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Purpose: To evaluate the precision of image-guided radiotherapy (IGRT) using cone-beam computed tomography (CB-CT) for volume imaging and a robotic couch for correcting setup errors in six degrees of freedom., Patients and Methods: 47 consecutive patients with 372 fractions were classified according to whether a patient fixation device was used (pat(fix): n = 28) or not (pat(non-fix): n = 19). Prior to treatment a CB-CT was acquired and translational and rotational setup errors were corrected online without an action level using a robotic couch (HexaPOD). A second CB-CT was acquired after the correction process and after treatment in 134 and 238 fractions, respectively., Results: In 17 fractions (4.6%) rotational errors > 3 degrees exceeded the motion range of the HexaPOD. Errors (3D vector) after the correction process were significantly smaller for pat(fix) compared to pat(non-fix) (p < 0.001): 0.9 mm +/- 0.5 mm and 1.6 mm +/- 0.8 mm, respectively. For pat(non-fix) the correction of rotational errors resulted in displacements of the patients on the angled couch of 0.6 mm/1 degree. Intrafractional motion further decreased precision in pat(non-fix) but not in pat(fix)., Conclusion: Very high precision in cranial and extracranial treatment of immobilized patients was demonstrated. Without application of adequate immobilization the correction of rotational errors and intrafractional patient motion significantly decreased the accuracy of the online correction protocol.

Purpose: Intra-fractional variability of tumor position and breathing motion was evaluated in cone-beam CT (CB-CT) based image-guided radiotherapy (IGRT) of pulmonary tumors., Materials and Methods: Twenty-four patients (27 lesions: prim. NSCLC n=6; metastases n=21) were treated with stereotactic body radiotherapy (SBRT) (one to eight fractions). Prior to every treatment fraction (n=66) and immediately after treatment a CB-CT was acquired. Patient motion, absolute drift and drift of the tumor relative to the bony anatomy were measured. Tumor motion was investigated based on the density distribution in the CB-CT., Results: Absolute intra-fractional drift (3D vector) of the tumor position was 2.8 mm+/-1.6 mm (mean +/- SD), maximum 7.2 mm. Poor correlation between patient motion and absolute tumor drift was observed. Changes of the tumor position due to patient motion and due to drifts independently from the bony anatomy were of similar magnitude with 2.1 mm +/- 1.4 mm and 2.3 mm +/- 1.6 mm, respectively. No systematic increase or decrease of breathing motion was seen. The intra-fractional change of breathing motion was more than 2 mm and 3 mm in 39% and 16%, respectively., Conclusion: Intra-fractional tumor position and breathing motion were stable. In IGRT of pulmonary tumors we suggest an ITV-to-PTV margin of 5 mm to compensate intra-fractional changes.

Purpose: Respiratory correlated computed tomography has been shown to be effective for evaluation of breathing-induced motion of pulmonary tumors. This study investigated whether a single four-dimensional CT study (4D-CT) is representative and sufficient for treatment planning in stereotactic body radiotherapy (SBRT)., Methods and Materials: Four repeated helical 4D-CT studies were acquired every 10 min for 10 patients with 14 pulmonary metastases. Patients remained immobilized in a stereotactic body frame (SBF) for 30 min; abdominal compression was applied to seven patients. Using amplitude based sorting, eight phases equally distributed over the breathing cycle were reconstructed for each 4D-CT study. Tumor position was defined in a total of 406 CT series and variability of breathing motion and mean tumor position were evaluated., Results: Peak-to-peak tumor motion was 9.9 mm +/- 6.8 mm (mean +/- standard deviation) and 9.0 mm +/- 7.4 mm at time point 0 min (t(0)) and t(30), respectively. In one patient with poor pulmonary function, continuous increase of breathing motion from 17.4 mm at t(0) to 28.3 mm at t(30) was seen. In five and two lesions, respectively, a drift of the mean tumor position greater than 3 mm and 5 mm was observed. A borderline significance was calculated for larger tumor position variability in midventilation phases compared with peak-ventilation phases of the breathing cycle (p = 0.08)., Conclusion: Treatment planning based on a single 4D-CT study is reliable for the majority of patients. Increased intrafractional uncertainties were seen for patients with poor pulmonary function and with tumors located in the lower lobe.

Purpose: To scrutinize the positioning accuracy and reproducibility of a commercial hexapod robot treatment table (HRTT) in combination with a commercial cone-beam computed tomography system for image-guided radiotherapy (IGRT)., Methods and Materials: The mechanical stability of the X-ray volume imaging (XVI) system was tested in terms of reproducibility and with a focus on the moveable parts, i.e., the influence of kV panel and the source arm on the reproducibility and accuracy of both bone and gray value registration using a head-and-neck phantom. In consecutive measurements the accuracy of the HRTT for translational, rotational, and a combination of translational and rotational corrections was investigated. The operational range of the HRTT was also determined and analyzed., Results: The system performance of the XVI system alone was very stable with mean translational and rotational errors of below 0.2 mm and below 0.2 degrees , respectively. The mean positioning accuracy of the HRTT in combination with the XVI system summarized over all measurements was below 0.3 mm and below 0.3 degrees for translational and rotational corrections, respectively. The gray value match was more accurate than the bone match., Conclusion: The XVI image acquisition and registration procedure were highly reproducible. Both translational and rotational positioning errors can be corrected very precisely with the HRTT. The HRTT is therefore well suited to complement cone-beam computed tomography to take full advantage of position correction in six degrees of freedom for IGRT. The combination of XVI and the HRTT has the potential to improve the accuracy of high-precision treatments.

Purpose: To evaluate the delineation of either the rectal volume (RV) or the rectal wall (RW) in intensity-modulated radiotherapy (IMRT) for prostate cancer: influence on dose distribution to the targets and organs at risk (OARs) was investigated., Material and Methods: For ten patients with localized prostate cancer IMRT treatment plans were generated with the RV, wall including the filling, and the RW without the lumen as OAR (plan-RV and plan-RW), respectively. Two different IMRT treatment- planning systems (TPS) were utilized. The influence on target coverage and sparing of OARs was investigated., Results: No influence was seen on target coverage and sparing of the bladder and femoral heads. Doses to the RV were significantly reduced in plan-RV for all evaluated dose levels: maximum 26% and 17%, respectively, in both TPS. The dose distribution to the RW was not significantly different between plan-RV and plan-RW., Conclusion: The different delineation of the OAR rectum significantly affected the inverse IMRT treatment-planning process. The use of the RV as OAR resulted in improved dose distributions to the RV. Therefore, it is suggested using the RV as OAR in IMRT treatment planning of the prostate.

Background: The dose distribution to the rectum, delineated as solid organ, rectal wall and rectal surface, in 3D conformal (3D-CRT) and intensity-modulated radiotherapy treatment (IMRT) planning for localized prostate cancer was evaluated., Materials and Methods: In a retrospective planning study 3-field, 4-field and IMRT treatment plans were analyzed for ten patients with localized prostate cancer. The dose to the rectum was evaluated based on dose-volume histograms of 1) the entire rectal volume (DVH) 2) manually delineated rectal wall (DWH) 3) rectal wall with 3 mm wall thickness (DWH(3)) 4) and the rectal surface (DSH). The influence of the rectal filling and of the seminal vesicles' anatomy on these dose parameters was investigated. A literature review of the dose-volume relationship for late rectal toxicity was conducted., Results: In 3D-CRT (3-field and 4-field) the dose parameters differed most in the mid-dose region: the DWH showed significantly lower doses to the rectum (8.7% +/- 4.2%) compared to the DWH(3) and the DSH. In IMRT the differences between dose parameters were larger in comparison with 3D-CRT. Differences were statistically significant between DVH and all other dose parameters and between DWH and DSH. Mean doses were increased by 23.6% +/- 8.7% in the DSH compared to the DVH in the mid-dose region. Furthermore, both the rectal filling and the anatomy of the seminal vesicles influenced the relationship between the dose parameters: a significant correlation of the difference between DVH and DWH and the rectal volume was seen in IMRT treatment., Discussion: The method of delineating the rectum significantly influenced the dose representation in the dose-volume histogram. This effect was pronounced in IMRT treatment planning compared to 3D-CRT. For integration of dose-volume parameters from the literature into clinical practice these results have to be considered.

Purpose: To establish volume imaging using an on-board cone-beam CT (CB-CT) scanner for evaluation of three-dimensional patient setup errors., Methods and Materials: The data from 24 patients were included in this study, and the setup errors using 209 CB-CT studies and 148 electronic portal images were analyzed and compared. The effect of rotational errors alone, translational errors alone, and combined rotational and translational errors on target coverage and sparing of organs at risk was investigated., Results: Translational setup errors using the CB-CT scanner and an electronic portal imaging device differed <1 mm in 70.7% and <2 mm in 93.2% of the measurements. Rotational errors >2 degrees were recorded in 3.7% of pelvic tumors, 26.4% of thoracic tumors, and 12.4% of head-and-neck tumors; the corresponding maximal rotational errors were 5 degrees , 8 degrees , and 6 degrees . No correlation between the magnitude of translational and rotational setup errors was observed. For patients with elongated target volumes and sharp dose gradients to adjacent organs at risk, both translational and rotational errors resulted in considerably decreased target coverage and highly increased doses to the organs at risk compared with the initial treatment plan., Conclusions: The CB-CT scanner has been successfully established for the evaluation of patient setup errors, and its feasibility in day-to-day clinical practice has been demonstrated. Our results have indicated that rotational errors are of clinical significance for selected patients receiving high-precision radiotherapy.

Four different algorithms were investigated with the aim to determine their suitability to track an object in conventional megavoltage portal images. The algorithms considered were the mean of the sum of squared differences (MSSD), mutual information (MI), the correlation ratio (CR), and the correlation coefficient (CC). Simulation studies were carried out with various image series containing a rigid object of interest that was moved along a predefined trajectory. For each of the series the signal-to-noise ratio (SNR) was varied to compare the performance of the algorithms under noisy conditions. For a poor SNR of -6 dB the mean tracking error was 2.4, 6.5, 39.0, and 17.2 pixels for MSSD, CC, CR and MI, respectively, with a standard deviation of 1.9, 12.9, 19.5, and 7.5 pixels, respectively. The size of a pixel was 0.5 mm. These results improved to 1.1, 1.3, 1.3, and 2.0 pixels, respectively, with a standard deviation of 0.6, 0.8, 0.8, and 2.1 pixels, respectively, when a mean filter was applied to the images prior to tracking. The implementation of MSSD into existing in-house software demonstrated that, depending on the search range, it was possible to process between 2 and 15 images/s, making this approach capable of real-time applications. In conclusion, the best geometric tracking accuracy overall was obtained with MSSD, followed by CC, CR, and MI. The simplest and best algorithm, both in terms of geometric accuracy as well as computational cost, was the MSSD algorithm and was therefore the method of choice.

Background: Bleeding in hemophilic neonates has a low incidence. A possible explanation for this could be the peculiarities of the neonatal hemostatic system, especially low levels of the inhibitors tissue factor pathway inhibitor (TFPI) and antithrombin (AT)., Objective: We investigated the influence of an elevation of these inhibitors to adult levels on the thrombin generation (TG) in normal neonatal plasma and factor (F) VIII-depleted neonatal plasma by means of incubation with anti-FVIII-antibodies., Patients/methods: TG was measured after activation with low amounts of tissue factor (TF) by using Calibrated Automated Thrombography., Results: TG in FVIII-depleted neonatal plasma was nearly as high as in normal neonatal plasma. TG decreased after elevation of AT in both neonatal plasmas. After elevation of TFPI TG decreased much more in FVIII-depleted neonatal plasma than in normal neonatal plasma. After elevation of both inhibitors their synergistic effect led to a stronger decrease of TG in FVIII-depleted neonatal plasma. TG measured in plasma of one hemophilic newborn showed the same pattern as in FVIII-depleted neonatal plasma., Conclusion: Our observation provides a biochemical basis for the rare bleeding in hemophilic neonates and shows the important role of the natural inhibitors in the hemostatic system of hemophilic patients.

Background and Purpose: The retrospective planning study for intensity-modulated radiotherapy (IMRT) of prostate cancer evaluated whether proximal rectum and supra-anal rectum/anal canal should be delineated as separated organs-at-risk (OARs) to achieve optimal dose distributions to the anorectal region., Patients and Methods: For 10 patients with localized prostate cancer IMRT plans were generated with the rectum and anal canal as separated OARs (Rec-sep) and as one single OAR (Rec-tot). Two different treatment planning systems (TPS) were utilized. Influence on dose distributions to target and OARs was analyzed., Results: Results from both TPS showed significantly increased doses to the distal rectum/anal canal for plans Rec-tot compared with Rec-sep in case of a distended rectum in the planning CT study: doses were increased by up to mean 31% (P = 0.02) and 18% (P = 0.03), respectively, in both TPS. For the patient with the largest rectum, the maximum dose increase was 61%. No significant differences in doses to target, bladder, femoral head and proximal rectum were seen., Conclusions: For patients with a distended rectum in the planning CT, delineation of separated OARs for proximal rectum and distal rectum/anal canal resulted in superior dose distributions to the anorectal region and therefore, we recommend this as standard procedure for IMRT planning of prostate cancer.

Cyanobacterial light-harvesting complexes, the phycobilisomes, are proteolytically degraded when the organisms are starved for combined nitrogen, a process referred to as chlorosis or bleaching. Gene nblA, present in all phycobilisome-containing organisms, encodes a protein of about 7 kDa that plays a key role in phycobilisome degradation. The mode of action of NblA in this degradation process is poorly understood. Here we presented the 1.8-A crystal structure of NblA from Anabaena sp. PCC 7120. In the crystal, NblA is present as a four-helix bundle formed by dimers, the basic structural units. By using pull-down assays with immobilized NblA and peptide scanning, we showed that NblA specifically binds to the alpha-subunits of phycocyanin and phycoerythrocyanin, the main building blocks of the phycobilisome rod structure. By site-directed mutagenesis, we identified amino acid residues in NblA that are involved in phycobilisome binding. The results provided evidence that NblA is directly involved in phycobilisome degradation, and the results allowed us to present a model that gives insight into the interaction of this small protein with the phycobilisomes.

The purpose was to evaluate the clinical results of stereotactic radiotherapy in primary liver tumors and hepatic metastases. Five patients with primary liver cancer and 39 patients with 51 hepatic metastases were treated by stereotactic radiotherapy since 1997. Twenty-eight targets were treated in a "low-dose"-group with 3 x 10 Gy (n = 27) or 4 x 7 Gy (n = 1) prescribed to the PTV-encl. 65%-isodose. In a "high-dose"-group patients were treated with 3 x 12 - 12.5 Gy (n = 19; same dose prescription) or 1 x 26 Gy/PTV-enclosing 80%-isodose (n = 9). Median follow-up was 15 months (2-48 months) for primary liver cancer and 15 months (2-85 months) for hepatic metastases. While all primary liver cancers were controlled, nine local failures (3-19 months) of 51 metastases were observed resulting in an actuarial local control rate of 92% after 12 months and 66% after 24 months and later. A borderline significant correlation between dose and local control was observed (p = 0.077): the actuarial local control rate after 12 and 24 months was 86% and 58% in the low-dose-group versus 100% and 82% in the high-dose-group. In multivariate analysis high versus low-dose was the only significant factor predicting local control (p = 0.0089). Overall survival after 1 and 2 years was 72% and 32% for all patients and was impaired due to systemic progression of disease. No severe acute or late toxicity exceeding RTOG/EORTC-score 2 were observed. Stereotactic irradiation of primary liver cancer and hepatic metastases offers a locally effective treatment without significant complications in patients, who are not amenable for surgery. Patient selection is important, because those with low risk for systemic progression are more likely to benefit from this approach.

The effects of two modalities of dose-escalated radiotherapy on health-related quality of life (HRQOL) were compared. Forty-one consecutive patients were treated with a 3-D conformal (3-DC) boost to 74 Gy, and 43 with high-dose rate (HDR) brachytherapy boost (2x9 Gy), following 3-D conformal treatment to 46 Gy. Median age was 70 years in both groups, median initial PSA was 7.9 microg/l in 3-DC boost patients and 8.1 microg/l in HDR boost patients. Stage was or=7 in 52% and 47%, respectively. HRQOL was assessed cross-sectionally using EORTC QLQ-C30 and organ-specific PR25 modules 3--32 (median 19) and 4--25 (median 14) months after treatment, respectively. Questionnaires were completed by 93% and 97% of patients, respectively. Diarrhea and insomnia scores were significantly increased in both groups. In the PR25 module, scores of 3-DC boost and HDR boost patients for urinary, bowel and treatment-related symptoms were similar. Among responders, 34% of 3-DC boost patients and 86% of HDR boost patients had severe erectile problems. Dose escalation in prostate cancer by either 3-DC boost to 74 Gy or HDR brachytherapy boost appears to result in similar HRQOL profiles.

Cone-beam CT (CB-CT) based image-guidance was evaluated for extracranial stereotactic radiotherapy of intrapulmonary tumors. A total of 21 patients (25 lesions: prim. NSCLC n = 6; pulmonary metastases n = 19) were treated with stereotactic radiotherapy (1 to 8 fractions). Prior to every fraction a CB-CT was acquired in treatment position, errors between planned and actual tumor position were measured and corrected. Intra- and inter-observer variability of manual evaluation of tumor position error was investigated and this manual method was compared with automatic image registration. Based on CB-CTs from 66 fractions the discrepancy (3-D vector) between planned and actual tumor position was 7.7 mm +/-1.3 mm. Tumor position error relative to the bony anatomy was 5.3 mm +/-1.2 mm, the correlation between bony anatomy and tumor position was poor. Intra-observer and inter-observer variability of manual evaluation of tumor position error was 0.9 mm +/-0.8 mm and 2.3 mm +/-1.1 mm, respectively. Automatic image registration showed highly reproducible results (<1 mm). However, compared with manual registration a systematic error was found in direction of predominant tumor breathing motion (2.5 mm vs 1.4 mm). Image-guidance using CB-CT was validated for high precision radiotherapy of intrapulmonary tumors. It was shown that both the planning reference and the verification image study have to consider tumor breathing motion.

The aim of this research was to investigate whether a spatial correlation could be found between an external 3-D respiratory signal and the tumour trajectory. The respiratory signal was obtained by tracking the abdominal movement and the tumour trajectory was obtained by automatically determining the tumour position in a series of portal images. Three different models, based on Systems Identification, are presented to model the correlation using a 1-D respiratory signal, a 3-D respiratory signal and a 3-D respiratory signal together with previously determined tumour positions. Adequate correlation was found for all models in the direction of the tumour movement with standard deviations of 0.89 mm, 0.72 mm and 0.75 mm, respectively, and model fit of Rt2 = 0.19, 0.63 and 0.82, respectively. Increasing the frame rate for the acquisition of portal images from 3 to 15 frames per second improved the standard deviation and model fit. In summary, it is possible to spatially correlate a 3-D respiratory signal with the tumour trajectory using this approach. The models presented provide a framework that can be extended to include more information if required. A 3-D respiratory signal is preferable to a 1-D signal in modelling the tumour motion that is not along the main axis of tumour movement.

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

The dose-response for local tumor control after stereotactic radiotherapy of 92 pulmonary tumors (36 NSCLC and 56 metastases) was evaluated. Short course irradiation of 1-8 fractions with different fraction doses was used. After a median follow-up of 14 months (2-85 months) 11 local recurrences were observed with significant advantage for higher doses. When normalization to a biologically effective dose (BED) is used a dose of 94Gy at the isocenter and 50Gy at the PTV-margin are demonstrated to give 50% probability of tumor control (TCD50). Multivariate analysis revealed the dose at the PTV-margin as the only significant factor for local control.

Background: 1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25[OH](2)D(3)) exerts its effects on the immune system, particularly through suppression of T helper/cytotoxic cell 1 (T(H)/T(C)1)-mediated reactions, although direct actions of 1 alpha,25(OH)(2)D(3) on human T lymphocytes have not yet been studied in detail., Objective: We evaluated the effect of 1 alpha,25(OH)(2)D(3) on basal and cytokine-driven T-cell functions at the single-cell level., Methods: We used 4-color flow cytometry for simultaneous detection of intracellular cytokines in CD4(+) and CD8(+) human PBMCs that had been cultured in the presence of 1 alpha,25(OH)(2)D(3) singly or in combination with either IL-12 or IL-4. According to the exploratory nature of these investigations, the Bonferroni correction was not applied for data analysis and presentation., Results: 1 alpha,25(OH)(2)D(3) had little effect on T(H)/T(C)1 cytokines but significantly inhibited IL-12-induced IFN-gamma production. Constitutive synthesis of T(H)/T(C)2-related cytokines was also only modestly affected by 1 alpha,25(OH)(2)D(3) alone. When T(H)/T(C)2 differentiation was induced by IL-4, 1 alpha,25(OH)(2)D(3) significantly expanded the percentages of IL-4(+) and IL-13(+) cells. However, the predominant effect of 1 alpha,25(OH)(2)D(3) on T lymphocytes, particularly in the presence of IL-4, was the induction of separate CD4(+) and CD8(+) subpopulations with almost exclusive expression of IL-6. This might be an important facet of the immunomodulatory action of 1 alpha,25(OH)(2)D(3) because IL-6 might act in parallel with 1 alpha,25(OH)(2)D(3) in modulation of T(H)/T(C) effector cell functions., Conclusions: Our data imply that the specific actions of 1 alpha,25(OH)(2)D(3) on cytokine-stimulated T-cell functions could play a role in the prevention of T(H)/T(C)1-related autoimmune diseases but also predispose toward T(H)/T(C)2-mediated allergic reactions.

Neonatal plasma clots slower than adult plasma, and only 30-50% of peak adult thrombin activity can be produced in neonatal plasma when high amounts of tissue factor (TF) are added to trigger clotting, as used in standard clotting assays. Plasma activation by addition of low amounts of TF probably better reflects conditions in vivo. Under these conditions, cord plasma clots faster than adult plasma. In the present study, we show that after activation with low amounts of TF, higher amounts of the anticoagulants heparin and hirudin are required in cord plasma for effective inhibition of thrombin generation compared with adult plasma. After strong activation with high amounts of TF (30 microM), the thrombin potential was significantly more suppressed in cord plasma compared with adult plasma in the presence of 0.4 IE/mL heparin (-92 versus -75%; p < 0.01) and in the presence of 2 IE/mL hirudin (-18 versus -8%; p < 0.01). In contrast, after weak activation with low amounts of TF (30 pM), the thrombin potential was significantly more suppressed in adult plasma compared with neonatal plasma in the presence of 0.025 IE/mL heparin (-93 versus -8%; p < 0.01) and in the presence of 2 IE/mL hirudin (-89 versus -48%; p < 0.01). Our results show that in neonates, effects of anticoagulants very much depend on the type of activation used to initiate clotting, and doses of anticoagulants should not be derived from studies done in adults, because potentially higher doses of anticoagulants may be required.

Loss of heterozygosity of the small arm of chromosome 3 is one of the most common alterations in human cancer. Most notably, a segment in 3p21.3 is frequently lost in lung cancer and several other carcinomas. We and others have identified a novel Ras effector at this segment, which was termed Ras Association Domain family 1 (RASSF1A) gene. RASSF1 consists of two main variants (RASSF1A and RASSF1C), which are transcribed from distinct CpG island promoters. Aberrant methylation of the RASSF1A promoter region is one of the most frequent epigenetic inactivation events detected in human cancer and leads to silencing of RASSF1A. Hypermethylation of RASSF1A was commonly observed in primary tumors including lung, breast, pancreas, kidney, liver, cervix, nasopharyngeal, prostate, thyroid and other cancers. Moreover, RASSF1A methylation was frequently detected in body fluids including blood, urine, nipple aspirates, sputum and bronchial alveolar lavages. Inactivation of RASSF1A was associated with an advanced tumor stage (e.g. bladder, brain, prostate, gastric tumors) and poor prognosis (e.g. lung, sarcoma and breast cancer). Detection of aberrant RASSF1A methylation may serve as a diagnostic and prognostic marker. The functional analyses of RASSF1A reveal an involvement in apoptotic signaling, microtubule stabilization and mitotic progression. The tumor suppressor RASSF1A may act as a negative Ras effector inhibiting cell growth and inducing cell death. Thus, RASSF1A may represent an epigenetically inactivated bona fide tumor suppressor in human carcinogenesis.

The present paper describes the fast acquisition and processing of portal images directly from a TV camera-based portal imaging device (Siemens Beamview Plus). This approach employs not only hard- and software included in the standard package installed by the manufacturer (in particular the frame grabber card and the Matrox Intellicam interpreter software), but also a software tool developed in-house for further processing and analysis of the images. The technical details are presented, including the source code for the Matrox interpreter script that enables the image capturing process. With this method it is possible to obtain raw images directly from the frame grabber card at an acquisition rate of 15 images per second. The original configuration by the manufacturer allows the acquisition of only a few images over the course of a treatment session. The approach has a wide range of applications, such as quality assurance (QA) of the radiation beam, real-time imaging, real-time verification of intensity-modulated radiation therapy (IMRT) fields, and generation of movies of the radiation field (fluoroscopy mode).

Introduction: This study was performed to compare the anticoagulant activity of melagatran, the active form of the oral direct thrombin inhibitor ximelagatran, in umbilical cord plasma with that in adult plasma. In contrast with the most frequently administered anticoagulants, the heparins, melagatran acts independently of antithrombin (AT). As a consequence, administration of melagatran is of special interest in neonates, who have physiologically low levels of AT., Materials and Methods: Plasma samples were activated under high (as used in standard clotting assays) and low (more comparable with the physiological milieu) coagulant challenge. In the absence of melagatran, adult plasma clotted significantly faster than umbilical cord plasma under high coagulant challenge. Conversely, under low coagulant challenge, clotting of adult plasma was significantly delayed compared with umbilical cord plasma. For both high and low coagulant challenges, clotting times increased and prothrombin fragment 1.2 and thrombin-antithrombin (TAT) formation decreased with melagatran in a concentration-dependent fashion in umbilical cord and adult plasma. With increasing melagatran concentrations, the quotient between prothrombin fragment 1.2 and TAT formation increased in adult and umbilical cord plasma under both high and low coagulant challenges., Results and Conclusions: Our in vitro results cannot be directly extrapolated to clinical efficacy, but assessing the degree of inhibition of thrombin generation may be a useful surrogate for selecting effective doses of ximelagatran for in vivo studies in neonates with thromboembolic complications.

Phycobilisomes (PBS) are the major light-harvesting complexes of cyanobacteria. These usually blue-coloured multiprotein assemblies are rapidly degraded when the organisms are starved for combined nitrogen. This proteolytic process causes a colour change of the cyanobacterial cells from blue-green to yellow-green ('bleaching'). As is well documented for the unicellular, non-diazotrophic cyanobacteria Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803, a gene termed nblA plays a key role in PBS degradation. Filamentous, diazotrophic cyanobacteria like Anabaena adapt to nitrogen deprivation by differentiation of N(2)-fixing heterocysts. However, during the first hours after nitrogen deprivation all cells degrade their PBS. When heterocysts mature and nitrogenase becomes active, vegetative cells resynthesize their light-harvesting complexes while in heterocysts the phycobiliprotein content remains very low. Expression and function of nblA in Anabaena sp. PCC 7120 was investigated. This strain has two nblA homologous genes, one on the chromosome (nblA) and one on plasmid delta (nblA-p). Northern blot analysis indicated that only the chromosomal nblA gene is up-regulated upon nitrogen starvation. Mutants with interrupted nblA and nblA-p genes, respectively, grew on N(2) and developed functional heterocysts. Mutant DeltanblA-p behaved like the wild-type. However, mutant DeltanblA was unable to degrade its PBS, which was most obvious in non-bleaching heterocysts. The results show that NblA, encoded by the chromosomal nblA gene, is required for PBS degradation in Anabaena but is not essential for heterocyst differentiation.

Background: The present in vitro study of human plasma investigated the anticoagulant effects of recombinant human activated protein C (rhAPC; drotrecogin alfa [activated, Xigris]), combined with either unfractionated heparin (UH) or the direct thrombin inhibitor melagatran., Methods: Prolongation of clotting time and generation of prothrombin fragments 1 and 2 (F1 + 2) and of thrombin-antithrombin (TAT) complex were measured in vitro. Clot formation was induced by adding low levels (final concentration, 20 pmol/L) of lipidated tissue factor (TF) to citrated venous plasma samples from healthy human volunteers (n = 16). It has been suggested that experimental activation of plasma with low levels of TF more closely simulates conditions in vivo., Results: rhAPC, melagatran, or UH alone concentration-dependently prolonged the clotting time and suppressed F1 + 2 and TAT generation. rhAPC-mediated prolongation of clotting time concentration-dependently increased with the addition of either UH or melagatran, the effect being more pronounced with the addition of melagatran. Similarly, rhAPC-mediated generation of F1 + 2 and TAT also increased concentration-dependently with the addition of either UH or melagatran; the effect being, in this case, more pronounced with the addition of UH., Conclusions: This study demonstrated the effects of rhAPC, alone and combined with either UH or melagatran, on clotting time and markers of thrombin generation in human plasma. These results may guide facilitate estimation of appropriate doses of rhAPC and melagatran in future clinical trials.

Purpose: This study assessed the proportion of children with dental fear, the proportion of children with negative behavior and the relationship of children's dental fear and children's negative behavior in private pediatric dentistry practices in western Washington state., Methods: A total of 421 children seen in 21 private pediatric dentistry practices in western Washington State participated. The average age of the children was 6.8+/-2.8 years (range=0.8-12.8 years). An average of 21 children were studied per practice (range=7-25 children). Dental fear was measured using the parents' version of the Dental Subscale of the Child Fear Survey Schedule (CFS). Behavior of the child during treatment was rated using the Frankl scale. Additional data were collected regarding the child's previous experiences and parental fear., Results: The children's average item score on the 15-item CFS was 2+/-0.7, corresponding to a total score of 29.6 out of 75, where 75 indicates maximum fear. The proportion of children with dental fear, defined as an average item score of > or = 2.5 (corresponding to a total score of > or = 38), was 20% (85/421, 95% CI=16.3, 24%). The proportion of children who displayed negative behavior during treatment was 21% (95% CI=17.5, 24%). A prevalence ratio of 2.4 was calculated to describe the relationship between children's behavior during treatment and children's dental fear prior to dental treatment. Multivariate logistical regression analysis showed children with dental fear, younger children, and children exposed to treatment involving local anesthesia have higher odds of displaying negative behavior., Conclusions: The proportion of children with dental fear in private pediatric dentistry practices was 20%, and the proportion of children with negative behavior during treatment was 21%. Children with negative behavior had greater odds of having dental fear and children with dental fear had greater odds of having negative behavior. Screening for dental fear may allow pediatric dentists to prepare children more adequately for positive treatment experiences.

The present study was performed to compare the anti-coagulant efficiency of recombinant human activated protein C (rhAPC) in cord with that in adult plasma. RhAPC is a promising candidate to improve the outcome of severe sepsis. However, different anticoagulant efficiency of rhAPC in cord compared with adult plasma has to be expected due to physiological low plasma levels of tissue factor pathway inhibitor (TFPI) and antithrombin (AT) present in neonates, two inhibitors known to markedly influence the anticoagulant action of APC. Clot formation was induced in our experiments by addition of high (30 micro M) or low (20 pM) amounts of lipidated tissue factor (TF). High amounts of TF are conventionally applied in standard clotting assays, whereas plasma activation with low amounts of TF probably better matches the conditions in vivo. We demonstrate that under low coagulant challenge increasing amounts of rhAPC (0.1-0.5 micro g/ml final plasma concentration) dose-dependently prolonged clotting time and suppressed thrombin potential and prothrombin fragment 1+2 generation in both cord and adult plasma. The same was true for experiments performed under high coagulant challenge when 4-16 micro g/ml of rhAPC were added. Whereby, cord plasma was significantly more susceptible to addition of rhAPC in the presence of high amounts of TF and adult plasma was significantly more susceptible to addition of rhAPC in the presence of low amounts of TF. We demonstrate that increased anticoagulant efficiency of rhAPC in adult plasma under low coagulant challenge is attributable to the physiological high levels of TFPI and AT present in adults.

Purpose: Evaluation of the inter- and intraindividual applicator variability of multiple high-dose-rate (HDR) brachytherapy applications for primary treatment of cancer of the uterine cervix., Material and Methods: Retrospective analysis of 460 pairs of orthogonal X-ray films for conventional treatment in 92 patients with five intrauterine applications using an HDR tandem applicator. Measurement of the position of the applicator origin relative to a bony reference system in three dimensions. Evaluation of the differences of the applicator position in all 460 applications (interindividual variability), of the five applications in a single patient (intraindividual variability) and of the intraindividual variability relative to the applicator position at the first application., Results: The position of the applicator origin in the pelvis ranged from 23 mm cranial and 55 mm caudal to the top of femoral heads, 23 mm right and 27 mm left to the pelvic midline, and 6-53 mm dorsal to the mid of the femoral heads. Standard deviation (SD) of interindividual applicator variability was 12.9 mm (minimum/maximum -55/+23 mm, mean -13.6 mm) in longitudinal, 5.1 mm (-27/+23 mm, mean 1.6 mm) in lateral, and 7.6 mm (6/53 mm, mean 26 mm) in anterior-posterior [AP] direction. SD of intraindividual variability was 5.5 mm (-21/+23 mm, mean 0 mm) in longitudinal, 2.5 mm (-17/+19 mm, mean 0 mm) in lateral, and 4.2 mm (-15/+18 mm, mean 0 mm) in AP direction compared to intraindividual variability relative to the first insertion with an SD of 8.9 mm (-23/+36 mm, mean 2.8 mm) in longitudinal, 4.0 mm (-11/+23 mm, mean 0 mm) in lateral, and 6.8 mm (-27/+17 mm, mean -0.8 mm) in AP direction., Conclusion: Intraindividual applicator variability is significantly smaller than interindividual variability. Applicator-related procedures such as midline shielding or dose matching of tele- and brachytherapy should be performed with information on at least one individual applicator position.

We have produced biologically active recombinant (r) LTB, the nontoxic B subunit of heat-labile toxin (LT) of Escherichia coli in tobacco mosaic virus (TMV)-infected Nicotiana benthamiana plants. We amplified the LTB encoding sequence with its leader and introduced a hexahistidyl tag and an endoplasmic reticulum retention signal. The resulting product was ligated into a TMV-based plant viral expression vector that was used for the generation of recombinant viral RNA. Eighty-nine percent of N. benthamiana plants inoculated with the recombinant viral RNA were systemically infected as determined by anti-TMV enzyme-linked immunosorbent assay (ELISA) experiments. The rLTB monomer was identified by LT-specific as well as by histidyl-tag-specific immunoblots. rLTB from plant extracts of TMV-infected N. benthamiana leaves was purified to give 75 microg rLTB pentamers per gram fresh plant material and was capable of binding G(M)1 ganglioside. The immunogenicity of the plant-produced rLTB was tested in mice and showed that intranasal application of rLTB (15 microg per mouse) induced LTB-specific IgG1 antibodies. To prove its adjuvanticity, rLTB was intranasally co-administered with the Hevea latex allergen Hev b 3, leading to allergen-specific IgG1 and IgG2a antibody production. The fact that intranasal application of rLTB and Hev b 3 prior to systemic challenge with the allergen enhanced the Th2 responses at the humoral and cellular level indicated that rLTB promoted immune responses that were naturally induced by the antigen/allergen. In conclusion, these results indicate that the plant viral expression system is suitable for the rapid large-scale production of biologically active LTB with strong mucosal adjuvant capacity.

Purpose: The transcription factor subunit hypoxia-inducible factor-1 alpha (HIF-1 alpha) is currently discussed as a potential endogenous marker of tumor hypoxia to select patients for modified treatment. Despite an association of immunohistochemical HIF-1 alpha overexpression and poor prognosis after radiotherapy in many tumor entities, the reported pattern of HIF-1 alpha staining was often not consistent with tumor hypoxia. To explain this discrepancy, we studied the in vitro conditions under which HIF-1 alpha protein accumulates., Methods and Materials: FaDu human pharyngeal carcinoma and HT 1080 human fibrosarcoma cells were treated with different schedules of in vitro hypoxia at 5%, 1%, and 0.1% O(2) and reoxygenation. HIF-1 alpha protein levels were determined in nuclear extracts. Cellular radiation sensitivity was assessed by clonogenic survival assay after single-dose irradiation at the above oxygen concentrations., Results: In both cell lines, weak HIF-1 alpha expression was observed at 20% O(2) and after 10 min of hypoxia. Increased HIF-1 alpha protein levels were observed at 1 h of hypoxia, remained stable over 24 h, and decreased to baseline within 15 min of reoxygenation. HIF-1 alpha protein at 5% O(2) was half-maximal in FaDu but indistinguishable from 0.1% O(2) in HT 1080. A good correlation of HIF-1 alpha protein level and hypoxic radiation resistance, with equal ranking of data points by both assays, was observed in FaDu cells but not in HT 1080 cells., Conclusions: The ability of HIF-1 alpha to indicate radiobiologically relevant levels of tumor hypoxia seems to be cell type specific. This finding may explain the inconsistent results regarding the pattern of HIF-1 alpha expression in tumor sections.

A radiotherapy table top was to be designed and manufactured, suitable for computer tomography in the same room. The aim was a distortion of the dose distribution by the table top of not more than 4% in small subunits of the target volume (TV) and less than 2% for the mean dose in the TV. Only negligible artifacts in the CT slices should occur in order to assure the exact relocalization of the TV. The table top manufactured of carbon fiber reinforced plastic was constructed by means of finite-element-algorithms. The trapezoidal shaped sandwich plate is supported with tapered beams. The small side of the table can penetrate a 60 cm CT-gantry. When turned, the table top allows large ap-pa opposed fields without irradiating through the beams. Transmission measurements were performed with an ionization chamber under different irradiation angles to determine the influence of the beams and the covering plate. Additionally, a portal imaging device (PID) was used for comparison. It was found that the deflection and torsion of the carbon fiber table top was smaller than for the original. The transmission of the sandwich plate was 97.5% (18 MV) and 96.5% (6 MV photons) respectively. If the irradiation of the beam walls can not be avoided the transmission is not in a critical range. The CT-slices show only minor artifacts along the side walls of the beams. The localization of the tumor or the TV is not restricted.

The prevalence of type I allergy to Hevea brasiliensis latex is particularly high among individuals with frequent exposure to latex products, such as health-care workers (HCW) and patients with spina bifida (SB). Treatment of latex allergy seems problematic as preventive measures, such as allergen avoidance, are not always possible and conventional immunotherapy with standardized latex extracts is not performed routinely. Thus, the aim of the present study was to establish a mouse model of latex allergy using two major latex allergens for HCWs and SB patients, Hev b 1 and Hev b 3, for sensitization. Prophylactic measures on the basis of mucosal tolerance induction with the recombinant allergens were tested in this model. Female BALB/c mice immunized intraperitoneally with recombinant (r)Hev b 1 or rHev b 3 displayed strong immune responses in vivo and in vitro. Intranasal treatment with rHev b 1 and rHev b 3 prior to sensitization led to reduced allergen-specific IgG1/IgE levels and significantly suppressed allergen-induced basophil degranulation. Moreover, lymphocyte proliferation and cytokine production (IL-4, IL-5, IFN-gamma) in vitro were significantly suppressed after pretreatment with both allergens. Suppressive cytokines, such as interleukin (IL)-10 and transforming growth factor (TGF)-beta, remained unchanged after the intranasal pretreatment, indicating mechanism of anergy rather than active immunosuppression. Taken together, these results suggest that mucosal tolerance induction with recombinant allergens could present a promising prevention strategy against latex allergy.

Purpose: To determine whether a little dose to a large normal lung volume or a high dose to a small lung volume is more critical for induction of clinical pneumonitis. The second question is if dose-volume histogram (DVH) parameters are more reliable, if the lungs are analyzed as separate organs or as a whole organ., Patients and Methods: We analyzed the clinical and DVH data from 49 patients treated for a thoracic malignancy using 3-D conformal treatment plans. 18 patients had developed a clinical pneumonitis (CTC II or III). The majority of patients (n = 48) received radiochemotherapy for non-small-cell lung cancer (NSCLC) with a combination of paclitaxel and carboplatin. Patients were generally treated 5 fx/week, single dose 2 Gy, using a two-series approach (shrinking field) up to a total dose of 60-70 Gy. For every individual patient, the overall dose distribution was recalculated in the Helax-TMS by means of adding dose plans according to the total dose applied in each series. The lungs were defined both as separate organs and as a whole organ. Low-dose volume (< or = 10 Gy, Vlow), moderate-dose volume (> 10-40 Gy, Vmod) and high-dose volume (> 40 Gy, Vhigh), as well as V10-V40 and mean lung dose (MLD) were defined from the cumulative DVH. Dose-effect relationships were fitted with a logistic regression model., Results: Manifestation of clinical pneumonitis was within 3 months from termination of irradiation in all cases. For the ipsilateral lung, the incidence of pneumonitis was closely correlated to Vhigh. The pneumonitis rate increased from 13% up to 60%. By contrast, with increasing Vlow the pneumonitis rate dropped to < 10%. A similar but less pronounced effect was seen for the total lung. The lung volumes Vlow, Vmod and Vhigh of the ipsilateral, contralateral and whole lung were significantly correlated to the corresponding MLD. The incidence of pneumonitis increased with increasing MLD for the ipsilateral lung with a D50 of 32 Gy and a gamma 50 of 0.98. For the whole lung, the observed increase was less steep. MLD showed a close correlation to NTCP calculated by the Kutcher model. However, NTCP calculation overestimated the pneumonitis risk for the ipsilateral lung and underestimated the risk for the whole lung due to the steeper gradient. The logistic regression curve for the DVH parameters V10-V40 showed an increase of steepness toward higher doses. From the logistic regression curves, a DVH template indicating critical borders of V10-V40 was generated for the ipsilateral as well as for the total lung., Conclusion: Our data indicate that it is reasonable to disperse the dose outside the target volume over large areas in order to reduce the volumes of lung receiving > 40 Gy. Reducing the high-dose volume reduces the pneumonitis rate more than a corresponding reduction in the low-dose regions of the DVH. Landmarks for DVH optimization as defined in this analysis may serve as a basis for DVH contrains in IMRT planning. Separate organ analysis produced more reliable results and should be preferred to whole-organ analysis, if techniques mainly involving one side of the lung are applied. Further validation of these constraints is necessary prior to general recommendation.

The protozoan parasite Entamoeba histolytica, which is responsible for intestinal amebiasis and amebic liver abscess, is causing significant morbidity and mortality worldwide. Proteophosphoglycans (PPGs, also known as lipophosphoglycans, LPGs, or lipopeptidophosphoglycans, LPPGs) are major surface components of E. histolytica. Passive immunization with a monoclonal antibody (EH5) directed against the PPGs protected severe combined immune-deficient mice from amebic liver abscess. The structure of the PPGs is very complex and only known in part. To find peptide mimics of E. histolytica PPG antigens, we had screened phage-displayed random peptide libraries with the antibody EH5. We identified various peptide mimics of E. histolytica PPGs, all sharing a consensus sequence Gly-Thr-His-Pro-X-Leu. Several of the phage clones induced a significant, specific IgG response against membrane antigens of E. histolytica after immunization of mice with whole phage particles. In the present work, in order to avoid the use of phage particles for immunization, we coupled two selected chemically synthesized peptides to keyhole limpet hemocyanin (KLH). The two KLH-conjugated peptides were immunogenic in mice and induced the production of high titers of anti-peptide antibodies, and one of the two peptides was also able to induce significant titers of antibodies against E. histolytica PPGs. Our results demonstrate that the KLH-conjugated peptides are able to mimic the EH5 epitope without the M13 phage sequences flanking the peptide inserts and independent of the structural framework of the phage.

Purpose: Our organ procurement organization (OPO) evaluated the clinical and financial efficacy of point-of-care testing (POCT) in management of our deceased organ donors., Methods: Before we implemented point-of care testing with the i-STAT into routine clinical donor management, we compared the i-STAT result with the result from the respective donor hospital lab (DHL) for certain analytes on 15 consecutive donors in our OPO from 26 March to 14 May 2001. The financial impact was studied by reviewing 77 donors from July 2001 to March 2002., Results: There was a strong correlation for each analyte between the POC and DHL test results with r-values as follows: pH 0.86; PCO2 = 0.96; PO2 = 0.98; sodium = 0.98; potassium = 0.95; chloride = 0.94; BUN = 0.98; glucose = 0.92; haematocrit = 0.87 and creatinine = 0.95. Since our OPO coordinators began using i-STAT in their routine clinical management of organ donors, they can now more quickly maximize oxygenation and fluid management of the donor and make extra-renal placement calls sooner. Finally, since we are no longer being billed for the testing performed on the i-STAT, average financial savings to our OPO are US dollars 733 per case., Conclusions: Point-of-care testing in management of our OPO donors provides a result that is equivalent to that of the donor hospital lab, has quicker turn-around time than the donor hospital laboratory, allowing more immediate clinical management decisions to be made so that extra-renal offers may begin sooner.

Background and Objective: In previous studies we have generated mimotopes of Bet v 1, the major birch pollen allergen, by biopannings of phage-display random peptide libraries. In the present study, we analysed the humoral and cellular immune response to Bet v 1-mimotopes., Methods: The mimotope CFPYCYPSESA, designated Bet mim 1, was used for intraperitoneal immunizations of BALB/c mice in phage-displayed form. For examination of the humoral immune response, enzyme-linked immunosorbent assay (ELISA) experiments were applied. Stimulation capacities were investigated in cultured mouse splenocytes and in humoral Bet v 1-specific T cell clones., Results: We demonstrated that the Bet mim 1-induced murine antibody response against Bet v 1 was predominated by the IgG1 isotype. In these mice only the phage-displayed mimotopes, but neither the allergen nor the synthetic Bet mim 1-mimotopes were able to stimulate proliferation of cultured splenocytes. Using Bet v 1-specific T cell clones of allergic patients, phage-displayed and synthetic mimotopes were unable to stimulate T cell proliferation. Moreover, tolerance induction to Bet v 1 in mice by intranasal administration of Bet mim 1-phages or Bet mim 1-peptide failed., Conclusion: Taking these results together, our data indicate that Bet mim 1 mimics a Bet v 1-epitope on the B cell but not on the T cell level. We suggest that the phage itself is responsible for the recruitment of T cells providing bystander help in the formation of a mimotope-specific humoral response.

The nblA family of genes encodes for small proteins necessary for the ordered degradation of phycobilisomes under certain stress conditions, a process known as chlorosis. Genes homologous to nblA seem to occur in all phycobilisome-containing organisms. However, to date, no molecular mechanism is known for the action of NblA, nor have the gene products been characterized to understand the physical properties of the molecule and thus help elucidate the mechanism on a structural basis. In this study we report on the first characterization of an NblA-homologous gene product. The chromosomal gene from the cyanobacterium Anabaena sp. PCC 7120 was cloned, heterologously expressed in Escherichia coli and purified to apparent homogeneity. This allowed the protein to be characterized by analytical ultracentrifugation and CD spectroscopy. These experiments show that the NblA protein has a mostly alpha-helical structure, undergoing an association reaction of folded monomers to form trimers in solution. No dimers are detectable.

Background: To evaluate the influence of total dose and tumor volume on local control and survival in primary radiotherapy of non-small-cell lung cancer (NSCLC)., Methods and Materials: We retrospectively analyzed the clinical course and CT-derived pre- and post-therapeutic tumor volume data of 135 patients with NSCLC undergoing primary radiotherapy at our department between 1989 and 1996. Among these, a total of 192 spatially separated tumor volumes (135 primary tumors, 1 additional intrapulmonary tumor, and 56 involved lymph nodes) were available for analysis. In all patients, treatment was planned using CT-based three-dimensional treatment planning. The dose to each tumor volume was derived from the individual dose plans. Mean total dose was 59.9 Gy (range: 30-80 Gy). All but 3 patients were followed until death. For local control analysis, each tumor was analyzed separately, and its remission status was determined in serial follow-up CT scans. A total of 784 CT scans were analyzed. Actuarial local control analysis was performed for the 192 separated tumor volumes, and survival analysis was performed for the 135 patients. Tumor control probability was calculated using a Poisson statistical model., Results: Overall 1- and 2-year local control rate was 50% and 37%, respectively. The 2-year local control rate for tumors <50 ccm, 50-200 ccm, and >200 ccm was 51%, 22%, and 10%, respectively (p = 0.02). The 2-year local control rate for dose levels < or = 60 Gy and >60 Gy was 28% and 43% (p < 0.001). For the subgroup of 147 tumors smaller than 100 ccm, the local control rate increased up to 70% (1 year) and 51% (2 years) with doses of more than 60 Gy. For tumors larger than 100 ccm, no dose effect was seen. Only 2 of 45 tumors >100 ccm were controlled more than 2 years. Multivariate analysis revealed tumor volume, total dose, histopathologic type, and grading as significant and independent prognostic factors for local control. The number of delay days by split course (if used) and application of chemotherapy was not found to influence local control. Overall 1- and 2-year survival rate was 42% and 13%. Total radiation dose, chemotherapy, and T and N stage---but not tumor volume---were found to be independent and significant prognostic factors for survival in multivariate analysis., Conclusion: Tumor volume is an important predictor of local control in NSCLC. We found a clear dose effect for local control and survival in NSCLC. Long-term local control for a significant proportion of patients seems possible for small tumors only (<100 ccm, i.e., maximum diameter 6 cm) with doses of 70 Gy and more. Tumors of > or = 100 ccm are unlikely to be controlled long term by conventional doses up to 70 Gy. These results support dose escalation in patients with NSCLC.

Background: Several studies have demonstrated that mucosal administration of soluble antigens can prevent the onset or reduce the severity of certain autoimmune diseases or allergies. Few studies exist showing the efficacy of mucosal tolerance for therapy of such diseases., Objective: The aim of the present study was to modulate an allergic immune response by intranasal antigen administration in an already sensitized organism., Methods: A murine model of allergic asthma to birch pollen (BP) and its major allergen Bet v 1 was utilized. Sensitized mice were intranasally treated with recombinant (r)Bet v 1 in different concentrations and at different intervals. On the day the mice were killed, blood and bronchoalveolar lavage fluids were taken and immediate type I skin tests were performed. T cell proliferation and cytokine production (interleukin (IL)-5, interferon (IFN)-gamma) were measured in spleen and lung cell cultures., Results: Mucosal treatment with rBet v 1 (3 x 50 microg in 4 day intervals) led to a reduction of type I skin reactions, suppressed immunoglobulin (Ig)G1/IgE antibody levels and markedly decreased IL-5 and IFN-gamma production in vitro in spleen and lung cell cultures. Moreover, lung inflammation (i.e. eosinophilia and IL-5 levels in bronchoalveolar lavage fluids) was significantly suppressed by the treatment., Conclusion: Our results demonstrate that intranasal treatment with rBet v 1 reduced systemic allergic immune responses as well as airway inflammation in BP-sensitized mice. We therefore suggest that mucosal tolerance induction with recombinant allergens could be a promising concept for the therapy of allergic diseases.

We conducted a study in a squatter settlement in Karachi, Pakistan where residents report commonly washing their hands to determine if providing soap, encouraging hand washing, and improving wash-water quality would improve hand cleanliness. We allocated interventions to 75 mothers and collected hand-rinse samples on unannounced visits. In the final model compared with mothers who received no hand-washing intervention, mothers who received soap would be expected to have 65% fewer thermotolerant coliform bacteria on their hands (95% CI 40%, 79%) and mothers who received soap, a safe water storage vessel, hypochlorite for water treatment, and instructions to wash their hands with soap and chlorinated water would be expected to have 74% fewer (95% CI 57%, 84%). The difference between those who received soap alone, and those who received soap plus the safe water vessel was not significant (P = 0.26). Providing soap and promoting hand washing measurably improved mothers' hand cleanliness even when used with contaminated water.

Background: The major birch pollen allergen Bet v 1 represents one of the most prevalent environmental allergens responsible for allergic airway inflammation., Objective: In the present study we sought to compare the complete recombinant Bet v 1 allergen molecule with genetically produced hypoallergenic fragments of Bet v 1 regarding mucosal tolerance induction in a mouse model of allergic asthma., Methods: BALB/c mice were intranasally treated with recombinant Bet v 1 or with two recombinant Bet v 1 fragments (F I: aa 1-74; F II: aa 75-160) prior to aerosol sensitization with birch pollen and Bet v 1., Results: Intranasal application of F II, containing the major T cell epitope, led to significant reduction of IgE/IgG1 antibody responses, in vitro cytokine production (IL-5, IFN-gamma, IL-10) and negative immediate cutaneous hypersensitivity reactions comparable to the pretreatment with the complete rBet v 1 allergen. Moreover, airway inflammation (eosinophilia, IL-5) was inhibited by the pretreatment with either the complete Bet v 1 or F II. However, for prevention of airway hyperresponsiveness the complete molecule was required. The mechanisms leading to immunosuppression seemed to differ in their dependence on the conformation of the molecules, since tolerance induced with the complete Bet v 1, but not with F II, was transferable with spleen cells and associated with increased TGF-beta mRNA levels., Conclusion: We conclude that mucosal tolerance induction with recombinant allergens and genetically engineered hypoallergenic derivatives thereof could provide a convenient and safe intervention strategy against type I allergy., (Copyright 2001 S. Karger AG, Basel)

Mutation of the zebrafish lakritz (lak) locus completely eliminates the earliest-born retinal cells, the ganglion cells (RGCs). Instead, excess amacrine, bipolar, and Müller glial cells are generated in the mutant. The extra amacrines are found at ectopic locations in the ganglion cell layer. Cone photoreceptors appear unaffected by the mutation. Molecular analysis reveals that lak encodes Ath5, the zebrafish eye-specific ortholog of the Drosophila basic helix-loop-helix transcription factor Atonal. A combined birth-dating and cell marker analysis demonstrates that lak/ath5 is essential for RGC determination during the first wave of neurogenesis in the retina. Our results suggest that this wave is skipped in the mutant, leading to an accumulation of progenitors for inner nuclear layer cells.

Background: Carcinoma of the external auditory canal are tumours considered to have a poor prognosis. Improvement of the survival rate by surgical means alone is not possible. Individual therapy modalities as a result of an interdisciplinary approach between otosurgeon and radiotherapist are necessary., Patients and Methods: A series of 30 patients (3 patients pretreated at other institutions) with carcinoma of the external auditory canal and middle ear treated between 1978 and 1997 in our institutions was analysed with particular reference to tumour size and its relation to surrounding tissues, patterns of neck node involvement, surgical procedures, and radiation techniques. Clinical endpoints were freedom from local failure, overall survival, disease-free survival. The mean follow-up was 4.7 years (range: 0.1 to 18.8 years), median 3 years., Results: Treatment by surgery and radiotherapy resulted in an overall 5-year survival rate of 51%. According to Pittsburgh classification the 5-year survival rate for early disease (T1- and T2-tumours) was 89%, for stage III 67% and for stage IV 39%. Most important prognostic factors were dural infiltration (all patients with dural invasion died within 2.2 years) and the infiltration of surgical margins (the 5-year survival rate of patients with complete tumour resection was 100%, but 54% in patients with tumour beyond surgical margins). 192-iridium HDR afterloading brachytherapy based on 3D CT-treatment planning is an effective tool in the management of local recurrences following surgery and a full course of external beam radiotherapy., Conclusions: Surgical resection followed by radiotherapy adapted to the stage of disease and grade of resection is the preferred treatment of cancer of the external auditory canal and middle ear.

One of the responses exhibited by cyanobacteria when they are limited for an essential nutrient is the rapid degradation of their light-harvesting complex, the phycobilisome. Phycobilisome degradation is an ordered proteolytic process, visible by a color change of the cyanobacterial cell from blue-green to yellow-green (chlorosis). The small polypeptide NblA plays a key role in degradation of phycobilisomes in Synechococcus sp. PCC7942. Unlike Synechococcus, Synechocystis sp. PCC6803 has two nblA-homologous genes, nblA1 and nblA2, which are contiguous on the genome. Here we show that nblA1 and nblA2 are simultaneously expressed in Synechocystis 6803 upon nitrogen deprivation, and are both required for phycobilisome degradation.

Objective: To pilot test an inexpensive, home-based water decontamination and storage system in a low-income neighborhood of Karachi., Methods: Fifty households received a 20-L plastic water storage vessel with a high-quality spout and a regular supply of diluted hypochlorite solution. Twenty-five control households were recruited. Water samples were collected at baseline and during unannounced follow-up visits 1, 3, 6, and 10 weeks later., Results: Baseline drinking water samples among intervention households were contaminated with a mean 9397 colony-forming units (cfu)/100 mL of thermotolerant coliforms compared with a mean 10,990 cfu/100 mL from controls. After intervention the mean concentration of thermotolerant coliforms decreased by 99.8% among the intervention households compared with an 8% reduction among controls. Two years after vessel distribution, 34 (68%) of the families were still using the vessel. Thirteen of the households had stopped using their vessel because it had broken after more than 6 months of use, a pattern most consistent with ultraviolet radiation-induced degradation of the plastic., Conclusions: In a highly contaminated environment, a specifically designed water storage container and in-home water chlorination was acceptable and markedly improved water quality. Where plastic water vessels will be exposed to substantial sunlight, ultraviolet light stabilizers should be incorporated into the plastic.

Biosynthesis of the cyanobacterial nitrogen reserve cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is catalysed by cyanophycin synthetase, an enzyme that consists of a single kind of polypeptide. Efficient synthesis of the polymer requires ATP, the constituent amino acids aspartic acid and arginine, and a primer like cyanophycin. Using synthetic peptide primers, the course of the biosynthetic reaction was studied. The following results were obtained: (a) sequence analysis suggests that cyanophycin synthetase has two ATP-binding sites and hence probably two active sites; (b) the enzyme catalyses the formation of cyanophycin-like polymers of 25-30 kDa apparent molecular mass in vitro; (c) primers are elongated at their C-terminus; (d) the constituent amino acids are incorporated stepwise, in the order aspartic acid followed by arginine, into the growing polymer. A mechanism for the cyanophycin synthetase reaction is proposed; (e) the specificity of the enzyme for its amino-acid substrates was also studied. Glutamic acid cannot replace aspartic acid as the acidic amino acid, whereas lysine can replace arginine but is incorporated into cyanophycin at a much lower rate.

We studied the possible regulatory effects of 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] on cytokine production and differentiation of subsets of CD4+ [T helper 1 (Th1) and Th2] and CD8+ [T cytotoxic 1 (Tc1) and Tc2] lymphocytes at the single cell level. PBMC from healthy donors were cultured with or without 1alpha,25-(OH)2D3 for up to 21 days. On days 0, 7, 14, and 21, the percentage of cytokine-producing T lymphocytes was analyzed by intracellular cytokine detection with mAb and flow cytometry. Simultaneous staining for cell surface markers allowed discrimination of CD4+ and CD8+ T cell subsets. After culture with 1alpha,25-(OH)2D3 (10(-8) mol/L), no significant effects on the proportion of interferon-gamma (IFNgamma)- or interleukin-4 (IL-4)-producing cells were detected, whereas reduced frequencies of IL-2-producing cells in the CD4+ as well as in the CD8+ population were found. An increase in the low percentage of CD4+ and CD8+ T cells producing the Th2 cytokine IL-13 was noticed. Most interestingly, IL-6-producing CD4+ and CD8+ T cells could only be detected in cultures with 1alpha,25-(OH)2D3, reaching a plateau after 14 days. The percentage of IL-6-producing T cells induced by 1alpha,25-(OH)2D3 after a given time period remained stable for at least 7 weeks. Studies of cytokine coexpression revealed that about 70% of IL-6-producing CD4+ and CD8+ cells were also positive for IL-2, but more than 90% were negative for IFNgamma, IL-4, or IL-13, respectively. This suggests that the IL-6-producing population does not match the Th1/Tc1-like (IFNgamma+) or Th2/Tc2-like (IL-4+ or IL-13+) subset. The influence of 1alpha,25-(OH)2D3 on cytokine production by lymphocytes is probably an important point of intersection between the endocrine and the immune system.