESULTS: Both methods found no significant expression of IGFBP2 in normal glandular cells and hyperplasia (group I). Atypical hyperplasia showed a slightly increased cytoplasmic expression of IGFBP2, and carcinoma in situ showed a distinctive, membrane associated and cytoplasmic expression (group II). Infiltrating carcinomas strongly expressed cytoplasmic IGFBP2 (group III). There were significant differences between group I and II, and between group II and III. There were no significant differences between invasive lobular and invasive ductal carcinoma, or between grades I, II, and III within these entities. There was no significant correlation between IGFBP2 immunostaining and oestrogen or progesterone receptor positivity within the malignant group. CONCLUSIONS: IGFBP2 mitogenic signals of autocrine/paracrine regulatory mechanisms may be responsible for the growth of breast carcinomas and IGFBP2 may be an independent indicator of malignancy.     

Increase in vascular permeability induced by leukotriene b4 and the role of polymorphonuclear leukocytes

Leukotriene B₄ (LTB₄), a metabolite of arachidonic acid, is known to be a potent chemotactic and chemokinetic substance. We have used the hamster cheek pouch microcirculation model to study the effect of LTB₄ on vascular permeability and the involvement of neutrophil granulocytes in this response. Intravascular fluorescein-labeled dextran (mol wt 150,000) was used as a tracer of macromolecular permeability. Topical application of LTB₄ (150 nM-5 M) to the hamster cheek pouch resulted in an immediate increase in adhering leukocytes in postcapillary venules and later larger venules. Leukocyte accumulation was reversible, but continued longer the higher the dose of LTB₄ used. Subsequently, a dose-dependent increase in vascular permeability was seen at postcapillary and larger venules, with a maximum 10–20 min after application; the maximum occurred later the higher the dose of LTB₄. Depletion of neutrophil granulocytes by pretreatment of the animals with antineutrophil serum obtained from immunized rabbits significantly decreased the permeability response to LTB₄, whereas the response to histamine was unaffected. These results suggest that neutrophil granulocytes play a role in LTB₄-mediated permeability increase. LTB₄ may be of importance both for the leukocyte accumulation and for the edema formation seen in inflammatory reactions.     

Characterisation of recombinant human IgE-Fc fragments expressed in baculovirus-infected insect cells

IgE mediates its effector functions through the Fc region and it has been demonstrated that structures in the Cvarepsilon3-domain are crucial for FcvarepsilonR-binding. In order to further study structures of importance for the function of IgE, such as the carbohydrates, fragments with unmodified amino acid sequence were blunt-end cloned and expressed in baculovirus-infected Sf9 cells. Two fragments of human IgE, one encompassing the entire Fc-region (rCvarepsilon2-4) and a smaller one comprising the second and third domain (rCvarepsilon2-3), were produced and characterised with respect to epitope expression, glycosylation and FcvarepsilonR-binding. N-terminal analysis showed the expected VCSRDF-sequence of the Cvarepsilon2-domain, confirming correct cleavage of the secretion signal. Immunoblotting and gel permeation chromatography demonstrated that rCvarepsilon2-4 mainly formed a dimer, whereas rCvarepsilon2-3 also existed as monomers and oligomers. Endoglycosidase-treatment revealed that both fragments were N-glycosylated. In inhibition ELISA, rCvarepsilon2-4 and myeloma protein IgE(DES) reacted in a near equimolar way with monoclonal antibodies against the Cvarepsilon2-, Cvarepsilon3- and Cvarepsilon4-domains, whereas rCvarepsilon2-3 only reacted with anti-Cvarepsilon2 mAbs. Moreover, in FACS analysis rCvarepsilon2-4 interacted with two cell-lines constitutively expressing FcvarepsilonRI or FcvarepsilonRII, whereas rCvarepsilon2-3 lacked reactivity. A substantial reduction in the ability of rCvarepsilon2-4, following endoglycosidase treatment, to react with recombinant alpha-chain of the high affinity receptor for IgE in sandwich ELISA, indicated a role of N-linked oligosaccharides in stabilising receptor binding structures. Taken together, our results show that rCvarepsilon2-4, but not rCvarepsilon2-3, will be useful in studies of structure-function relationships of IgE, including the role of N-glycosylation, since it demonstrated appropriate epitope expression, conformation and ability to bind Fcvarepsilon-receptors.     

Characterization of the major secreted zinc metalloprotease- dependent glycerophospholipid:cholesterol acyltransferase, PlaC, of Legionella pneumophila

Legionella pneumophila, an intracellular pathogen causing a severe pneumonia, possesses distinct lipolytic activities which have not been completely assigned to specific enzymes so far. We cloned and characterized a gene, plaC, encoding a protein with high homology to PlaA, the major secreted lysophospholipase A of L. pneumophila and to other hydrolytic enzymes belonging to the GDSL family. Here we show that L. pneumophila plaC mutants possessed reduced phospholipase A and lysophospholipase A activities and lacked glycerophospholipid:cholesterol acyltransferase activity in their culture supernatants. The mutants' reduced phospholipase A and acyltransferase activities were complemented by reintroduction of an intact copy of plaC. Additionally, plaC conferred increased lysophospholipase A and glycerophospholipid:cholesterol acytransferase activities to recombinant Escherichia coli. Furthermore, PlaC was shown to be another candidate exported by the L. pneumophila type II secretion system and was activated by a factor present in the bacterial culture supernatant dependent on the zinc metalloprotease. Finally, the role of plaC in intracellular infection of Acanthamoeba castellanii and U937 macrophages with L. pneumophila was assessed, and plaC was found to be dispensable. Thus, L. pneumophila possesses another secreted lipolytic enzyme, a protein with acyltransferase, phospholipase A, and lysophospholipase A activities. This enzyme is distinguished from the previously characterized phospholipases A and lysophospholipases A by its capacity not only to cleave fatty acids from lipids but to transfer them to cholesterol. Cholesterol is an important compound of eukaryotic membranes, and an acyltransferase might be a tool for host cell modification to fit the needs of the bacterium.     

Gene expression of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase in skeletal muscle from type 2 diabetic subjects

The gene of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase gives rise to several splice variants. We hypothesized that the expression of p85alpha splice variants may be altered in skeletal muscle from subjects with type 2 diabetes mellitus. Skeletal muscle biopsies were obtained from nine type 2 diabetic and eight healthy men, matched for age, body mass index (BMI) and physical fitness. PI 3-kinase activity in skeletal muscle following in vitro insulin stimulation was reduced in subjects with type 2 diabetes. p85alpha mRNA was elevated fourfold in type 2 diabetic as compared to healthy control subjects ( P<0.05). p85alpha mRNA abundance was positively correlated with plasma insulin concentration ( P<0.01) and serum glucose concentration ( P<0.01). Despite this, protein levels of p85alpha, p55alpha, and the novel human p50alpha were not altered in type 2 diabetic subjects. Thus, although gene expression of full-length p85alpha is increased in skeletal muscle from type 2 diabetics, this is not reflected by increased protein levels. Therefore, defects in PI 3-kinase activity are likely due to impaired activation of the enzyme rather than changes in protein expression of the isoforms of the regulatory subunit.     

Antibody responses to honey-bee venom and monomethoxy-polyethylene glycol-modified honey-bee venom in mice

Antibody responses of the IgE isotype were raised in mice with honey-bee venom (HBV) administered in alum or by daily injections without adjuvant. The sensitized mice were treated with single injections of HBV modified with monomethoxy-polyethylene glycol. By such treatment with modified but not with natural HBV, suppression of the IgE antibody responses was achieved. The IgG antibody responses, in contrast, were unchanged or enhanced.     

Specific IgE, IgG, and IgA antibody response to oral immunotherapy in birch pollinosis

Thirty-nine patients with birch pollinosis participated in a double-blind, placebo-controlled trial of oral immunotherapy (OIT) for 18 months. They were treated with increasing doses of freeze-dried birch-pollen antigens for 16 months, reaching a cumulative dose of 280 x 10(6) biologic units. This is about 200 times more than the dose used in conventional subcutaneous immunotherapy (IT). In the placebo-treated group, but not in the actively treated group, there was a rise in postseasonal birch-specific IgE antibody levels. A significant decline in postseasonal values after 1 year of treatment was recorded in the actively treated, but not in the placebo-treated, group. Compared to the placebo treatment, there was a significant rise in birch-specific IgG antibodies in patients administered active treatment; however, the rise was less than that usually observed during subcutaneous IT. No significant change in birch-specific serum IgA was found in either group. The changes in IgE and IgG antibody levels demonstrate that OIT affects the immune system. This supports our recent findings that OIT demonstrates a beneficial effect in the treatment of birch pollinosis in adults. But, as with subcutaneous IT, there was no clear relationship between antibody response and clinical findings in the patients. The underlying mechanisms responsible for the relief of symptoms thus remain unknown.     

Drug development strategies for asthma: in search of a new paradigm

The spiraling costs of asthma treatment seem set to continue rising, given the equivocal performance of the latest generation of specific anti-inflammatory drugs in trials in adult asthmatics. We argue that the continuation of this trend is inevitable unless there is a substantial realignment of entrenched drug development policy in the pharmaceutical industry and a parallel shift in licensing policy by regulatory authorities to encourage the development of drugs capable of halting the progression from acute to chronic asthma when the disease first manifests in childhood. The theoretical framework for such an approach, including proof-of-principle data from studies in children with early-stage disease and a range of candidate drugs, already exists. What is needed is informed debate on the risks versus potential benefits of this approach.     

Use of protein G for preparation and characterization of rabbit antibodies against rat adipose tissue hormone-sensitive lipase

The newly described immunoglobulin G-binding streptococcal surface protein, protein G, was used to prepare and characterize rabbit antibodies. The antibodies were directed against rat hormone-sensitive lipase, the rate-limiting enzyme in the hydrolysis of the triacylglycerols stored in adipose tissue. Antiserum was obtained after two injections with 20 micrograms enzyme protein, and the immunoglobulin fraction was obtained using a protein G-based solid-phase radioimmunoassay. The hydrolysis of acylglycerols by the enzyme was inhibited by the antibodies, and the enzyme could be efficiently removed from a solution using the antibodies and heat-killed streptococci expressing surface protein G. By Western blot and detection with 125I-protein G, the antibodies were found to selectively bind to hormone-sensitive lipase and to a smaller extent to two minor contaminants, possibly proteolytic fragments of the lipase. The amount of 125I-labelled protein G bound to the lipase on the blot was quantitatively related to the amount of enzyme protein down to the detection limit 10 ng.