Search

Glycosylation is an attractive approach to enhance biological properties of pharmaceutical proteins; however, the precise installation of glycans for structure-function studies remains challenging. Here, we describe a chemoenzymatic methodology for glyco-tagging of proteins by peptidoligase catalyzed modification of the N -terminus of a protein with a synthetic glycopeptide ester having an N -acetyl-glucosamine (GlcNAc) moiety to generate an N -GlcNAc modified protein. The GlcNAc moiety can be elaborated into complex glycans by trans -glycosylation using well-defined sugar oxazolines and mutant forms of endo β- N -acetylglucosaminidases (ENGases). The glyco-tagging methodology makes it possible to modify on-demand therapeutic proteins, including heterologous proteins expressed in E. coli , with diverse glycan structures. As a proof of principle, the N -terminus of interleukin (IL)-18 and interferon (IFN)α-2a was modified by a glycopeptide harboring a complex N -glycan without compromising biological potencies. The glyco-tagging methodology was also used to prepare several glycosylated insulin variants that exhibit reduced oligomerization, aggregation, and fibrillization yet maintained cell signaling properties, which are attractive for the development of insulins with improved shelf-lives. It was found that by employing different peptidoligases, it is possible to modify either the A or both chains of human insulin.

Access to defined glycans and glycoconjugates is pivotal for discovery, dissection, and harnessing of a range of biological functions orchestrated by cellular glycosylation processes and the glycome. We previously employed genetic glycoengineering by nuclease-based gene editing to develop sustainable production of designer glycoprotein therapeutics and cell-based glycan arrays that display glycans in their natural context at the cell surface. However, access to human glycans in formats and quantities that allow structural studies of molecular interactions and use of glycans in biomedical applications currently rely on chemical and chemoenzymatic syntheses associated with considerable labor, waste, and costs. Here, we develop a sustainable and scalable method for production of glycans in glycoengineered mammalian cells by employing secreted Glycocarriers with repeat glycosylation acceptor sequence motifs for different glycans. The Glycocarrier technology provides a flexible production platform for glycans in different formats, including oligosaccharides, glycopeptides, and multimeric glycomodules, and offers wide opportunities for use in bioassays and biomedical applications., (© 2024. The Author(s).)

Glucagon-like peptide receptor (GLP-1R) agonists (e.g., semaglutide, liraglutide, etc.) are efficient treatment options for people with type 2 diabetes and obesity. The manufacturing method to produce semaglutide, a blockbuster GLP-1 drug on the market, involves multistep synthesis. The large peptide has a hydrophobic fatty acid side chain that makes it sparingly soluble, and its handling, purification, and large-scale production difficult. The growing demand for semaglutide that the manufacturer is not capable of addressing immediately triggered a worldwide shortage. Thus, we have developed a potential alternative analogue to semaglutide by replacing the hydrophobic fatty acid with a hydrophilic human complex-type biantennary oligosaccharide. Our novel glycoGLP-1 analogue was isolated in an ∼10-fold higher yield compared with semaglutide. Importantly, our glycoGLP-1 analogue possessed a similar GLP-1R activation potency to semaglutide and was biologically active in vivo in reducing glucose levels to a similar degree as semaglutide.

Small molecule therapeutics represent the majority of the FDA-approved drugs. Yet, many attractive targets are poorly tractable by small molecules, generating a need for new therapeutic modalities. Due to their biocompatibility profile and structural versatility, peptide-based therapeutics are a possible solution. Additionally, in the past two decades, advances in peptide design, delivery, formulation, and devices have occurred, making therapeutic peptides an attractive modality. However, peptide manufacturing is often limited to solid-phase peptide synthesis (SPPS), liquid phase peptide synthesis (LPPS), and to a lesser extent hybrid SPPS/LPPS, with SPPS emerging as a predominant platform technology for peptide synthesis. SPPS involves the use of excess solvents and reagents which negatively impact the environment, thus highlighting the need for newer technologies to reduce the environmental footprint. Herein, fourteen American Chemical Society Green Chemistry Institute Pharmaceutical Roundtable (ACS GCIPR) member companies with peptide-based therapeutics in their portfolio have compiled Process Mass Intensity (PMI) metrics to help inform the sustainability efforts in peptide synthesis. This includes PMI assessment on 40 synthetic peptide processes at various development stages in pharma, classified according to the development phase. This is the most comprehensive assessment of synthetic peptide environmental metrics to date. The synthetic peptide manufacturing process was divided into stages (synthesis, purification, isolation) to determine their respective PMI. On average, solid-phase peptide synthesis (SPPS) (PMI ≈ 13,000) does not compare favorably with other modalities such as small molecules (PMI median 168-308) and biopharmaceuticals (PMI ≈ 8300). Thus, the high PMI for peptide synthesis warrants more environmentally friendly processes in peptide manufacturing.

Exenatide was the first marketed GLP-1 receptor agonist for the treatment of type 2 diabetes. Modification to the chemical structure or the formulation has the potential to increase the stability of exenatide. We introduced human complex-type sialyloligosaccharide to exenatide at the native Asn28 position. The synthesis was achieved using both solid phase peptide synthesis (SPPS) and Omniligase-1-mediated chemoenzymatic ligation. The results demonstrate that glycosylation increases the proteolytic stability of exenatide while retaining its full biological activity.

Targeted molecular radiation therapy is a promising emerging treatment modality in oncology, and peptide synthesis may shorten the time to reach the clinical stage. In this study, we have explored Chemo-Enzymatic Peptide Synthesis, or CEPS, as a new means of producing a therapeutic HER2 targeted Affibody ® molecule, comprising a C-terminal albumin binding domain (ABD) for half-life extension and a total length of 108 amino acids. In addition, a DOTA moiety could be incorporated at N-terminus directly during the synthesis step and subsequently utilized for site-specific radiolabeling with the therapeutic radionuclide 177 Lu. Retained thermodynamic stability as well as retained binding to both HER2 and albumin was verified. Furthermore, HER2 binding specificity of the radiolabeled Affibody molecule was confirmed by an in vitro saturation assay showing a significantly higher cell-bound activity of SKOV-3 (high HER2 expression) compared with BxPC3 (low HER2 expression), both in the presence and absence of HSA. In vivo evaluation in mice bearing HER2 expressing xenografts also showed specific tumor targeting as well as extended time in circulation and reduced kidney uptake compared with a HER2 targeted Affibody molecule without the ABD moiety. To conclude, we have demonstrated that CEPS can be used for production of Affibody-fusion molecules with retained in vitro and in vivo functionality.

Purpose: To benchmark and improve, through means of a targeted intervention, the quality of intensity modulated radiation therapy treatment planning for locally advanced head and neck cancer (HNC) in the Netherlands. The short and long-term impact of this intervention was assessed., Methods and Materials: A delineated computed tomography-scan of an oropharynx HNC case was sent to all 15 Dutch radiation therapy centers treating HNC. Aims for planning target volume and organ-at-risk (OAR) dosimetry were established by consensus. Each center generated a treatment plan. In a targeted intervention, OAR sparing of all plans was discussed, and centers with the best OAR sparing shared their planning strategies. Impact of the intervention was assessed by (1) short-term (half a year after intervention) replanning of the original case and (2) long-term (1 and 3 years after intervention) planning of new cases., Results: Benchmarking revealed substantial difference in OAR doses. Initial mean doses were 22 Gy (range, 15-31 Gy), 35 Gy (18-49 Gy), and 37 Gy (20-46 Gy) for the contralateral parotid gland, contralateral submandibular gland, and combined swallowing structures, respectively. Replanning after targeted intervention significantly reduced mean doses and variation, but clinically relevant differences still remained: 18 Gy (14-22 Gy), 28 Gy (17-45 Gy), and 29 Gy (18-39 Gy), respectively. One and 3 years later the variation remained stable., Conclusions: Despite many years of HNC intensity modulated radiation therapy experience, initial treatment plans showed surprisingly large variations. The simple targeted intervention used in this analysis improved OAR sparing, and its impact was durable; however, fairly large dose differences still continue to exist. Additional work is needed to understand these variations and to minimize them. A national radiation oncology platform can be instrumental for developing and maintaining high-quality planning protocols., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The purification of heparin from offal is an old industrial process for which commercial recipes date back to 1922. Although chemical, chemoenzymatic, and biotechnological alternatives for this production method have been published in the academic literature, animal-tissue is still the sole source for commercial heparin production in industry. Heparin purification methods are closely guarded industrial secrets which are not available to the general (scientific) public. However by reviewing the academic and patent literature, we aim to provide a comprehensive overview of the general methods used in industry for the extraction of heparin from animal tissue., Competing Interests: All authors hold positions in an enterprise which commercially manufactures heparin.

A modular approach toward the synthesis of all possible trimer repeating units of the type 1 capsular polysaccharide of Streptococcus pneumonia, Sp1, is described. This zwitterionic polysaccharide is built up from trisaccharide repeats, which in turn are composed of two galacturonic acid monomers and a 2,4,6-trideoxy-4-amino-2-acetamido-D-galactose moiety. All monomeric constituents are linked through cis-glycosidic bonds. To overcome the difficulty associated with the efficient stereoselective introduction of the α-galacturonic acid bonds, we have employed galacturonic acid-[3,6]-lactone building blocks. Not only did these building blocks perform well when used as donor galactosides, they were also shown to be reactive acceptor glycosides when equipped with a free hydroxyl function. All three frame-shifted trimer repeats were constructed via highly stereoselective glycosylation reactions, with one exception. The epimeric mixture of trisaccharides, formed in the nonselective glycosylation event, could be readily separated after global deprotection using high performance anion exchange chromatography (HPEAC).

The search for stereoselective glycosylation reactions has occupied synthetic carbohydrate chemists for decades. Traditionally, most attention has been focused on controlling the S(N)2-like substitution of anomeric leaving groups as highlighted by Lemieux's in situ anomerization protocol and by the discovery of anomeric triflates as reactive intermediates in the stereoselective formation of beta-mannosides. Recently, it has become clear that also S(N)1-like reaction pathways can lead to highly selective glycosylation reactions. This review describes some recent examples of stereoselective glycosylations in which oxacarbenium ions are believed to be at the basis of the selectivity. Special attention is paid to the stereodirecting effect of substituents on a pyranosyl ring with an emphasis on the role of the C-5 carboxylate ester in the condensations of mannuronate ester donors., (Copyright 2010 Elsevier Ltd. All rights reserved.)

In this paper the synthesis of an Enterococcus Faecalis teichoic acid (TA) hexamer is presented. The key kojibiosyl-glycerol phosphoramidite building block was obtained by condensation of thioglucose donors, provided with various protecting groups at the C2 hydroxyl function with an orthogonally protected glycerol acceptor. After selective deprotection, the resulting 1,2-cis-linked pseudodisaccharide acceptor was coupled to an alpha-directing thioglucose donor, giving the corresponding pseudotrisaccharide, which is then transformed to a phosphoramidite synthon. The kojibiosyl-glycerol phosphoramidite in combination with a glycerolphosphoramidite, an aminohexylphosphoramidite and dibenzylglycerol were coupled to a fully protected glycerol TA hexamer, using chemistry that can be amended for future automated synthesis. Global deprotection afforded the target hexamer kojibiosyl-glycerol containing TA (1).

Glycosylations of mannuronate ester donors proceed highly selectively to produce the 1,2-cis-linked products. We here forward a mechanistic rationale for this counterintuitive selectivity, based on the remote stereodirecting effect of the C5-carboxylate ester, which has been demonstrated using pyranosyl uronate ester devoid of ring substituents other than the C5- carboxylate ester. It is postulated that the C5-carboxylate ester prefers to occupy an axial position in the oxacarbenium intermediate, thereby favoring the formation of the (3)H4 half-chair over the (4)H3 conformer. Nucleophilic attack on the (3)H4 half-chair intermediate occurs in a beta-fashion, providing the 1,2-cis-mannuronates with excellent stereoselectivity. The potential of the mannuronate ester donors in the formation of the beta-mannosidic linkage has been capitalized upon in the construction of a mannuronic acid alginate pentamer using a convergent orthogonal glycosylation strategy.

The glycosylation properties of gulopyranosides have been mapped out, and it is shown that gulose has an intrinsic preference for the formation of 1,2-cis-glycosidic bonds. It is postulated that this glycosylation behaviour originates from nucleophilic attack at the oxacarbenium ion, which adopts the most favourable 3H4 conformation. Building on the stereoselectivity of gulose, a guluronic acid alginate trisaccharide was assembled for the first time by using gulopyranosyl building blocks.

The synthesis of hyaluronic acid (HA) oligomers using a stepwise glycosylation strategy is described. This method employs protected 1-hydroxyuronic acid and 1-phenylthio glucosamine donors, both of which are activated with the Ph2SO/Tf2O activator system.

A historical overview is presented on stereo-directing effects of cis- and trans-fused diol protective groups used on both donor and acceptor glycosides. Attention is focused on the use of cyclic carbonates and carbamates, diacetals and acetals and finally the special case of 1,2-O-orthoesters and 1,2-O-cyanoalkylidene functionalised residues.

A facile synthesis route toward beta-linked mannuronic acid oligomers using the corresponding 1-thiomannuronic acid esters in combination with the Ph2SO/Tf2O or NIS/TMSOTf reagent combinations is presented. The presence of the remotely attached carboxylic ester sufficiently influences the electronic environment to allow good to excellent beta-selectivities.

A variety of thioglycosides are chemoselectively hydrolyzed to the corresponding 1-hydroxy glycosides using equimolar amounts of NIS/TFA as promoter systems.

This tutorial review surveys the use of thioglycosides in the development of sequential glycosylation methodologies, with a focus on chemoselective, orthogonal and iterative glycosylation strategies reported since the beginning of this century. Both fundamental aspects of glycosidic bond formation and ingenious state-of-the-art methodologies are presented.

Background and Purpose: To determine the effect of organ motion and set-up uncertainties on IMRT dose distributions for prostate., Methods: For five patients, IMRT techniques were designed to irradiate the CTV (prostate plus seminal vesicles). Technique I delivered 78 Gy to PTV1 (CTV+10 mm margin). Technique II delivered 68 Gy to PTV1, and a 10 Gy boost to PTV2 (CTV+an anisotropic margin of 0 to 5 mm). Technique III delivered 68 Gy to PTV1 and simultaneously 78 Gy to PTV2. Uncertainties were simulated using population statistics of organ motion and set-up accuracy. The average TCP (TCPpop) of the CTV and average NTCP (NTCPpop) of the rectal wall were calculated., Results: The planning TCP was a good predictor for TCPpop for Techniques I and II. Technique III was sensitive for geometrical uncertainties, reducing TCPpop by 0.8 to 2.4% compared to planning. NTCPpop was reduced for Technique III by a factor 2.6 compared to Technique I. For all plans, the planning NTCP was strongly correlated with NTCPpop., Conclusions: Dose distributions created with Techniques I and II are insensitive for geometrical uncertainties, while Technique III resulted in a reduction of TCPpop. This reduction can be compensated by a small dose escalation, while still resulting in an NTCPpop of the rectal wall that is lower or comparable to Technique I.

[reaction: see text] Partially protected thioglycuronic acids are prepared efficiently by chemo- and regioselective oxidation of the corresponding thioglycosides using the TEMPO/BAIB reagent combination. After esterification, the thioglycuronic acids proved to be useful as both donor and acceptor in sulfonium-mediated condensations toward acidic di- and trisaccharides.

Persistent left superior vena cava (LSVC) is the most common congenital systemic venous anomaly, which may give rise to several problems. We present a case in which a persistent LSVC was an unsuspected finding. A 70-year-old male presented with intracerebral empyema which may have been caused by venous septic emboli from the left arm and facilitated by a persistent LSVC draining directly into the left atrium. Visualisation of the anomaly was performed with echocardiography and magnetic resonance angiography. In addition we present a brief review of the literature concerning this disorder.

Purpose: To compare two methods to generate treatment plans for intensity-modulated radiotherapy (IMRT) of prostate cancer, delivered in a step-and-shoot mode. The first method uses fluence optimization (inverse planning) followed by conversion of the fluence weight map into a limited number of segments. In the second method, segments are manually assigned using a class solution (forward planning), followed by computer optimization of the segment weights., Methods: Treatment plans for IMRT, utilizing a simultaneous integrated boost, were created. Plans comprise a five-field technique to deliver 78 Gy to the prostate plus seminal vesicles. Five patients were evaluated. Optimization objectives of both planning approaches concerned dose coverage of the target volumes and the dose distribution in the rectal wall. The two methods were evaluated by comparing dose distributions, the complexity of the resulting plan and the time expenditure to generate and to deliver the plan., Results: For both planning approaches 99% of the target volumes received 95% of the prescribed dose, which complies with our planning objectives. Inverse planning resulted in more conformal dose distributions than forward planning (conformity index: 1.37 versus 1.51). Inverse planning reduced the dose to the rectal wall compared to a manually designed plan, albeit to a small extent. The theoretical probability of severe rectal proctitis and/or stenosis was reduced on average by 1.9% with inverse planning. Maximal sparing of the rectal wall was achieved with inverse planning for a patient whose target volume was partly wrapped around the rectum. The number of segments generated with inverse planning ranged between 33 and 52, and between 9 and 13 segments for manually created segments., Conclusion: Dose coverage of the planning target volumes is adequate for both approaches of planning. Inverse planning results in slightly better dose distributions with respect to the rectal wall compared to manual planning, at the cost of an increase of the number of segments by a factor of 3.

A tethered nucleophilic substitution strategy for the stereoselective introduction of axially oriented amino functions on suitably protected gluco- and mannopyranosides is presented. The obtained oxazine is a versatile building block, which after some manipulation, could be used in the construction of highly functionalised oligosaccharides.

Background and Purpose: To compare intensity-modulated treatment plans of patients with head and neck cancer generated by forward and inverse planning., Materials and Methods: Ten intensity-modulated treatment plans, planned and treated with a step&shoot technique using a forward planning approach, were retrospectively re-planned with an inverse planning algorithm. For this purpose, two strategies were applied. First, inverse planning was performed with the same beam directions as forward planning. In addition, nine equidistant, coplanar incidences were used. The main objective of the optimisation process was the sparing of the parotid glands beside an adequate treatment of the planning target volume (PTV). Inverse planning was performed both with pencil beam and Monte Carlo dose computation to investigate the influence of dose computation on the result of the optimisation., Results: In most cases, both inverse planning strategies managed to improve the treatment plans distinctly due to a better target coverage, a better sparing of the parotid glands or both. A reduction of the mean dose by 3-11Gy for at least one of the parotid glands could be achieved for most of the patients. For three patients, inverse planning allowed to spare a parotid gland that had to be sacrificed by forward planning. Inverse planning increased the number of segments compared to forward planning by a factor of about 3; from 9-15 to 27-46. No significant differences for PTV and parotid glands between both inverse planning approaches were found. Also, the use of Monte Carlo instead of pencil beam dose computation did not influence the results significantly., Conclusion: The results demonstrate the potential of inverse planning to improve intensity-modulated treatment plans for head and neck cases compared to forward planning while retaining clinical utility in terms of treatment time and quality assurance.

Background and Purpose: To assess the effect of differences in the calculation of the dose outside segment edges on the overall dose distribution and the optimisation process of intensity modulated radiation therapy (IMRT) treatment plans., Patients and Methods: Accuracy of dose calculations of two treatment planning systems (TPS1 and TPS2) was assessed, to ensure that they are both suitable for IMRT treatment planning according to published guidelines. Successively, 10 treatment plans for patients with prostate and head and neck tumours were calculated in both systems. The calculations were compared in selected points as well as in combination with volumetric parameters concerning the planning target volume (PTV) and organs at risk., Results: For both planning systems, the calculations agree within 2.0% or 3 mm with the measurements in the high-dose region for single and multiple segment dose distributions. The accuracy of the dose calculation is within the tolerances proposed by recent recommendations. Below 35% of the prescribed dose, TPS1 overestimates and TPS2 underestimates the measured dose values, TPS2 being closer to the experimental data. The differences between TPS1 and TPS2 in the calculation of the dose outside segments explain the differences (up to 50% of the local value) found in point dose comparisons. For the prostate plans, the discrepancies between the TPS do not translate into differences in PTV coverage, normal tissue complication probability (NTCP) values and results of the plan optimisation process. The dose-volume histograms (DVH) of the rectal wall differ below 60 Gy, thus affecting the plan optimisation if a cost function would operate in this dose region. For the head and neck cases, the two systems give different evaluations of the DVH points for the PTV (up to 22% differences in target coverage) and the parotid mean dose (1.0-3.0 Gy). Also the results of the optimisation are influenced by the choice of the dose calculation algorithm., Conclusions: In IMRT, the accuracy of the dose calculation outside segment edges is important for the determination of the dose to both organs at risks and target volumes and for a correct outcome of the optimisation process. This aspect should therefore be of major concern in the commissioning of a TPS intended for use in IMRT. Fulfilment of the accuracy criteria valid for conformal radiotherapy is not sufficient. Three-dimensional evaluation of the dose distribution is needed in order to assess the impact of dose calculation accuracy outside the segment edges on the total dose delivered to patients treated with IMRT.

[reaction: see text] A novel sequential glycosylation procedure is described that combines the use of 1-hydroxyl and thiodonors. The Ph(2)SO/Tf(2)O-mediated dehydrative condensation of 1-hydroxyl donors with thioglycosides affords in good yield the thiodisaccharides, which in turn can be activated by the same activator system to furnish trisaccharides. The alpha-Gal epitope and a hyaluronan trisaccharide were efficiently assembled in a one-pot procedure.

Depth and field size dependence of the sensitometric curves of radiographic films have been studied by various groups. Limited information is, however, available on the magnitude of the variations in sensitometric curves applied in clinical practice in different institutions. In this study we assessed in a systematic way the effect of the various parameters influencing the shape of the sensitometric curve: batch composition, irradiation conditions, film processing, and film scanning. Two types of film, Kodak X-Omat V and CEA TVS, were irradiated, processed, and analyzed in three different institutions. The interinstitutional variation of the sensitometric curves, expressed as the OD variation at 50 cGy, can be up to 32% and is mainly caused by differences in film processing and to a lesser degree to differences in batch composition, film scanning, and irradiation conditions. For the Kodak films, the average OD difference at 50 cGy between the three institutions is 17% as a result of differences in batch composition and 25% due to differences in processing conditions. For the CEA films these data are 6% and 24%, respectively. The long-term variation of the sensitometric curves of KODAK films in one institution was smaller than the differences in batch composition between the three institutions. The sensitometric curves of CEA films showed in one institution a large variation with time; the shape gradually varied from sigmoidal to quasilinear. By using relative OD values rather than absolute OD values, variations in sensitometric curves of KODAK films can be reduced to 2%. Consequently, one sensitometric curve is sufficient to derive relative dose values. If processing conditions are well controlled, it might therefore be advantageous to determine the absolute OD only at one or two dose values, in combination with a "universal" relative sensitometric curve.

Purpose: To reduce the dose in the rectal wall from prostate irradiation at high dose levels., Methods and Materials: Treatment plans in which the boost fields were integrated into the large fields (simultaneous integrated boost [SIB]) were compared with plans in which the large fields and boost fields were planned individually and applied in a sequential manner (sequential boost). Two target volumes were delineated: PTV1, the target volume of the large fields that is irradiated to 68 Gy, and PTV2, the target volume of the boost fields that is irradiated to 10 Gy. The sequential boost and the SIB were normalized to the mean dose in PTV2, being 78 Gy. We used a five-field intensity-modulated radiotherapy (IMRT) technique, applied in a step and shoot mode, and included beam weight optimization. A set of 5 patients with varying degree of overlap between PTV1 and the rectal wall was used for analysis., Results: The SIB resulted in a reduction of the dose in the rectal wall. Rectal normal tissue complication probability (NTCP) decreased for the SIB, on average, by a factor of almost 2, compared with the sequential boost., Conclusion: The SIB reduced the dose in the rectal wall, compared with the sequential boost technique.

Purpose: To assess for locoregional irradiation of breast cancer patients, the dependence of cardiac (cardiac mortality) and lung (radiation pneumonitis) complications on treatment technique and individual patient anatomy., Materials and Methods: Three-dimensional treatment planning was performed for 30 patients with left-sided breast cancer and various breast sizes. Two locoregional techniques (Techniques A and B) and a tangential field technique, including only the breast in the target volume, were planned and evaluated for each patient. In both locoregional techniques tangential photon fields were used to irradiate the breast. The internal mammary (IM)-medial supraclavicular (MS) lymph nodes were treated with an anterior mixed electron/photon field (Technique A) or with an obliquely incident mixed electron/photon IM field and an anterior electron/photon MS field (Technique B). The optimal IM and MS electron field dimensions and energies were chosen on the basis of the IM-MS lymph node target volume as delineated on CT-slices. The position of the tangential fields was adapted to match the IM-MS fields. Dose-volume histograms (DVHs) and normal tissue complication probabilities (NTCPs) for the heart and lung were compared for the three techniques. In the beam's eye view of the medial tangential fields the maximum distance of the heart contour to the posterior field border was measured; this value was scored as the Maximum Heart Distance., Results: The lymph node target volume receiving more than 85% of the prescribed dose was on average 99% for both locoregional irradiation techniques. The breast PTV receiving more than 95% of the prescribed dose was generally smaller using Technique A (mean: 90%, range: 69-99%) than using Technique B (mean: 98%, range: 82-100%) or for the tangential field technique (mean: 98%, range: 91-100%). NTCP values for excess cardiac mortality due to acute myocardial ischemia varied considerably between patients, with minimum and maximum values of 0.1 and 7.5% (Technique A), 0.1 and 5.8% (Technique B) and 0.0 and 6.1% (tangential tech.). The NTCP values were on average significantly higher (P<0.001) by 1.7% (Technique A) and 1.0% (Technique B) when locoregional breast irradiation was given, compared with irradiation of the left breast only. The NTCP values for the tangential field technique could be estimated using the Maximum Heart Distance. NTCP values for radiation pneumonitis were very low for all techniques; between 0.0 and 1.0%., Conclusions: Technique B results in a good coverage of the breast and locoregional lymph nodes, while Technique A sometimes results in an underdosage of part of the target volume. Both techniques result in a higher probability of heart complications compared with tangential irradiation of the breast only. Irradiation toxicity for the lung is low in all techniques. The Maximum Heart Distance is a simple and useful parameter to estimate the NTCP values for cardiac mortality for tangential breast irradiation.

It has been shown in vitro that the time-intensity data of echo contrast agents may be influenced by the background intensity of the myocardium and attenuation at high contrast agent concentrations. In the present study, these effects are evaluated from in vivo data. An effect of background intensity of the myocardium on the determination of the transit rate of the contrast agent could not be demonstrated unambiguously. A statistically significant relation between transit rate and background intensity was found only for intermediate flows in the transmural region. The magnitude of this relation was such that it does not provide a serious source of error. Attenuation and shadowing typically underestimate the transit rate of the contrast agent, which results in overestimation of flow. It is recommended that the lowest doses of contrast agent inducing myocardial opacification should be applied.

In the present study, cardiac contraction was regionally impaired to investigate the relationship between contractility [maximum first time derivative of left ventricular pressure (dPLV/dtmax)] and PLV on epicardial lymph pressure (Plymph) generation. Measurements were performed in open-chest anesthetized dogs under control conditions and while local contraction was abolished by intracoronary administration of lidocaine. Lidocaine significantly lowered dPLV/dtmax and PLV pulse to 77 +/- 9 (SD; n = 5) and 82 +/- 5% of control, respectively, whereas Plymph pulse increased to 186 +/- 101%. The relative increase of maximum Plymph to PLV related inversely to the change in dPLV/dtmax after lidocaine administration. Additional data were obtained when PLV was transiently increased by constriction of the descending aorta. The ratio of pulse Plymph to PLV during aortic clamping increased after lidocaine administration, from 0.063 +/- 0.03 to 0.15 +/- 0.09. The results suggest that transmission of PLV to the cardiac lymphatic vasculature is enhanced when regional contraction is impaired. These findings imply that during normal, unimpaired contraction lymph vessels are shielded from high systolic PLV by the myocardium itself.

Literature states that the inability of myocardial contrast echocardiography to assess differences in flow between regions could be related to myocardial shadowing. In this study, we evaluate this effect on the time-intensity curves. In a perfusion phantom, shadowing was induced by high concentrations of a contrast agent (approximately 1 x 10(8) bubbles/mL), and evaluated for different flows (50-270 mL/min). The high concentration resulted in an increase of the video-intensity in regions of interest close to the transducer (2 mm) and a marked reduction in remote areas (20 mm). The peak intensity of the time-intensity curves did not correlate with flow (range r = 0.1, 0.37), while the inverse area under the curve correlated strongly (r = 0.98). The inverse curve width and the decay after peak intensity also correlated excellently with flow (r = 0.99 and range r = 0.97, 0.99). We opt for the decay as transmural flow indicator using contrast echocardiography, since this parameter is least affected by shadowing.

Background subtraction for contrast echocardiography is considered a prerequisite for an appropriate assessment of flow from the time-intensity curves. We evaluated the influence of different background intensities on flow determinations with contrast echocardiography. Experiments were performed with a mixing chamber as a phantom for myocardial perfusion. Three regions of interest with different background intensities but equal flow were examined. Time-intensity curves were obtained and, after background subtraction, a number of curve parameters were determined: peak intensity, area under the curve, curve width and decay after peak intensity. The values of these parameters differed significantly between a region with a low and a region with a high background. In addition, a model is presented to describe these findings. It is concluded that subtraction of the background intensity from a time-intensity curve induces errors that make a quantitative interpretation of the curves ambiguous. These drawbacks are avoided if regions of interest with equal background intensities are compared.