Towards fast online intrafraction replanning for free-breathing stereotactic body radiation therapy with the MR-linac.

The hybrid MRI-radiotherapy machines, like the MR-linac (Elekta AB, Stockholm, Sweden) installed at the UMC Utrecht (Utrecht, The Netherlands), will be able to provide real-time patient imaging during treatment. In order to take advantage of the system's capabilities and enable online adaptive treatments, a new generation of software should be developed, ranging from motion estimation to treatment plan adaptation. In this work we present a proof of principle adaptive pipeline designed for high precision stereotactic body radiation therapy (SBRT) suitable for sites affected by respiratory motion, like renal cell carcinoma (RCC). We utilized our research MRL treatment planning system (MRLTP) to simulate a single fraction 25 Gy free-breathing SBRT treatment for RCC by performing inter-beam replanning for two patients and one volunteer. The simulated pipeline included a combination of (pre-beam) 4D-MRI and (online) 2D cine-MR acquisitions. The 4DMRI was used to generate the mid-position reference volume, while the cine-MRI, via an in-house motion model, provided three-dimensional (3D) deformable vector fields (DVFs) describing the anatomical changes during treatment. During the treatment fraction, at an inter-beam interval, the mid-position volume of the patient was updated and the delivered dose was accurately reconstructed on the underlying motion calculated by the model. Fast online replanning, targeting the latest anatomy and incorporating the previously delivered dose was then simulated with MRLTP. The adaptive treatment was compared to a conventional mid-position SBRT plan with a 3 mm planning target volume margin reconstructed on the same motion trace. We demonstrate that our system produced tighter dose distributions and thus spared the healthy tissue, while delivering more dose to the target. The pipeline was able to account for baseline variations/drifts that occurred during treatment ensuring target coverage at the end of the treatment fraction.

On-line MR imaging for dose validation of abdominal radiotherapy.

For quality assurance and adaptive radiotherapy, validation of the actual delivered dose is crucial.Intrafractional anatomy changes cannot be captured satisfactorily during treatment with hitherto available imaging modalitites. Consequently, dose calculations are based on the assumption of static anatomy throughout the treatment. However, intra- and interfraction anatomy is dynamic and changes can be significant.In this paper, we investigate the use of an MR-linac as a dose tracking modality for the validation of treatments in abdominal targets where both respiratory and long-term peristaltic and drift motion occur.The on-line MR imaging capability of the modality provides the means to perform respiratory gating of both delivery and acquisition yielding a model-free respiratory motion management under free breathing conditions.In parallel to the treatment, the volumetric patient anatomy was captured and used to calculate the applied dose. Subsequently, the individual doses were warped back to the planning grid to obtain the actual dose accumulated over the entire treatment duration. Ultimately, the planned dose was validated by comparison with the accumulated dose.Representative for a site subject to breathing modulation, two kidney cases (25 Gy target dose) demonstrated the working principle on volunteer data and simulated delivery. The proposed workflow successfully showed its ability to track local dosimetric changes. Integration of the on-line anatomy information could reveal local dose variations -2.3-1.5 Gy in the target volume of a volunteer dataset. In the adjacent organs at risk, high local dose errors ranging from -2.5 to 1.9 Gy could be traced back.

In situ transmission electron microscopy imaging of grain growth in a platinum nanobridge induced by electric current annealing.

We report an in situ transmission electron microscopy (TEM) imaging of grain growth in a Pt nanobridge induced by a high electric current density. The change in morphology at the nanoscale was recorded in real time together with the electrical characterization of the Pt nanobridge. We find a drop in the differential resistance as the voltage across the bridge is increased; TEM inspection shows that this coincides with thermally induced grain growth, indicating that a reduction of grain boundary scattering is the cause of the resistance decrease.

Microparticle-associated tissue factor activity: a link between cancer and thrombosis?

BACKGROUND: Cancer, in particular mucinous adenocarcinoma, is associated with venous thromboembolism (VTE). Tissue factor (TF), initiator of coagulation, plays a central role in the paradigm that clotting and tumor growth form a vicious circle, in which hypercoagulability facilitates the aggressive biology of cancer and vice versa. Expression of TF in tumors is associated with poor differentiation and poor prognosis. PATIENT/METHODS: We investigated the association between clinically manifest VTE and procoagulant properties of circulating microparticles (MP) isolated from blood of unselected pancreatic and breast adenocarcinoma patients' consecutive subjects, who presented with ultrasound or CT-scan confirmed VTE, and healthy subjects. RESULTS: Patients with disseminated breast and pancreatic cancer had significantly increased levels of MP-associated TF activity compared with healthy controls, subjects with idiopathic acute VTE and non-metastatic cancer patients. Patients with both high MP-associated TF-activity and MP-associated epithelial mucin (MUC1) had a lower survival rate at 3-9 months follow-up than those with low TF-activity and no MUC1 expression: the likelihood of survival was 0.42 (95% CI: 0.19- 0.94) for an individual with these two predictor variables present, after adjustment for other factors (age cohort, type of cancer, VTE) in the Cox proportional hazards model. CONCLUSIONS: Our results suggest an important role for MP-associated TF and MUC1 in the pathogenesis of thrombosis in disseminated mucinous adenocarcinoma patients. Future studies should reveal the mechanism underlying the observed associations.

A new model for limbal transplantation using E-GFP for follow-up of transplant survival.

Limbal transplants in humans show a high rate of rejection even under local and systemic immunotherapy. In order to test immunomodulatory treatments a new limbal transplant model in the rat was developed using enhanced green fluorescent protein (E-GFP) as marker for follow-up. Sixty E-GFP-positive limbal transplants from Sprague-Dawley TgN(act-EGFP)Osb4 rats were transplanted onto 18 wild-type inbred Sprague-Dawley (isografts) rats, six wild-type litter mate Sprague-Dawley (sibling) rats, 18 Fischer 344 (allografts) rats, and 18 Fischer 344 rats depleted from monocytes and macrophages by subconjunctival treatment with clodronate liposomes. All rats were monitored three times a week with fluorescence microscopy, until fluorescence had disappeared. At postoperative days 6, 9, 12, and 15, three rats of all groups were killed for immunohistochemical analysis of infiltrating cells. Using a modified digital fluorescence microscope, we were able to monitor transplant behavior over time without disturbance of the ocular surface. The average days of rejection were 14 days in the isograft group, the sibling group, and the untreated allograft group. However, the average day of rejection in the allogeneic macrophage-depleted group was 27 days. Marked infiltration of macrophages and lymphocytes was seen in the untreated isografts and allografts. In the clodronate liposome-treated allografts infiltration was minor. A successful new limbal transplant model is described. The transplant can be accurately followed up in vivo by E-GFP labeling of the donor tissue without disturbing the corneal surface. Although E-GFP itself proved to be immunogenic, local clodronate liposome injections significantly increased graft survival. So the model seems to be useful for testing immunosuppressive or modulatory agents in limbal transplantation studies.

Reflection contrast microscopy: The bridge between light and electron microscopy.

Reflection contrast microscopy (RCM) is a light microscopic method to image cells at high definition and enhanced sensitivity compared to conventional bright-field microscopy. RCM images have very high contrast, which makes them easily applicable for digital image analysis. Because ultrathin sections are mostly used in this method, RCM also functions by bridging light with electron microscopy: the combination of ultrastructural with histochemical studies. RCM can also replace electron microscopy for rapid and simple screening of large quantities of samples for immunocytochemical staining. Special attention is paid to small biological objects, which have to be processed for RCM. If you encounter the limits of brightfield microscopy, in resolution, sensitivity or handling of the specimen, RCM will be a feasible option. Reflection contrast microscopy methods use only slightly adjusted electron microscopy methods for specimen preparation. Therefore, many familiar techniques for ultrathin specimen preparation can be applied. It is essential that only refractive index differences exist in those areas that are of interest and that the further specimen is as optically homogenic as possible, with a refractive index as close to that of glass as possible. Therefore, plastic embedding is recommended.

SDHD mutations in head and neck paragangliomas result in destabilization of complex II in the mitochondrial respiratory chain with loss of enzymatic activity and abnormal mitochondrial morphology.

Hereditary head and neck paragangliomas are tumours associated with the autonomic nervous system. Recently, mutations in genes coding for subunits of mitochondrial complex II, succinate-ubiquinone-oxidoreductase (SDHB, SDHC, and SDHD), have been identified in the majority of hereditary tumours and a number of isolated cases. In addition, a fourth locus, PGL2, has been mapped to chromosome 11q13 in an isolated family. In order to characterize phenotypic effects of these mutations, the present study investigated the immunohistochemical expression of the catalytic subunits of complex II (flavoprotein and iron protein), SDH enzyme activity, and mitochondrial morphology in a series of 22 head and neck paragangliomas. These included 11 SDHD-, one SDHB-, two PGL2-linked tumours, and eight sporadic tumours. In the majority of the tumours (approximately 90%), the enzyme-histochemical SDH reaction was negative and immunohistochemistry of catalytic subunits of complex II showed reduced expression of iron protein and enhanced expression of flavoprotein. Ultrastructural examination revealed elevated numbers of tightly packed mitochondria with abnormal morphology in SDHD-linked and sporadic tumours. Immuno-electron microscopy showed localization of the flavoprotein on the remnants of the mitochondrial inner membranes, whereas virtually no signal for the iron protein was detected. These results indicate that the function of mitochondrial complex II is compromised in the majority of head and neck paragangliomas.

Central venous catheter complications in 447 patients on home parenteral nutrition: an analysis of over 100.000 catheter days.

RATIONALE: The purpose of this study is to report the frequency of central venous catheter (CVC) complications and to analyze the potential risk factors for complications requiring CVC removal in home parenteral nutrition (HPN) patients. METHODS: A questionnaire developed by the ESPEN HAN WORKING GROUP was distributed to 12 European centers to investigate the complications occurring during the period between January 1995 and December 2000 when HPN patients used their first CVC. The questionnaire collected informations related to the Home Parenteral Nutrition technique and the underlying disease. Factors affecting the time of CVC removal were jointly investigated using Cox's multivariable regression models. RESULTS: The study was performed on 447 patients for a total of 110869 CVC-days. Complications occurred in about 1/4 of patients, approximately half were infections and about half required Central Venous Catheter removal. The Cox analysis showed that using the CVC 7 times/week and implanted ports were associated with a hazard ratio of 3 and 2.8, respectively. A reduced risk of removal (of about 40%) was associated with using CVC also for non-nutritional purposes (P = 0.0016). CONCLUSIONS: Within the limits of this retrospective investigation, the type of CVC, the type of administration of HPN and the type of training are important factors associated with occurrence of complications or with CVC removal. However, in our opinion, proper care of the CVC, of preparation and administration of the nutritive admixture seem to be paramount for a safe management of HPN.

Towards implementation of optimum nutrition and better clinical nutrition support.

Clinical Nutrition Support--defined as nutrition for hospitalized patients suffering from metabolic stress--plays a limited role in the therapeutic routine of the physician. This is not surprising as most research in the field of clinical nutrition is disappointing with regard to the objective outcomes: morbidity and mortality. These reflections advocate a more 'pharmaceutical approach' to nutrition in order to perform more proper studies on the potential effectiveness of this treatment modality. To provide all patients in the Academic Medical Centre (AMC) in Amsterdam, The Netherlands, with optimum clinical nutrition support, a Nutrition Support Team (NST) was established in 1996. This NST is coaching the dieticians and physicians in the AMC regarding clinical nutrition support. In practice this coaching consists of providing clear guidelines on what is supposed to be optimum nutrition, a basic course in parenteral nutrition and further continuous education. The concept of optimum nutrition is spread by the NST through various ways of education, both nationally and internationally. For adults, optimum nutrition is defined as the amount of protein, that stimulates whole body protein synthesis maximally (1.7 g/kg actual body weight) and covers anabolic energy need (35 kcal/kg actual body weight). The dietician is considered to be the expert in the field of optimum nutrition by oral, enteral or parenteral route. The Dietetic Department has increased its influence in the care of the patient by placing nutritional status and care on the chart of the patient's treatment. To provide optimal Nutrition Support for children and severe ill patients (Intensive care department) specialized teams were started which were co-ordinated by the central NST. The central NST has a co-ordinating and educating role, while the Specialized Nutrition Support Teams (Specialized NST) construct guidelines, undertake research and provide continuous optimum nutrition care.

Epidermal growth factor-like repeats mediate lateral and reciprocal interactions of Ep-CAM molecules in homophilic adhesions.

Ep-CAM is a new type of cell adhesion molecule (CAM) which does not structurally resemble the members of the four major families (cadherins, integrins, selectins, and CAMs of the immunoglobulin superfamily) and mediates Ca(2+)-independent, homophilic adhesions. The extracellular domain of Ep-CAM consists of a cysteine-rich region, containing two type II epidermal growth factor (EGF)-like repeats, followed by a cysteine-poor region. We generated mutated Ep-CAM forms with various deletions in the extracellular domain. These deletion mutants, together with monoclonal antibodies recognizing different epitopes in the extracellular domain, were used to investigate the role of the EGF-like repeats in the formation of intercellular contacts mediated by Ep-CAM molecules. We established that both EGF-like repeats are required for the formation of Ep-CAM-mediated homophilic adhesions, including the accumulation of Ep-CAM molecules at the cell-cell boundaries, and the anchorage of the Ep-CAM adhesion complex to F-actin via alpha-actinin. Deletion of either EGF-like repeat was sufficient to inhibit the adhesion properties of the molecule. The first EGF-like repeat of Ep-CAM is required for reciprocal interactions between Ep-CAM molecules on adjacent cells, as was demonstrated with blocking antibodies. The second EGF-like repeat was mainly required for lateral interactions between Ep-CAM molecules. Lateral interactions between Ep-CAM molecules result in the formation of tetramers, which might be the first and necessary step in the formation of Ep-CAM-mediated intercellular contacts.

Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy.

The authors recently showed variable subcellular immunoreactivity of the Bcl-2 and Bax proteins after fixation of cell monolayers with acetone, methanol, or paraformaldehyde (PF) followed by methanol (PF/methanol). Here, the authors demonstrate by reflection contrast microscopy and transmission electron microscopy that acetone or methanol fixation result in complete loss of integrity of intracellular structures in contrast with PF or glutaraldehyde fixation. Scanning electron microscopy revealed poor preservation of plasma membrane integrity after fixation in acetone or methanol. Fixation with PF before methanol reduced damage to intracellular and plasma membranes. In addition, Western blot analysis demonstrated loss of Bcl-2 and Bax protein during acetone or methanol fixation, whereas PF fixation before methanol permeabilization markedly reduced this loss. For studies on the intracellular localization of soluble or unknown types of antigen, the authors discourage the use of acetone and methanol as single fixatives.

Polycystin-1, the product of the polycystic kidney disease 1 gene, co-localizes with desmosomes in MDCK cells.

Polycystin-1 is a novel protein predicted to be a large membrane-spanning glycoprotein with an extracellular N-terminus and an intracellular C-terminus, harboring several structural motifs. To study the subcellular localization, antibodies raised against various domains of polycystin-1 and against specific adhesion complex proteins were used for two-color immunofluorescence staining. In Madine Darby canine kidney (MDCK) cells, polycystin-1 was detected in the cytoplasm as well as co-localizing with desmosomes, but not with tight or adherens junctions. Using confocal laser scanning and immunoelectron microscopy we confirmed the desmosomal localization. By performing a calcium switch experiment, we demonstrated the sequential reassembly of tight junctions, subsequently adherens junctions and finally desmosomes. Polycystin-1 only stained the membrane after incorporation of desmoplakin into the desmosomes, suggesting that membrane-bound polycystin-1 may be important for cellular signaling or cell adhesion, but not for the assembly of adhesion complexes.

Amyloid beta protein (Abeta) starts to deposit as plasma membrane-bound form in diffuse plaques of brains from hereditary cerebral hemorrhage with amyloidosis-Dutch type, Alzheimer disease and nondemented aged subjects.

To clarify where and how beta-amyloid begins to deposit in senile plaques, we examined the ultrastructural localization of amyloid beta protein (Abeta) in diffuse plaques of brains with hereditary cerebral hemorrhage with amyloidosis-Dutch type. Alzheimer disease (AD), and from nondemented aged subjects. Serial ultrathin sections of osmium-plastic blocks were immunogold-labeled for Abetax-42 (Abeta42), and sections on grids were observed under the electron microscope (EM) after observing the exact localization of the diffuse plaques in sections on glass slides by the reflection contrast microscope. Abeta42 deposition, which was decollated with gold particles, appeared in 3 forms in all subjects under the EM: 1) Scattered small bundles of amyloid fibrils between cell processes, frequently seen in the densely stained area of diffuse plaques. 2) Scattered small foci of nonfibrillar materials between cell processes as a relatively minor form. 3) Abeta42 on a part of the cell surface plasma membrane of normal appearing cell processes, a major form in weakly immunostained areas. The last form was not associated with degenerative neurites or reactive glia. Abeta42 deposition on the cell surface plasma membrane appears to be an initial event in diffuse plaques, and then it develops into amorphous/fibrillar amyloid between cell processes.

Ultrastructural evidence of early non-fibrillar Abeta42 in the capillary basement membrane of patients with hereditary cerebral hemorrhage with amyloidosis, Dutch type.

The C-terminal profile and ultrastructure of small and presumably early capillary amyloid beta protein (Abeta) deposits were investigated in four patients with hereditary cerebral hemorrhage with amyloidosis, Dutch type. The C terminus of the 40 (Abeta40) or the 42 (Abeta42) amino acid form of Abeta was gold labeled in serial, ultrathin sections on glass slides for reflection contrast microscopy and on grids for electron microscopy. In all studied subjects, reflection contrast microscopy revealed capillaries with focal Abeta42 immunolabeling in the absence of Abeta40 labeling. In the adjacent electron microscopic section, Abeta42 labeling was confined to the capillary basement membrane. The majority of Abeta42(+)40(-) deposits showed no amyloid fibrils. Abeta42(+)40(-) deposits were sometimes observed in an unremarkable basement membrane but usually showed increased electron density and reticular structures. A small subset of Abeta42(+)40(-) deposits with basement membrane changes showed few amyloid fibrils. Abeta42(+)40(+) capillary deposits always showed definite fibrils and were larger than Abeta42(+)40(-) capillary deposits. The present findings suggest that in capillaries the accumulation and subsequent polymerization of Abeta42, possibly in conjunction with basement membrane changes, precedes the definite fibril formation with Abeta40.

The terminal sequence of complement plays an essential role in antibody-mediated renal cell apoptosis.

Mesangial cell (MC) injury is a characteristic feature in the early phase of Thy.1 nephritis. The present study investigates the contribution of complement to MC apoptosis in this experimental model of kidney disease in rats. Thy.1 nephritis was induced by injection of mouse anti-Thy.1 monoclonal antibody (ER4G). To assess the contribution of the terminal sequence of complement on apoptosis, the studies were performed in complement-sufficient PVG/c (PVG/c+) rats and in rats deficient in complement C6 (PVG/c-). Apoptosis was monitored by assessment of the number of condensed nuclei in kidney sections stained with periodic acid-Schiff (PAS) and by the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) method and expressed as number of apoptotic cells per 50 glomerular cross sections. In the PAS method, 1 h after intravenous injection of ER4G, PVG/c+ rats exhibited 160.9 +/- 49.5 apoptotic cells, whereas PVG/c- rats had only 3.2 +/- 1.4 apoptotic cells. Control rats exhibited 0.9 +/- 0.6 apoptotic cells. These findings were confirmed with the TUNEL method. In PVG/c- rats, a maximum number of 8.8 +/- 3.1 TUNEL-positive (TUNEL+) cells was found at 6 h followed by a decline thereafter. In PVG/c+ rats, apoptosis was associated with deposition of C6 and C5b-9. Restoration of the complement system of PVG/c- rats with purified human C6 resulted in an increase of apoptosis at 1 h after injection of ER4G from minimal numbers to 239.9 +/- 52.4 TUNEL+ cells. These studies appear to indicate for the first time that the terminal sequence of complement is involved in induction of apoptosis.

Thromboxane-dependent platelet activation in vivo precedes arterial thrombosis in thrombocythaemia: a rationale for the use of low-dose aspirin as an antithrombotic agent.

The clinical course of patients with polycythaemia vera (PV) and essential thrombocythaemia (ET) is frequently complicated by arterial thrombotic events. The pathogenesis is not clearly understood but attributed to abnormalities in platelet function. An increase in platelet thromboxane formation has been described in the majority of asymptomatic patients with thrombocythaemia, probably reflecting spontaneous platelet activation in vivo. In the present study we prospectively investigated whether an increase in platelet thromboxane formation actually precedes arterial microvascular thrombosis. In addition, we studied the effect of selective inhibition of platelet thromboxane formation on clinical outcome by reinstitution of low-dose aspirin (50 mg/ d). Six ET patients and one PV patient participated in this study. Within 10 d after withdrawal of aspirin, three patients developed arterial microvascular thrombosis of extremities (erythromelalgia), which was preceded by a 3-30-fold increase in urinary thromboxane excretion as compared with patients who remained asymptomatic. The increased urinary thromboxane excretion and clinical signs could be inhibited by a platelet-specific aspirin regimen of 50 mg/d without affecting vascular cyclooxygenase, indicating that platelets were the main source of the increased thromboxane generation. These data suggest that in symptomatic patients an enhanced formation of thromboxane by platelets, reflecting platelet activation in vivo, precedes the development of arterial microvascular thrombosis. These data provide a rationale for using low-dose aspirin as an antithrombotic agent in thrombocythaemia.

The structural analysis of adhesions mediated by Ep-CAM.

The epithelial cell adhesion molecule Ep-CAM is capable of mediating Ca2+-independent homotypic cell-cell adhesion when introduced into cells lacking their own means of cell-cell interactions. We used (confocal) immunofluorescent and (immuno-) electron microscopy to investigate the structural organization of Ep-CAM-mediated adhesions and their relation to other types of intercellular adhesions. Ep-CAM-transfected cell lines, cells of epithelial origin, and epithelial tissues were analyzed. In transfected L cells Ep-CAM brings the opposing intercellular membranes into a close proximity (approximately 10-14 nm) at sporadic contacts; however, no structures resembling junctional complexes were observed. In L cells cotransfected with Ep-CAM and E-cadherin, both molecules localize at the sites of cell-cell contact, forming independent adhesion sites with no Ep-CAM detectable within the structurally distinguishable cadherin-mediated adherens junctions. In well-differentiated carcinoma cell lines Ep-CAM colocalized with E-cadherin practically along the whole lateral domain; however, no colocalization was observed between Ep-CAM and the components of the tight junction complex (occludin and ZO-1), desmosomes (desmoplakins I/II), or cell-substrate adhesions (beta1 integrins). This was confirmed by analysis of polarized epithelium of normal colon where Ep-CAM was present at the lateral membrane including the adherens junction areas, but was fully excluded from the apical cell membrane (microvilli), tight junctions, and desmosomes. We conclude that (1) Ep-CAM does not form junctional complexes in L cells, (2) in epithelial cells, cell surface Ep-CAM is present at the lateral cell membrane, but is excluded from tight junctions and desmosomes, and (3) in epithelial cells, Ep-CAM is present within adhesions mediated by the classic cadherins (especially E-cadherin) with both types of molecules remaining as independent clusters. The colocalization with cadherins might be important for the modulating effect of Ep-CAM on cadherin-mediated adhesions.