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.     

The developmentally regulated neural crest-associated glycotope HNK-1 predicts metastasis in cutaneous malignant melanoma

Aberrant glycosylation is a common feature of metastatic sub-clones of malignant tumours and in uveal melanoma in particular, the HNK-1 glycotope has been positively correlated with poor prognosis. So far, no such correlation has been investigated in cutaneous melanoma. In order to do so, HNK-1 expression was evaluated immunohistochemically in 100 primary cutaneous melanomas and correlated with metastasis after up to 10-years' follow-up. Furthermore, HNK-1 expression was analysed in metastatic deposits (19 distant cutaneous metastases and six sentinel lymph node metastases), as well as in benign nevi. Kaplan-Meier analysis revealed a positive association between HNK-1 expression and metastasis (p < 0.005) and multivariate Cox regression analysis adjusted for the standard prognostic markers ulceration and vertical tumour thickness confirmed HNK-1 expression as an independent prognostic marker. HNK-1 expression was preserved in 42% of the distant cutaneous metastases, but metastatic cells in lymph nodes were devoid of HNK-1 immunoreactivity. None of the benign pigmented lesions exhibited HNK-1 immunoreactivity. Expression of the HNK-1 glycotope in cutaneous malignant melanoma is an independent prognostic marker of metastasis. Differential HNK-1 expression at the metastatic sites implies that its expression is modulated by the surrounding environment. As HNK-1 is also transiently expressed during migration of melanocyte precursor cells derived from the neural crest, recapitulation of this transient expression might occur during metastatic spread of cutaneous malignant melanoma.     

Spectral signature and heterodyne efficiency for different wavelengths in laser Doppler flowmetry

Laser Doppler perfusion monitoring and imaging technologies generate time traces and two-dimensional flow maps of the microcirculation. With the goal of reaching different tissue depths, these technologies are equipped with lassers operating at different wavelengths λ. The fact that the average scattering angle, at a single scattering event, between a photon and a red blood cell increases with λ is compensated for by a 1/λ effect in the scattering vector, rendering the average frequency shift virtually independent of the choice of wavelength. Monte Carlo simulations showed that the corresponding spectral signature of the Doppler signals for λ=632.8nm and 780nm were close to identical. The theoretical predictions were verified by calculating the centre-of-gravity (COG) frequency of the laser Doppler power spectral density for the two wavelengths from forearm and finger skin, representing a low and high perfusion area, respectively (forearm COG=123 against 121Hz, finger COG=220 against 212 Hz). When the wavelength changes from 632.8nm to 780nm, the heterodyne efficiency of the detector and, thereby, the inherent system amplifcation increase. For tissues with identical microvascular flow conditions, the output signal therfore tends to increase in magnitude when shifting to longer wavelengths.     

Neutrophilic granulocytes are the predominant cell type infiltrating pancreatic islets in contact with ABO-compatible blood

The poor outcome of intraportal islet transplantation may be explained by the instant blood-mediated inflammatory reaction (IBMIR), characterized by islet entrapment in blood clots, leucocyte infiltration and disruption of islet morphology. Here we employ a newly developed in vitro system to identify the blood cells involved in this process. Islets were mixed with ABO-compatible blood in heparinized tubes and incubated for various times up to 6 h. Clots were analysed immunohistochemically for detection of platelets (CD41a), leucocytes/lymphocytes (CD11b), granulocytes (CD16, lysozyme), neutrophilic granulocytes (neutrophil elastase), eosinophilic granulocytes (NaCN + H(2)O(2)), macrophages (CD68), dendritic cells (CD209/DC-SIGN), B cells (CD20) and T cells (CD4, CD8). Platelets were rapidly deposited around the islets in contact with the blood, reaching a maximum by 30 min. The first neutrophilic granulocytes appeared in the islets after 15 min, increased at 1 h and peaked at 2 h. Small numbers of macrophages were found infiltrating the islets already after 5 min, with a slight increase over time. However, control stainings of cultured islets and pancreas biopsies identified these cells as being largely of donor origin. No T cells, B cells, dendritic cells or eosinophilic granulocytes were detected during the 6 h observation time. Neutrophilic granulocytes were identified as the main infiltrating blood cell in islets exposed to blood, implying that these cells play a key role in clinical islet transplantation. Because islets are known to be exquisitely susceptible to oxidative stress, development of drugs targeting neutrophilic cytotoxicity could markedly improve the outcome of islet transplantation.     

Confounding factors in HLA-DW 2 typing of human leucocytes.

Three healthy HLA-B7 homozygous subjects were found with similar but not identical HLA-D antigens; one was DW 2 homozygous according to independent typing results. This could be an expression of "long" and "short" HLA-D antigens or be due to differences in weak antigens outside the HLA-D region. Two further healthy HLA-B7 homozygous subjects were studied; one was apparently heterozygous for DW 2, the other apparently carried no DW 2 antigen. Both could discriminate between different DW 2 homozygous test cells. Two such test cells--one from a patient with multiple sclerosis (MS) and the other from a man with two children with MS--gave variable and absurb reactions with cells from the two subjects in question. It is tentatively suggested that genes exist which, when present in both moities in a mixed leucocyte reaction (MLR), can impair the MLR and give false "typing" reactions. This might be more common among patients with MS and perhaps also some other diseases (certain arthritides, e.g. rheumatoid arthritis) than among healthy subjects and can complicate or make impossible the interpretation of HLA-D typing data. It could also explain the previously-described impaired MLR between cells from patients with these diseases.     

Synthesis and secretion of IgE by an established human myeloma cell line

The rate of IgE production in vitro and the cell population proliferation rate by the established human myeloma line 266 Bl have been studied quantitatively under various tissue culture conditions. The rate of extracellular IgE accumulation depended on the type of medium used, the cell density and the period of time elapsed after explantation. The maximum production rate of 8·1 × 10⁻¹² g IgE/cell/48 hr was noticed at cell densities <10⁶/30 ml and with the presence of feeder human skin fibroblasts or glia-like cells or with the use of conditioned media harvested from such cells. The rate of cell proliferation and secretion of IgE to the medium ran parallel suggesting that the IgE production is highest when cells are in the best physiological condition. During more than one year the rate of synthesis of IgE remained stable. This functionally stable human myeloma line is suitable for further studies on immunoglobulin biosynthesis at the cellular and subcellular level under the tissue culture conditions found optimal in this study.     

Die Bedeutung des Natriums für den renal-tubulären Calcium-Transport bei Rana ridibunda

Zusammenfassung In Anlehnung an frühere Experimente zur Na⁺-Abhängigkeit der renal-tubulären Transporte von Glucose, PAH und Urea wurden Versuche über die Bedeutung des Na⁺ für die Ca⁺⁺-Reabsorption an der isolierten künstlich perfundierten Niere von Rana ridibunda durchgeführt.Erniedrigung des Na⁺-Angebotes von 76,5 auf 20,0 mMol/l vermindert sowohl die Menge reabsorbierten Natriums als auch die des reabsorbierten Calciums, letztere allerdings weniger als erstere. Der Quotient reabsorbiertes Na⁺/reabsorbiertes Ca⁺⁺ verschiebt sich zugunsten des Ca⁺⁺.Fehlendes Na⁺-Angebot auf der Lumen- oder der Blutseite der Tubulusepithelien hat keinen sicheren Einfluß auf die Menge reabsorbierten Calciums, dagegen sinkt die Reabsorptionsrate von Ca⁺⁺ auf weniger als 10% des filtrierten, wenn Na⁺ weder auf der Lumen- noch auf der Blutseite angeboten wird.Die Ca⁺⁺-Reabsorptionsrate ist gesteigert bei Angebot von Ca⁺⁺ ausschließlich auf der Lumenseite, vermutlich wegen des unter physiologischen Bedingungen nicht existenten Konzentrationsgradienten, der blutwärts gerichtet ist. Bei Angebot von Ca⁺⁺ nur von der Blutseite erscheint kein Ca⁺⁺ im Harn: die Tubulusepithelien sind für Ca⁺⁺ im Richtungssinne der Sekretion undurchlässig.Durch die Hemmstoffe der Na⁺-Reabsorption Convallatoxin und Furosemid wird auch die Reabsorption von Ca⁺⁺ — sogar relativ stärker als die von Na⁺ — gehemmt. Da in den Versuchen mit Hemmstoffen das Na⁺-Angebot (extracelluläre Konzentration) konstant bleibt, wird geschlossen, daß die Ca⁺⁺-Reabsorption mit dem Na⁺ über dessen Transport gekoppelt ist.Im Prinzip folgt die Verknüpfung der Ca⁺⁺-Reabsorption mit dem Na⁺ den gleichen Gesetzmäßigkeiten wie die Verknüpfung des Na⁺ mit den Transporten von Glucose, PAH und Harnstoff. Damit ist ein weiteres Indiz für die Vermutung gewonnen, daß die Na⁺-Abhängigkeit ein zellphysiologisches Prinzip von weiter Gültigkeit ist.

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Summary With regard to earlier experiments about the influence of Na⁺ on the tubular transport of glucose, PAH and urea, the effect of Na⁺ on the reabsorption of Ca⁺⁺ in the isolated artificially perfused kidney of Rana ridibunda was studied.Lowering of the Na⁺-concentration from 76.5–20.0 mMol/l results in a decrease of the reabsorbed amounts of both Na⁺ and Ca⁺⁺, the decrease being less marked in the latter. The quotient Na⁺ reabsorbed/Ca⁺⁺ reabsorbed changes in favour of the calcium.Lack of Na⁺ on the luminal or the contraluminal side of the tubular wall does not significantly influence the amount of Ca⁺⁺ being reabsorbed. If, however, Na⁺ is missing on both sides the rate of reabsorption of Ca⁺⁺ falls below 10% of the filtered amount.The rate of Ca⁺⁺-reabsorption increases if Ca⁺⁺ is offered on the luminal side only. Presumably this increase is due to a concentration gradient for Ca⁺⁺ which does not exist under physiological conditions. If Ca⁺⁺ is offered on the contraluminal side only, no Ca⁺⁺ appears in the urine. Apparently the tubular epithelium is impermeable for Ca⁺⁺ from the blood to the tubular lumen.The decrease of the reabsorption of Ca⁺⁺ brought about by substances inhibiting the reabsorption of Na⁺ such as Convallatoxin and Furosemid is relatively more marked than the concomitant inhibition of the reabsorption of Na⁺.Since in the experiments with the above mentioned inhibitors the extracellular Na⁺-concentration remains constant, it is concluded that the Ca⁺⁺-reabsorption is coupled with the transport of Na⁺.In principle the Na⁺-dependence of the Ca⁺⁺-reabsorption follows the same pattern as the Na⁺-dependence of the transports of glucose, PAH and urea, thus supporting our contention that the Na⁺-dependence of active transports is a physiological mechanism of general importance.

     

Epidermal growth factor receptor expression correlates with poor survival in gastric adenocarcinoma from Mexican patients: a multivariate analysis using a standardized immunohistochemical detection system.

The aim of the study was to determine epidermal growth factor receptor (EGFR) expression in gastric adenocarcinoma by standardized immunohistochemistry and to correlate EGFR expression with clinical features and patient survival. EGFR expression was investigated in paraffin sections of resection specimens of 89 gastric carcinomas from Mexican Mestizo patients using standardized immunohistochemistry with antigen retrieval (Dako EGFRpharmDx assay detection system). Membrane staining of EGFR was evaluated in the neoplastic cells and graded using a semiquantitative score (0-3+). Of the 89 carcinomas examined, staining of neoplastic cells was weak in 17 (19.1%, score 1+), moderate in 16 (18.0%, score 2+), and strong in nine cases (10.1%, score 3+). EGFR reactivity was heterogeneous, frequently showing completely negative up to 3+ positive areas within an individual tumor. EGFR reactivity score correlated with distant metastases (P=0.002) and clinical stage (P=0.033). EGFR score 0/1+ was significantly associated with an increase in patient survival when compared to score 2+/3+ (P=0.0006). In a multivariate analysis, EGFR positive cells in muscularis or subserosa (P=0.004), distant metastases (P=0.016) and residual disease (P=0.039) were significantly correlated with decreased survival. The prognosis was associated with the EGFR reactivity score (P=0.003), distant metastases (P=0.0001) and residual disease (P=0.012) in a univariate analysis. EGFR reactivity in neoplastic cells is an independent prognostic factor in gastric adenocarcinoma. The relevance of the heterogeneity in EGFR expression with regard to tumor progression, metastasis and anti-EGFR therapy needs to be studied.     

Monitoring of basophil activation using CD63 and CCR3 in allergy to muscle relaxant drugs

Allergic or pseudoallergic reactions that occur during anesthesia have been increasing for the last few years. To date, the diagnosis of allergy to muscle relaxants remains difficult. In this respect, we developed a flow cytometric method for the study of drug-induced basophil degranulation using CD63 and CCR3. Fifty patients who developed clinical features evocative of allergic reactions immediately after induction of anesthesia were included and classified into two groups. Group 1 (n = 39) comprised true allergic patients, who developed typical signs of shock associated to positive skin testing. Group 2 (n = 11) consisted of patients whose clinical history was not typical and skin testing was negative or nonconclusive. Seventeen control subjects were also studied in this report. We compared data from flow cytometry to skin tests, specific IgE, and histamine release results. Flow cytometry showed a sensitivity of 54%, while that of specific IgE was similar, at 62%. Interestingly, when considering the sensitivity of IgE + CD63 for diagnosis, we reached a sensitivity value of 80%. Of 15 negative results for specific IgE, we found 7 positive CD63 tests, while histamine release gave positive results in only 2 cases. Furthermore, the CD63 protocol showed good specificity (100%). We conclude that our flow cytometry protocol is a promising tool in allergy diagnosis since it is specific and complementary to specific IgE detection.