Different endurance exercises modulate NK cell cytotoxic and inhibiting receptors.

PURPOSE: Induction of IDO depends on the activation of AhR forming the AhR/IDO axis. Activated AhR can transcribe various target genes including cytotoxic and inhibiting receptors of NK cells. We investigated whether AhR and IDO levels as well as activating (NKG2D) and inhibiting (KIR2DL1) NK cell receptors are influenced by acute exercise and different chronic endurance exercise programs. METHODS: 21 adult breast and prostate cancer patients of the TOP study (NCT02883699) were randomized to intervention programs of 12 weeks of (1) endurance standard training or (2) endurance polarized training after a cardiopulmonary exercise test (CPET). Serum was collected pre-CPET, immediately post-CPET, 1 h post-CPET and after 12 weeks post-intervention. Flow cytometry analysis was performed on autologous serum incubated NK-92 cells for: AhR, IDO, KIR2DL1 and NKG2D. Differences were investigated using analysis-of-variance for acute and analysis-of-covariance for chronic effects. RESULTS: Acute exercise: IDO levels changed over time with a significant increase from post-CPET to 1 h post-CPET (p = 0.03). KIR2DL1 levels significantly decreased over time (p < 0.01). NKG2D levels remained constant (p = 0.31). Chronic exercise: for both IDO and NKG2D a significant group × time interaction, a significant time effect and a significant difference after 12 weeks of intervention were observed (IDO: all p < 0.01, NKG2D: all p > 0.05). CONCLUSION: Both acute and chronic endurance training may regulate NK cell function via the AhR/IDO axis. This is clinically relevant, as exercise emerges to be a key player in immune regulation.

Prospective PED-study of intravitreal aflibercept for refractory vascularized pigment epithelium detachment due to age-related macular degeneration: morphologic characteristics of non-responders in optical coherence tomography.

PURPOSE: The aim of this study was to investigate the outcomes of a fixed intravitreal aflibercept regimen in patients with vascular pigment epithelium detachment (vPED) secondary to age-related macular degeneration with refractory subretinal fluid. METHODS: A prospective, interventional case series involved 20 eyes of 20 patients with refractory subretinal fluid and vPED treated with at least three injections of intravitreal anti-VEGF prior to study inclusion. After study inclusion, patients were treated with three injections of intravitreal aflibercept 2 mg/0.05 mL monthly followed by injections every 8 weeks. Best-corrected visual acuity (BCVA) and spectral-domain optical coherence tomography (SD-OCT) were evaluated at all visits. Fluorescein angiography and indocyanine green angiography were performed at baseline and quarterly. Primary outcomes were effectivity of a fixed treatment as measured in change in BCVA, PED greatest linear diameter (GLD), and PED height from baseline to month 12. In an additional post hoc analysis, vPED patients were differentiated into two groups: (1) vPED lesions that showed persistence of subretinal fluid throughout 1 year of treatment and (2) vPED lesions that showed complete resolution of subretinal fluid at least at one of the monthly performed OCT volume scans. Reflectivity values were determined in the subretinal pigment epithelium (RPE) compartment in OCT scans at baseline, month 6 and 12. RESULTS: A total of 18 patients completed the study protocol. The mean age was 74.8 ± 10.6 years, and six patients were female. The median BCVA of all patients was 72.0 ± 8.0 EDTRS letters at baseline and 72.5 ± 9.5 EDTRS letters at 12-month follow-up (p = 0.7420). The median PED height in all patients as measured in the OCT images significantly decreased from 372.0 ± 140.0 µm to 149.0 ± 142.0 µm after 12 months of treatment (p = 0.0020). Persistent subretinal fluid was present at every OCT control in six patients (group 1). Twelve patients showed resolution of subretinal fluid at least at one OCT control (group 2). Reflectivity values in the sub-RPE compartment in OCT scans were 41.48 ± 4.48 (group 1) and 42.62 ± 12.34 (group 2) at baseline (p = 0.854) and 65.88 ± 6.74 and 50.87 ± 14.11 at month 12 (p = 0.038). CONCLUSIONS: Intravitreal aflibercept in refractory vPED leads to a significant reduction in PED height and disease activity as well as preservation of BCVA over 1 year. Persistent subretinal fluid was present in PED lesions with high values of reflectivity under the RPE, suggesting both a diffusion barrier and an increasing fibrovascular maturization of the choroidal neovascularization. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03370380.

Resistance training as supportive measure in advanced cancer patients undergoing TKI therapy-a controlled feasibility trial.

PURPOSE: While there is growing evidence for positive effects of progressive resistance training in curatively treated cancer patients, data on advanced cancer patients are scarce. This pilot study aimed at investigating for the first time feasibility and effects of progressive resistance training in advanced cancer patients undergoing tyrosine kinase inhibitor (TKI) therapy. METHODS: Patients starting a TKI-based anti-tumor therapy were assigned to a resistance training group (RT, 12 weeks of progressive machine-based resistance training 2×/week) or a control group (CON, treatment as usual) until 10 patients had finished in each group (RT 80% males, 90% renal cell carcinoma, 65 ± 11 years, CON 80% males, 70% renal cell carcinoma, 61 ± 6 years). Primary endpoint was feasibility. Furthermore, fatigue (MFI), quality of life (QoL, EORTC QLQC30), and muscle strength were assessed. Testing occurred at baseline and after 12 weeks. RESULTS: Training was feasible in 9 out of 10 participants and no serious adverse events occurred. It had beneficial effects on muscle strength (maximum voluntary isometric contraction of the quadriceps: RT +11 ± 9 Nm, CON -13 ± 25 Nm, p = 0.005), but not on fatigue (general fatigue score RT +0.3 ± 4.1, CON -1.5 ± 3.0, p = 0.223) or QoL (global QoL score RT -5.6 ± 16.1, CON -2.0 ± 18.2, p = 0.617). CONCLUSIONS: Progressive machine-based resistance training appears feasible in the majority of advanced cancer patients undergoing TKI therapy. However, its positive effects on muscle strength do not seem to be associated with positive effects on fatigue or quality of life. Future studies should therefore compare whether home-based training is more beneficial for patient-reported outcomes. TRIAL REGISTRATION: NCT01645150.

Limited benefit of Fatmax-test to derive training prescriptions.

The intensity that elicits maximal fat oxidation (Fatmax) is recommended for training fat metabolism. However, it remains unclear whether Fatmax leads to the highest fat oxidation rates during prolonged exercise. It was hypothesized that there are no differences in fat oxidation rates among 3 different exercise intensities. Therefore, fat metabolism was compared among 1-h constant load tests at Fatmax, a higher and a lower intensity. A cohort of 16 male cyclists (28±6 yrs, BMI: 22.5±1.2 kg/m2; n=8 with maximal oxygen uptake [VO2max] of 50-60 ml/min/kg [ET]; n=8 with VO2max>60 ml/min/kg [HET]) completed a maximal incremental cycling test, a submaximal incremental Fatmax-test and, thereafter, three 1-h constant-load tests in randomized order at Fatmax, one exercise stage below (LOW) and one above (HIGH). LOW, Fatmax and HIGH were performed at 52±13, 60±13 and 70±12% VO2max. Heart rate and blood lactate were significantly different (p<0.001). However, the fat oxidation rate showed no difference (p=0.61). This was also true within each subgroup (ET: p=0.69, HET p=0.61). In conclusion, the fat oxidation rate of endurance trained cyclists shows no difference between 1-h constant load exercise bouts at about 50-70% VO2max. The precision and necessity of Fatmax-tests for controlling the training of fat oxidation are therefore debatable.

Blood lactate concentrations are mildly affected by mobile gas exchange measurements.

We sought to investigate the effects of wearing a mobile respiratory gas analysis system during a treadmill test on blood lactate (bLa) concentrations and commonly applied bLa thresholds. A total of 16 recreational athletes (31±3 years, VO2max: 58±6 ml · min(-1) · kg(-1)) performed one multistage treadmill test with and one without gas exchange measurements (GEM and noGEM). The whole bLa curve, the lactate threshold (LT), the individual anaerobic thresholds according to Stegmann (IATSt) and Dickhuth (IATDi), and a fixed bLa concentration of 4 mmol ∙ l(-1) (OBLA) were evaluated. The bLa curve was shifted slightly leftward in GEM compared to noGEM (P<0.05), whereas the heart rate response was not different between conditions (P=0.89). There was no difference between GEM and noGEM for LT (2.61±0.34 vs. 2.64±0.39 m · s(-1), P=0.49) and IATSt (3.47±0.42 vs. 3.55±0.47 m · s(-1), P=0.12). However, IATDi (3.57±0.39 vs. 3.66±0.44 m · s(-1), P<0.01) and OBLA (3.85±0.46 vs. 3.96±0.47 m · s(-1), P<0.01) occurred at slower running velocities in GEM. The bLa response to treadmill tests is mildly affected by wearing a mobile gas analysis system. This also applies to bLa thresholds located at higher exercise intensities. While the magnitude of the effects is of little importance for recreational athletes, it might be relevant for elite athletes and scientific studies.

Differences in adaptations to 1 year of aerobic endurance training: individual patterns of nonresponse.

Lacking responses to endurance training (ET) have been observed for several variables. However, detailed analyses of individuals' responses are scarce. To learn more about the variability of ET adaptations, patterns of response were analyzed for each subject in a 1-year ET study. Eighteen participants [42 ± 5 years, body mass index: 24 ± 3 kg/m(2), maximal oxygen uptake (VO(2max) ): 38 ± 5 mL/min/kg] completed a 1-year jogging/walking program on 3 days/week, 45 min/session at 60% heart rate (HR) reserve. VO(2max), resting HR (rHR), exercise HR (eHR) and individual anaerobic threshold (IAT) were determined by treadmill and cycling ergometry respectively. Intraindividual coefficients of variation were extracted from the literature to distinguish random changes from training responses. Eight participants showed improvements in all variables. In 10 participants, one or two variables did not improve (VO(2max), rHR, eHR and IAT remained unchanged in four, four, three and one cases, respectively). At least one variable improved in each subject. Data indicate that ET adaptations might be detected in each individual using multiple variables of different adaptation levels and intensity domains. Nonresponse seems to occur frequently and might affect all variables. Further studies should investigate whether nonresponders improve with altered training. Furthermore, associations between patterns of nonresponse and health benefits from ET are worth considering.

Effects of one year aerobic endurance training on resting metabolic rate and exercise fat oxidation in previously untrained men and women. Metabolic endurance training adaptations.

Although metabolic training adaptations are considered to be an important aim of recreational endurance exercise, effects of aerobic endurance training on metabolism have hardly been recorded over longer training periods. The aim of the study was therefore to record changes in resting metabolic rate (RMR), substrate oxidation at rest and maximal exercise fat oxidation rate (MFO) after one year of recreational endurance training within the ACSM-recommendations. Seventeen sedentary participants (7 male symbol/10 female symbol, 42+/-5 yr, pre-training characteristics: BMI: 24.6+/-2.2 kg.m (-2), VO(2max): 37.5+/-4.7 ml.min (-1).kg (-1)) completed a 12 months jogging/walking program 3 days/week for 45 min/session at a constant heart rate (HR) prescription of 60% HR-reserve. Resting measurements and maximal incremental treadmill tests were conducted before the training program, after 6 and 12 months of training. Indirect calorimetry was used to assess metabolic parameters. After 12 months of training, body weight remained unchanged ( P=0.16), however, body fat was significantly reduced by 3.4+/-2.1% ( P<0.001). Neither RMR ( P=0.42) nor substrate oxidation at rest ( P=0.25) changed significantly. MFO increased significantly over time by 0.07+/-0.08 g.min (-1) ( P<0.01) and occurred at significantly higher exercise intensities (35+/-6 vs. 44+/-15 vs. 50+/-14%VO(2max), P<0.01). In summary one year of recreational endurance training does therefore not appear to influence RMR or substrate oxidation at rest in previously untrained non-obese participants. In contrast, a constant training stimulus within the ACSM-recommendations elicits sustained improvements in MFO over at least one year of training.

The reliability of fat.

The exercise intensity eliciting maximal fat oxidation (Fat(max)) is typically determined during an incremental test. Its reproducibility, however, has not been thoroughly investigated so far. To address this issue, 21 healthy subjects (23.5+/-1.7 years; BMI 22.4+/-1.8 kg/m(2); VO(2peak) 47.4+/-11.3 mL/min/kg) carried out two identical cycling tests to determine Fat(max) after an initial incremental baseline test. The duration of each of five stages during the Fat(max) tests was 6 min. The first stage equalled the first increase in blood lactate during the baseline test; the highest stage corresponded to a respiratory exchange ratio of 1.00. Between these intensities the other three stages were distributed evenly. Fat(max) was 28.0+/-8.6 L/min (59.2+/-18.1% VO(2peak)) in the first test and 29.8+/-10.5 L/min (62.9+/-22.2% VO(2peak)) in the second one. There was no significant difference between both Fat(max) determinations [number of stage: P=0.31; total VO(2): P=0.20; VO(2) utilized for fat oxidation (VO(2Fat)): P=0.33]. Linear correlation coefficients between tests were r=0.84 (total VO(2); P<0.001) and r=0.83 (VO(2Fat); P<0.001). However, Bland-Altman plots revealed wide 95% limits of agreement of 0.91 L/min (total VO(2)) and 0.32 L/min (VO(2Fat)). In conclusion, spontaneous intraindividual variability in Fat(max) appears too large to recommend the use of this parameter for the prescription of training intensity.

Effects of microheterogeneity in hen egg-white lysozyme crystallization.

In earlier sodium dodecylsulfate-polyacylamide gel electrophoresis (SDS-PAGE) studies it has been found that commonly utilized commercial hen egg-white lysozyme (HEWL) preparations contained 0.2-0.4 mol% covalently bound dimers. Here it is shown, using high-performance capillary electrophoresis (HPCE), that HEWL contains, in addition, two differently charged monomers in comparable amounts. To explore the origin of these microheterogeneous contaminants, purified HEWL (PHEWL) has been oxidized with hydrogen peroxide (0.0026-0.88 M) at various pH levels between 4.5 and 12.0. Optical densitometry of oxidized PHEWL (OHEWL) bands in SDS-PAGE gels shows that hydrogen peroxide at 0.88 M in acetate buffer pH 4.5 increased the amount of dimers about sixfold over that in commercial HEWL. OHEWL had, in addition to one of the two monomer forms found in HEWL and PHEWL, three other differently charged monomer forms, each of them representing about 25% of the preparation. SDS-PAGE analysis of OHEWL yielded two closely spaced dimer bands with Mr = 28000 and 27500. In addition, larger HEWL oligomers with Mr = 1.7 million and 320000 were detected by gel-filtration fast protein liquid chromatography with multiangle laser light scattering detection. Non-dissociating PAGE in large pore size gels at pH 4.5 confirmed the presence of these large oligomers in HEWL and OHEWL. Increased microheterogeneity resulted in substantial effects on crystal growth and nucleation rate. On addition of 10 microgram-1 mg ml-1 OHEWL to 32 mg ml-1 HEWL crystallizing solutions, both the number and size of forming crystals decreased roughly proportionally to the concentration of the added microheterogeneity. The same effect was observed in HEWL solutions on addition of 0.03-0.3 M hydrogen peroxide. Repartitioning of the dimer during crystallization at various temperatures between 277 and 293 K was analyzed by SDS-PAGE. The crystals contained

A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts.

Recognition of antigen by T cells requires the formation of a specialized junction between the T cell and the antigen-presenting cell. This junction is generated by the recruitment and the exclusion of specific proteins from the contact area. The mechanisms that regulate these events are unknown. Here we demonstrate that ligand engagement of the adhesion molecule, CD2, initiates a process of protein segregation, CD2 clustering, and cytoskeletal polarization. Although protein segregation was not dependent on the cytoplasmic domain of CD2, CD2 clustering and cytoskeletal polarization required an interaction of the CD2 cytoplasmic domain with a novel SH3-containing protein. This novel protein, called CD2AP, is likely to facilitate receptor patterning in the contact area by linking specific adhesion receptors to the cytoskeleton.

X-ray topography of tetragonal lysozyme grown by the temperature-controlled technique.

Growth-induced defects in lysozyme crystals were observed by white-beam and monochromatic X-ray topography at the National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory (BNL). The topographic methods were non-destructive to the extent that traditional diffraction data collection could be performed to high resolution after topography. It was found that changes in growth parameters, defect concentration as detected by X-ray topography, and the diffraction quality obtainable from the crystals were all strongly correlated. In addition, crystals with fewer defects showed lower mosaicity and higher diffraction resolution as expected.

Real-time 3D dose calculation and display: a tool for plan optimization.

PURPOSE: Both human and computer optimization of treatment plans have advantages; humans are much better at global pattern recognition, and computers are much better at detailed calculations. A major impediment to human optimization of treatment plans by manipulation of beam parameters is the long time required for feedback to the operator on the effectiveness of a change in beam parameters. Our goal was to create a real-time dose calculation and display system that provides the planner with immediate (fraction of a second) feedback with displays of three-dimensional (3D) isodose surfaces, digitally reconstructed radiographs (DRRs), dose-volume histograms, and/or a figure of merit (FOM) (i.e., a single value plan score function). This will allow the experienced treatment planner to optimize a plan by adjusting beam parameters based on a direct indication of plan effectiveness, the FOM value, and to use 3D display of target, critical organs, DRRs, and isodose contours to guide changes aimed at improving the FOM value. METHODS AND MATERIALS: We use computer platforms that contain easily utilized parallel processors and very tight coupling between calculation and display. We ported code running on a network of two workstations and an array of transputers to a single multiprocessor workstation. Our current high-performance graphics workstation contains four 150-MHz processors that can be readily used in a shared-memory multithreaded calculation. RESULTS: When a 10 x 10-cm beam is moved, using an 8-mm dose grid, the full 3D dose matrix is recalculated using a Bentley-Milan-type dose calculation algorithm, and the 3D dose surface display is then updated, all in < 0.1s. A 64 x 64-pixel DRR calculation can be performed in < 0.1 s. Other features, such as automated aperture calculation, are still required to make real-time feedback practical for clinical use. CONCLUSION: We demonstrate that real-time plan optimization using general purpose multiprocessor workstations is a practical goal. Parallel processing technology provides this capability for 3D planning systems, and when combined with objective plan ranking algorithms should prove effective for optimizing 3D conformal radiation therapy. Compared to our earlier transputer work, multiprocessor workstations are more easily programmed, making software development costs more reasonable compared with uniprocessor development costs. How the dose calculation is partitioned into parallel tasks on a multiprocessor work station can make a significant difference in performance. Shared-memory multiprocessor workstations are our first choice for future work, because they require minimum programming effort and continue to be driven to higher performance by competition in the workstation arena.

Heterogeneity determination and purification of commercial hen egg-white lysozyme.

Hen egg-white lysozyme (HEWL) is widely used as a model protein, although its purity has not been adequately characterized by modern biochemical techniques. We have identified and quantified the protein heterogeneities in three commercial HEWL preparations by sodium dodecyl sulfate polyacrylamide gel electrophoresis with enhanced silver staining, reversed-phase fast protein liquid chromatography (FPLC) and immunoblotting with comparison to authentic protein standards. Depending on the source, the contaminating proteins totalled 1-6%(w/w) and consisted of ovotransferrin, ovalbumin, HEWL dimers, and polypeptides with approximate M(r) of 39 and 18 kDa. Furthermore, we have obtained gram quantities of electrophoretically homogeneous [> 99.9%(w/w)] HEWL by single-step semi-preparative scale cation-exchange FPLC with a yield of about 50%. Parallel studies of crystal growth kinetics, salt repartitioning and crystal perfection with this highly purified material showed fourfold increases in the growth-step velocities and significant enhancement in the structural homogeneity of HEWL crystals.

Repartitioning of NaCl and protein impurities in lysozyme crystallization.

Nonuniform precipitant and impurity incorporation in protein crystals can cause lattice strain and, thus, possibly decrease the X-ray diffraction resolution. To address this issue, a series of crystallization experiments were carried out, in which initial supersaturation, NaCl concentration, protein purity level and crystallized fraction were varied. Lysozyme and protein impurities, as well as sodium and chloride were independently determined in the initial solution, supernatant and crystals. The segregation coefficients for Na(+) and Cl(-) were found to be independent of supersaturation and NaCl concentration, and decreased with crystallized fraction/crystal size. Numerical evaluation of the extensive body of data, based on a nucleation-growth-repartitioning model, suggests a core of approximately 40 micro m in which salt is incorporated in much greater concentrations than during later growth. Small crystals containing higher amounts of incorporated NaCl also had higher protein impurity contents. This suggests that the excess salt is associated with the protein impurities in the core. X-ray topography revealed strain fields in the center of the crystals comparable in size to the inferred core. The growth rates of crystals smaller than 30-40 micro m in size were consistently 1.5-2 times lower than those of larger crystals, presumably due to higher chemical potentials in the core.

Advances in 3-dimensional radiation treatment planning systems: room-view display with real time interactivity.

PURPOSE: We describe our 3-dimensional (3-D) radiation treatment planning system for external photon and electron beam 3-D treatment planning which provides high performance computational speed and a real-time display which we have named "room-view" in which the simulated target volumes, critical structures, skin surfaces, radiation beams and/or dose surfaces can be viewed on the display monitor from any arbitrary viewing position. METHODS AND MATERIALS: We have implemented the 3-D planning system on a graphics superworkstation with parallel processing. Patient's anatomical features are extracted from contiguous computed tomography scan images and are displayed as wireloops or solid surfaces. Radiation beams are displayed as a set of diverging rays plus the polygons formed by the intersection of these rays with planes perpendicular to the beam axis. Controls are provided for each treatment machine motion function. Photon dose calculations are performed using an effective pathlength algorithm modified to accommodate 3-D off-center ratios. Electron dose calculations are performed using a 3-D pencil beam model. RESULTS: Dose distribution information can be displayed as 3-D dose surfaces, dose-volume histograms, or as isodoses superimposed on 2-D gray scale images of the patient's anatomy. Tumor-control-probabilities, normal-tissue-complication probabilities and a figure-of-merit score function are generated to aid in plan evaluation. A split-screen display provides a beam's-eye-view for beam positioning and design of patient shielding block apertures and a concurrent "room-view" display of the patient and beam icon for viewing multiple beam set-ups, beam positioning, and plan evaluation. Both views are simultaneously interactive. CONCLUSION: The development of an interactive 3-D radiation treatment planning system with a real-time room-view display has been accomplished. The concurrent real-time beam's-eye-view and room-view display significantly improves the efficacy of the 3-D planning process.