Increasing the Robustness of Deep Learning Models for Object Segmentation: A Framework for Blending Automatically Annotated Real and Synthetic Data.

Recent problems in robotics can sometimes only be tackled using machine learning technologies, particularly those that utilize deep learning (DL) with transfer learning. Transfer learning takes advantage of pretrained models, which are later fine-tuned using smaller task-specific datasets. The fine-tuned models must be robust against changes in environmental factors such as illumination since, often, there is no guarantee for them to be constant. Although synthetic data for pretraining has been shown to enhance DL model generalization, there is limited research on its application for fine-tuning. One limiting factor is that the generation and annotation of synthetic datasets can be cumbersome and impractical for the purpose of fine-tuning. To address this issue, we propose two methods for automatically generating annotated image datasets for object segmentation, one for real-world and another for synthetic images. We also introduce a novel domain adaptation approach called filling the reality gap (FTRG), which can blend elements from real-world and synthetic scenes in a single image to achieve domain adaptation. We demonstrate through experimentation on a representative robot application that FTRG outperforms other domain adaptation techniques, such as domain randomization or photorealistic synthetic images, in creating robust models. Furthermore, we evaluate the benefits of using synthetic data for fine-tuning in transfer learning and continual learning with experience replay using our proposed methods and FTRG. Our findings indicate that fine-tuning with synthetic data can produce superior results compared to solely using real-world data.

Towards robotic laboratory automation Plug & play: Survey and concept proposal on teaching-free robot integration with the lapp digital twin.

The Laboratory Automation Plug & Play (LAPP) framework is an over-arching reference architecture concept for the integration of robots in life science laboratories. The plug & play nature lies in the fact that manual configuration is not required, including the teaching of the robots. In this paper a digital twin (DT) based concept is proposed that outlines the types of information that must be provided for each relevant component of the system. In particular, for the devices interfacing with the robot, the robot positions must be defined beforehand in a device-attached coordinate system (CS) by the vendor. This CS must be detectable by the vision system of the robot by means of optical markers placed on the front side of the device. With that, the robot is capable of tending the machine by performing the pick-and-place type transportation of standard sample carriers. This basic use case is the primary scope of the LAPP-DT framework. The hardware scope is limited to simple benchtop and mobile manipulators with parallel grippers at this stage. This paper first provides an overview of relevant literature and state-of-the-art solutions, after which it outlines the framework on the conceptual level, followed by the specification of the relevant DT parameters for the robot, for the devices and for the facility. Finally, appropriate technologies and strategies are identified for the implementation.

Computationally Relaxed Unscented Kalman Filter.

Advanced robotics and autonomous vehicles rely on filtering and sensor fusion techniques to a large extent. These mobile applications need to handle the computations onboard at high rates while the computing capacities are limited. Therefore, any improvement that lowers the CPU time of the filtering leads to more accurate control or longer battery operation. This article introduces a generic computational relaxation for the unscented transformation (UT) that is the key operation of the Unscented Kalman filter-based applications. The central idea behind the relaxation is to pull out the linear part of the filtering model and avoid the calculations for the kernel of the nonlinear part. The practical merit of the proposed relaxation is demonstrated through a simultaneous localization and mapping (SLAM) implementation that underpins the superior performance of the algorithm in the practically relevant cases, where the nonlinear dependencies influence only an affine subspace of the image space. The numerical examples show that the computational demand can be mitigated below 50% without decreasing the accuracy of the approximation. The method described in this article is implemented and published as an open-source C++ library RelaxedUnscentedTransformation on GitHub.

PlatypOUs-A Mobile Robot Platform and Demonstration Tool Supporting STEM Education.

Given the rising popularity of robotics, student-driven robot development projects are playing a key role in attracting more people towards engineering and science studies. This article presents the early development process of an open-source mobile robot platform-named PlatypOUs-which can be remotely controlled via an electromyography (EMG) appliance using the MindRove brain-computer interface (BCI) headset as a sensor for the purpose of signal acquisition. The gathered bio-signals are classified by a Support Vector Machine (SVM) whose results are translated into motion commands for the mobile platform. Along with the physical mobile robot platform, a virtual environment was implemented using Gazebo (an open-source 3D robotic simulator) inside the Robot Operating System (ROS) framework, which has the same capabilities as the real-world device. This can be used for development and test purposes. The main goal of the PlatypOUs project is to create a tool for STEM education and extracurricular activities, particularly laboratory practices and demonstrations. With the physical robot, the aim is to improve awareness of STEM outside and beyond the scope of regular education programmes. It implies several disciplines, including system design, control engineering, mobile robotics and machine learning with several application aspects in each. Using the PlatypOUs platform and the simulator provides students and self-learners with a firsthand exercise, and teaches them to deal with complex engineering problems in a professional, yet intriguing way.

Towards robotic laboratory automation Plug & Play: The "LAPP" framework.

Increasing the level of automation in pharmaceutical laboratories and production facilities plays a crucial role in delivering medicine to patients. However, the particular requirements of this field make it challenging to adapt cutting-edge technologies present in other industries. This article provides an overview of relevant approaches and how they can be utilized in the pharmaceutical industry, especially in development laboratories. Recent advancements include the application of flexible mobile manipulators capable of handling complex tasks. However, integrating devices from many different vendors into an end-to-end automation system is complicated due to the diversity of interfaces. Therefore, various approaches for standardization are considered in this article, and a concept is proposed for taking them a step further. This concept enables a mobile manipulator with a vision system to "learn" the pose of each device and - utilizing a barcode - fetch interface information from a universal cloud database. This information includes control and communication protocol definitions and a representation of robot actions needed to operate the device. In order to define the movements in relation to the device, devices have to feature - besides the barcode - a fiducial marker as standard. The concept will be elaborated following appropriate research activities in follow-up papers.

Refractive outcomes of femtosecond laser-assisted secondary arcuate incisions in patients with residual refractive astigmatism after trifocal intraocular lens implantations.

PURPOSE: To evaluate the refractive and visual outcomes of arcuate incisions performed with the femtosecond laser in patients with a residual refractive astigmatism after refractive lens exchange (RLE) with trifocal intraocular lenses (IOLs). SETTING: EuroEyes Clinical Group, Hamburg, Germany. DESIGN: Retrospective interventional case series. METHODS: Pseudophakic patients with remaining refractive astigmatism after RLE with a trifocal IOL were treated with femtosecond laser-assisted corneal arcuate incisions. Patients who had a previous corneal treatment were excluded. Outcome measures were uncorrected (UDVA) and corrected distance visual acuities, manifest refraction, and a power vector analysis. RESULTS: The study enrolled 95 eyes of 70 patients. The mean follow-up was 5.6 months ± 4.9 (SD). Constructing an astigmatic power vector (APV) with Jackson cross-cylinder axes at 180 degrees and 90 degrees and Jackson cross-cylinder axes at 45 degrees and 135 degrees, the mean preoperative vector length was 0.46 ± 0.16 diopter (D). The mean postoperative APV was 0.17 ± 0.16 D. This difference was statistically significant (P < .001). The difference between the UDVA preoperatively (0.17 ± 0.15) and postoperatively (0.08 ± 0.10) was statistically significant (P < .001). No intraoperative or postoperative complications were observed. CONCLUSION: Femtosecond laser-assisted corneal arcuate incisions were safe, efficient, and feasible to reduce refractive astigmatism after trifocal IOL implantation.

[Tools for laparoscopic skill development - available trainers and simulators]. / A laparoszkópos készségfejlesztés eszközei ­ elérheto trénerek és szimulátorok.

The laparoscopic minimally invasive surgical technique is widely employed on a global scale. However, the efficient and ethical teaching of this technique requires equipment for surgical simulation. These educational devices are present on the market in the form of box trainers and virtual reality simulators, or some combination of those. In this article, we present a systematic overview of commercially available surgical simulators describing the most important features of each product. Our overview elaborates on box trainers and virtual reality simulators, and also touches on surgical robotics simulators, together with operating room workflow simulators, for the sake of completeness. Apart from presenting educational tools, we evaluated the literature of laparoscopic surgical education and simulation, to provide a complete picture of the unfolding trends in this field. Orv Hetil. 2017; 158(40): 1570-1576.

Temporal dynamics of object location processing in allocentric reference frame.

The spatial location of objects is processed in egocentric and allocentric reference frames, the early temporal dynamics of which have remained relatively unexplored. Previous experiments focused on ERP components related only to egocentric navigation. Thus, we designed a virtual reality experiment to see whether allocentric reference frame-related ERP modulations can also be registered. Participants collected reward objects at the end of the west and east alleys of a cross maze, and their ERPs to the feedback objects were measured. Participants made turn choices from either the south or the north alley randomly in each trial. In this way, we were able to discern place and response coding of object location. Behavioral results indicated a strong preference for using the allocentric reference frame and a preference for choosing the rewarded place in the next trial, suggesting that participants developed probabilistic expectations between places and rewards. We also found that the amplitude of the P1 was sensitive to the allocentric place of the reward object, independent of its value. We did not find evidence for egocentric response learning. These results show that early ERPs are sensitive to the location of objects during navigation in an allocentric reference frame.

LASIK versus photorefractive keratectomy for high myopic (> 3 diopter) astigmatism.

PURPOSE: To compare the efficacy, safety, predictability, and vector analysis indices of LASIK and photorefractive keratectomy (PRK) for correction of high cylinder of greater than 3 diopters (D) in myopic eyes. METHODS: The efficacy, safety, and predictability of LASIK or PRK performed in 114 consecutive randomly selected myopic eyes with an astigmatism of greater than 3 D were retrospectively analyzed at the 2- to 6-month follow-up visits. Vector analysis of the cylindrical correction was compared between the treatment groups. RESULTS: A total of 57 eyes receiving PRK and 57 eyes receiving LASIK of 114 refractive surgery candidates were enrolled in the study. No statistically significant difference in efficacy [efficacy index = 0.76 (±0.32) for PRK vs 0.74 (±0.19) for LASIK (P = .82)], safety [safety index = 1.10 (±0.26) for PRK vs 1.01 (±0.17) for LASIK (P = .121)], or predictability [achieved astigmatism < 1 D in 39% of PRK- and 54% of LASIK-treated eyes, and < 2 D in 88% of PRK- and 89% of LASIK-treated eyes (P = .218)] was demonstrated. Using Alpins vector analysis, the surgically induced astigmatism and difference vector were not significantly different between the surgery methods, whereas the correction index showed a slight and significant advantage of LASIK over PRK (1.25 for PRK and 1.06 for LASIK, P < .001). CONCLUSIONS: LASIK and PRK are comparably safe, effective, and predictable procedures for excimer laser correction of high astigmatism of greater than 3 D in myopic eyes. Predictability of the correction of the cylindrical component is lower than that of the spherical equivalent.

Expression of CD133 and other putative stem cell markers in uveal melanoma.

'Cancer stem cells' (CSCs) are tumor cells with stem cell properties hypothesized to be responsible for tumorigenesis, metastatis, and resistance to treatment, and have been identified in different tumors including cutaneous melanoma, using stem cell markers such as CD133. This study explored expression of CD133 and other putative stem cell markers in uveal melanoma. Eight uveal melanoma cell lines were subjected to flow-cytometric (fluorescence-activated cell sorting) analysis of CD133 and other stem cell markers. Eight paraffin-embedded tumors were analyzed by immunohistochemistry for CD133, Pax6, Musashi, nestin, Sox2, ABCB5, and CD68 expressions. Ocular, uveal melanoma, and hematopoietic stem cell distributions of C-terminal and N-terminal CD133 mRNA splice variants were compared by reverse-transcription PCR. Fluorescence-activated cell sorting analysis revealed a population of CD133-positive/nestin-positive cells in cell lines Mel270, OMM 2.3, and OMM2.5. All cell lines studied were positive for nestin, CXCR-4, CD44, and c-kit. Immunohistochemistry identified cells positive for CD133, Pax6, Musashi, nestin, Sox2, ABCB5, and CD68 predominantly at the invading tumor front. C-terminal primers interacting with CD133 splice variant s2 detected a novel variant lacking exon 27. Differential expression of CD133 splice variants was found in iris, ciliary body, retina, and retinal pigment epithelium/choroid as well as in uveal melanoma cell lines. mRNA for nestin, Sox2, and Musashi was present in all studied cell lines. Uveal melanoma such as cutaneous melanoma may therefore contain CSCs. Further experiments are needed to isolate stem cell marker-positive cells, to evaluate their functional properties and to explore therapeutical approaches to these putative CSCs in uveal melanoma.

Radial optic neurotomy in central retinal vein occlusion does not influence ocular hemodynamics.

PURPOSE: radial optic neurotomy (RON) is used for the treatment of central retinal vein occlusion. Its effects on visual acuity differ substantially between patients. Our study aims to evaluate if RON has an impact on ocular microcirculation and if analysis of ocular microcirculation might serve as a predictor for surgical success. PROCEDURES: a complete ophthalmologic examination including color Doppler imaging of the retrobulbar vessels was performed before and 2-4 months after RON in 12 patients. RESULTS: mean visual acuity was 0.09 ± 0.03 prior to and 0.24 ± 0.12 after RON. Visual acuity improved in 7 (+3.5 ± 0.9 lines), was stable in 3 (± 0 lines) and worsened in 2 cases (-6 and -2 lines). Doppler parameters were not affected by RON, and no correlations between visual acuity and perfusion parameters were found. CONCLUSIONS: Our data indicate that RON does not influence ocular microcirculation. None of the assessed hemodynamic parameters appears to be a predictor for surgical success.

A case of bilateral lacrimal fistula associated with Down syndrome.

INTRODUCTION: Congenital lacrimal fistulae are a developmental disorder and can be associated with other hereditary developmental diseases. CASE REPORT: A patient with Down syndrome presented with events of recurrent dacryocystitis and lacrimal fistulation on 1 eye. A DCR surgery with excision of a fistula had been performed 2 years before. The investigation under general anesthesia also revealed a clinically unrecognized lacrimal fistula on the contralateral side. COMMENT: The case report documents a unique case of bilateral congenital lacrimal fistulae in association with Down syndrome. Dacryocystitis in patients with Down syndrome could result from a congenital lacrimal fistula.

Neuronal activity influences hemodynamics in the paraoptic short posterior ciliary arteries: a comparison between healthy and glaucomatous subjects.

PURPOSE: Perfusion of the retina adapts to metabolic demand by neurovascular coupling. The present study was an investigation of the presence of neurovascular coupling in the anterior part of the optic nerve in healthy subjects and patients with glaucoma. METHODS: Retrobulbar blood flow velocities were determined by color Doppler imaging (CDI). Peak systolic and end diastolic velocities (PSVs and EDVs) in the central retinal artery (CRA) or the short posterior ciliary artery (SPCA) were the primary readout. CDI measurements were performed shortly before, during, immediately after, 60 seconds after, and 120 seconds after a 10-Hz flicker stimulation of the retina. RESULTS: Thirty-five patients with glaucoma and 44 healthy control subjects were included in the study. In the SPCA of healthy control subjects, flicker stimulation led to an increase in PSV from 9.7 +/- 0.8 to 12.5 +/- 0.8 cm/s (P < 0.001; n = 24) and of EDV from 2.4 +/- 0.3 to 3.6 +/- 0.3 cm/s (P < 0.001; n = 24). This effect was not detectable in patients with glaucoma. In the CRA, flicker light led to an increase in EDV from 2.1 +/- 0.2 to 3.0 +/- 0.3 cm/s (P = 0.002; n = 20) in healthy volunteers and from 1.3 +/- 0.2 to 2.0 +/- 0.2 cm/s in patients with glaucoma (P = 0.004; n = 15). PSV was not affected by flicker stimulation in either the healthy volunteers or patients with glaucoma. CONCLUSIONS: The data indicate the presence of neurovascular coupling in the vascular bed supplied by the paraoptic SPCA. The response pattern to the flicker stimulus differs between healthy subjects and individuals with glaucoma.

Protective effects of nebivolol on oxygen free radical-induced vasoconstrictions in vitro.

BACKGROUND: Disturbed ocular hemodynamics and vasospasms might be involved in the pathogenesis of glaucoma. On a clinical level there are indications for an optimization of ocular perfusion parameters in hypertensive glaucoma patients by switching a beta-adrenoceptor-antagonist therapy to nebivolol. Aim of the present study is to investigate vasoactive properties of nebivolol on ocular vasculature in vitro. Besides vasorelaxing effects, the impact of nebivolol on oxygen free radical-induced vasoconstrictions is studied. MATERIAL/METHODS: The experiments were carried out with ring preparations from porcine ciliary arteries. The preparations were placed in a myograph system and were kept under physiological conditions (pH 7.4, 37 degrees C, Krebs-Henseleit-Buffer, 1.75 mM Ca2+) and were stimulated by K+ depolarizations. The experiments were performed at a Nernst potential of -41 mV, which reflects half-maximal activation. For radical exposure, the preparations were superfused for 20 s in a specifically designed set-up with hydroxyl radicals generated by the Fenton reaction from H2O2 and Fe3+. NO synthase activity was modulated by adding L-arginine to the buffer. RESULTS: At a concentration of 10-5 M nebivolol leads to a reduction of vascular tone by -8.5+/-3.4% (n=11; P=0.016) vs. +2.6+/-1.9% (n=11; n.s.) in presence of its solvent DMSO. Nebivolol (10-5 M) reduces hydroxyl radical-induced vasoconstrictions by 53+/-10% (n=11; P<0.001). Stimulation of the NO synthase by L-arginine saturation potentiates this effect. CONCLUSIONS: Nebivolol combines vasorelaxing properties with protection against oxidative stress-induced vasoconstrictions. Both effects may be attributed to NO-releasing properties of nebivolol independently of its beta-adrenoceptor-blocking effect.

Effects of the systemic beta-adrenoceptor antagonist nebivolol on ocular hemodynamics in glaucoma patients.

BACKGROUND: Systemic antihypertensive treatment in glaucoma patients with hypertension carries the potential risk of an additional deterioration in ocular hemodynamics due to the reduction in ocular perfusion pressure. Nebivolol is a beta1-selective adrenoceptor antagonist with known peripheral vasodilatory effects due to NO-releasing properties. The effect of a switch in systemic beta-blocker treatment to nebivolol on retrobulbar hemodynamics in glaucoma patients with arterial hypertension was therefore investigated. MATERIAL/METHODS: Peak systolic (PSV) and end-diastolic (EDV) velocity in the short and long posterior ciliary arteries (SPCA, LPCA), central retinal artery (CRA), and ophthalmic artery (OA) were recorded by color Doppler imaging (CDI) in 23 glaucoma patients with arterial hypertension using their primary systemic beta-blocker medication and four weeks after a switch to nebivolol. RESULTS: Compared with the first recording under the primary antihypertensive medication, the CDI measurements after four weeks of nebivolol treatment revealed a significant acceleration of the PSV in the SPCA and LPCA and the EDV in the SPCA and CRA. No significant differences in flow velocities were found for the OA. Intraocular pressure and systemic blood pressure remained unchanged. CONCLUSIONS: Switching blood pressure treatment to nebivolol in glaucoma patients with hypertension leads to accelerated blood flow in the small retrobulbar vessels. A stabilization of ocular perfusion might be of particular importance in this group of co-morbid patients.

Influence of oxygen free radicals on the tone of ciliary arteries: a model of vasospasms of ocular vasculature.

BACKGROUND: Altered regulation of vascular tone and particularly vasospasms are thought to be a risk factor for the progression of primary open angle glaucoma (POAG). Apoptosis of retinal ganglion cells and possibly vascular tone regulation in glaucoma might be caused by oxidative stress. The aim of the present study was to investigate the influence of oxygen free radicals on the tone of ciliary arteries. METHODS: The experiments were carried out with fresh ring preparations from porcine ciliary arteries obtained from a slaughterhouse. The preparations were placed in a self-designed myograph system and were kept under physiologic conditions (pH 7.4, 37 degrees C, Krebs-Henseleit-Buffer, 1.75 mM Ca(2+)). The muscles were sub-maximally activated by depolarization to -41 mV Nernst potential for K(+). The pre-activated preparations were exposed to hydroxyl radicals generated by the Fenton reaction (4 mM H(2)O(2); 30 microM Fe(3+)). Exposure time varied between 10 s and 60 s in order to obtain different radical-time-doses. The developed force was evaluated relatively to the developed force at maximal depolarization to -4 mV. RESULTS: Ten seconds of radical exposure result in an additional increase of the relative developed force from 0.35 +/- .08 to 0.62 +/- 0.12 (P = 0.003; n = 8). Comparable results were obtained for 20 s and 60 s radical exposures. The developed force of a maximal activation to -4 mV was not reduced after a 10 s radical exposure (0.84 +/- 0.13; P = 0.25; n = 5), but was significantly reduced after 20 s exposure (0.25 +/- 0.21; P = 0.005; n = 6) and was virtually 0 after 60 s exposure. DISCUSSION: The data shows that oxygen free radicals induce transient contractions of isolated ciliary artery rings. The shape of these contractions shows parallels to vasospasms. Thus the established system may serve as an in vitro model of vasospasms.

Compromised autoregulatory control of ocular hemodynamics in glaucoma patients after postural change.

PURPOSE: The autoregulatory control of retrobulbar blood flow in response to postural challenge was investigated in normal-tension glaucoma (NTG) patients in comparison with primary open-angle glaucoma (POAG) patients and healthy volunteers. DESIGN: Prospective cohort study. PARTICIPANTS AND CONTROLS: Twenty POAG patients, 20 NTG patients, and 20 control subjects. METHODS: Peak systolic velocity (PSV), end diastolic velocity (EDV), and resistivity index (RI) in the short posterior ciliary artery (SPCA), central retinal artery (CRA) and ophthalmic artery (OA) were recorded after a change from sitting upright to a supine body position using color Doppler imaging. MAIN OUTCOME MEASURES: Peak systolic velocity, EDV, and RI. RESULTS: Ten minutes after postural change to a supine position, blood flow velocities in the SPCA remained unchanged in controls, whereas a significant increase of PSV and EDV was found in both glaucoma groups. The RI in the SPCA was significantly lowered in the NTG group. Recordings for the OA and CRA showed a significant increase in EDV and significant decrease in RI in all 3 groups; a significant increase in PSV in the CRA was detected only in the NTG group. CONCLUSIONS: The unaltered flow velocities in the SPCA of healthy controls may indicate tight autoregulatory control, whereas the flow velocities in the CRA and OA appeared to follow alterations in hydrostatic pressure. In contrast, NTG and POAG patients demonstrated an insufficient compensatory response to postural change, leading to accelerated flow in the SPCA. This compromised autoregulatory control could represent another contributing factor in the pathogenesis of glaucoma.

Effects of glaucoma drugs on ocular hemodynamics in normal tension glaucoma: a randomized trial comparing bimatoprost and latanoprost with dorzolamide [ISRCTN18873428].

BACKGROUND: Reduced choroidal perfusion is hypothesized to play a role in the pathogenesis of normal tension glaucoma. Thus the impact of antiglaucomatous eye drops on ocular perfusion has been the focus of recent research and the subject of intensive investigations. The present study investigates whether topically applied latanoprost or bimatoprost influence ocular perfusion in patients with normal tension glaucoma and compares these effects with that changes detected after the treatment with dorzolamide. METHODS: Ocular hemodynamics were assessed by color Doppler imaging (CDI) shortly before and after a one-month treatment with either latanoprost, bimatoprost or dorzolamide. Primary end-points of the study were peak systolic and end-diastolic blood flow velocities in the short posterior ciliary artery (SPCA) under the new therapy. Intraocular pressure (IOP) and additional perfusion parameters in the SPCA and other retrobulbar vessels were tracked as observational parameters. n = 42 patients with normal tension glaucoma were enrolled in the study. RESULTS: Systolic and diastolic blood flow velocities in the SPCA showed no significant alteration after the treatment with latanoprost or bimatoprost. Dorzolamide lead to increase of peak systolic velocity. IOP was reduced by all three agents in a range reported in the literature. CONCLUSION: Topically applied latanoprost and bimatoprost act in a hemodynamically neutral manner and have the capability to lower IOP even in patients with normal tension glaucoma and low initial IOP level. Dorzolamide accelerates blood flow in systole. None of the tested compounds has a negative impact on hemodynamics in the short posterior ciliary arteries.

Apoptotic pancreatic tumor cells are superior to cell lysates in promoting cross-priming of cytotoxic T cells and activate NK and gammadelta T cells.

Tumor vaccines using dendritic cells (DCs) have been shown to induce antitumor CTL responses. The choice of the tumor antigen preparation used for DC loading is still an unresolved issue. We compared DCs pulsed with cell lysates, whole apoptotic tumor cells or their supernatants of the HLA-A2(+) human pancreatic carcinoma cell line Panc-1 for their capacity to activate T cells. Monocyte-derived DCs from HLA-A2(+) donors were pulsed with tumor antigen, matured subsequently, and cocultured with autologeous peripheral blood mononuclear cells. After three weekly restimulations with DCs, T-cell activation was assessed by intracellular IFN-gamma staining and cytotoxicity assays. Compared with lysate, pulsing DCs with the supernatant of apoptotic tumor cells induced a higher frequency of activated CTLs and T-helper cells, as well as an enhanced MHC class I-restricted tumor cell lysis. No activation of natural killer (NK) or gammadelta T cells was detected. Pulsing DCs with whole apoptotic tumor cells induced an even more pronounced lytic effect. However, in this case, MHC class-I blocking was only partially effective, and unrelated cell lines were also killed. IFN-gamma staining revealed activation of CTLs and T-helper cells, as well as NK and gammadelta T cells. Trans-well cultures of NK cells, apoptotic tumor cells, and DCs showed that NK cell activation was dependent on direct cell-to-cell contact with tumor cells and the presence of interleukin-12 produced by DCs. These results indicate that the choice of antigen preparation is a critical determinant in the induction of antitumor immunity. Tumor vaccines consisting of DCs and apoptotic tumor cells may be able to activate CTLs, as well as effector cells of the innate immune system.