The polychromatism of postmortem cerebrospinal fluid.

Based on the assumption that postmortem cerebrospinal fluid (CSF) is contaminated depending on the chosen sampling technique in the forensic setting resulting in bloody or at least hemolytic CSF samples, we systematically documented a total of 183 postmortem CSF samples. These samples were all assessed for their quality and color, regardless of the cause of death or the postmortem interval. The investigations were carried out through subjective assessment of color and turbidity, as well as objective measurements of the optical density (OD) of the CSF supernatants after centrifugation of each sample, with standardized photographic documentation. The observations revealed that in 28 cases the CSF was absolutely (crystal-) clear and transparent. Most of our samples showed color changes ranging from xanthrochromic to rose. Intensive staining of the supernatants was only found in a small proportion of the examined collective. We found that postmortem CSF has no uniform appearance but rather a diverse range of color spectra, and the color, as well as the OD of the CSF, correlates significantly with the postmortem interval (p < 0.001) when sampled using the proposed standard procedure.

Effect of Blood Decontamination Procedures on the Microshear Bond Strength of Resin-modified Glass Ionomer Cement to Resin Composite.

OBJECTIVE: To investigate the effect of decontamination procedures on the microshear bond strength (µSBS) of blood-contaminated resin-modified glass ionomer cement (RMGIC) bonded to resin composite (RC). METHODS: Eighty RMGIC disc specimens were allocated into 5 groups (n=16). All groups except Group 2 were contaminated with blood. Group 1 had no decontamination procedure, Group 3 was decontaminated by rinsing, Group 4 was decontaminated by 34% phosphoric acid etching, and Group 5 was decontaminated by 5% sodium hypochlorite application. RMGIC specimens were subsequently bonded with RC using a universal adhesive in self-etch mode. µSBS tests were conducted using a universal testing machine at a crosshead speed of 1 mm/min. Failure mode analysis was conducted on RMGIC fracture surfaces under a scanning electron microscope. RESULTS: µSBS results indicated that Group 4 had the highest mean µSBS value of 6.22 ± 2.14 MPa, while Group 1 had the lowest mean µSBS value of 3.53 ±1.67 MPa. Significant differences were observed in the µSBS of Group 2 with no contamination (p=0.023) and Group 4 with decontamination by phosphoric acid-etching (p=0.003) when compared to Group 1 with blood contamination. No statistically significant differences (p>0.05) were observed between all other groups' µSBS. For all groups, the predominant mode of failure was adhesive failure between the RMGIC-RC interface, with a few mixed failures in RMGIC for Groups 2-5. CONCLUSIONS: Blood contamination before adhesive application significantly reduced the µSBS between RMGIC and RC. Phosphoric acid etching was the most effective blood decontamination procedure to improve the µSBS.

On the mechanical significance of vascular imprints of the human neurocranium when impacted at 11 m/s.

The course of the middle meningeal vessels can be traced through imprints on the inner table of the human neurocranium. It is as yet unexplored whether these notches lower the load-bearing capacity of the bone when compared to areas that are free of vascular imprints. Here, 310 temporo-parietal samples with and without vascular imprints, from 52 human Crosado-embalmed cadavers, were tested in a three-point bending setup with a half-cylindrical impactor (1 mm radius of curvature) contacting the sample at 11 m/s. The maximum forces before breaking, and the thicknesses of the samples, were statistically compared, including comparing the avascular group to several groups with vascular imprints of different orientations. Furthermore, the influence of sample length and impact location were investigated. To investigate structure and mechanical function of vascular imprints concomitantly, scanning electron microscopy was performed on selected samples in two different planes. The results showed that avascular samples were on average thicker (p < 0.001) and stronger (p ≤ 0.050) compared to samples with vascular imprints. When only thickness-matched samples were analysed, the observed maximum forces of vascular and avascular samples were statistically similar (p ≥ 0.531). Regarding the load-bearing capacity of samples with vascular imprints, it was irrelevant whether the imprint was placed parallel to and directly underneath the impactor, parallel to and offset from the impactor, or perpendicular to the impactor (p > 0.999). The overall results of this study were statistically unrelated to both sample length (p ≥ 0.720) and impact location (p > 0.999). Scanning electron microscopy revealed that vascular imprints are formed through a curve of the inner table. Perforating holes of the inner table are present in avascular areas, however, they are considerably larger in size and higher in number within vascular imprints. In conclusion, vascular imprints are formed through curving of the inner table. In numerical models of human head mechanics, vascular imprints can be accounted for through a simple thinning of the bone assuming the same load-bearing capacity as for the surrounding imprint-free areas.

The Influence of Saliva and Blood Contamination on Bonding Between Resin-modified Glass Ionomer Cements and Resin Composite.

OBJECTIVE: To investigate the influence of blood and saliva contamination on the microshear bond strength (µSBS) between resin-modified glass ionomer cement (RMGIC) and resin composite (RC). METHODS AND MATERIALS: Eighty RMGIC discs were allocated into four groups (n=20). Group 1 received universal dental adhesive application in a self-etch mode followed by a build-up with RC. Group 2 received saliva as a contaminant, Group 3 received blood as a contaminant, Group 4 received a 1:1 blood-saliva mixture as a contaminant. Specimens from Groups 2, 3, and 4 were submerged into their respective contaminants for 15 seconds and dried prior to the adhesive application, followed by the protocol for Group 1. All specimens were stored in distilled water for 24 hours. Subsequently, the bonded specimens were subjected to µSBS testing using a universal testing machine. Failure mode of the debonded RMGIC surfaces was examined using scanning electron microscopy. RESULTS: The µSBS from groups 1-4 were 10.76 ± 3.03 MPa, 9.36 ± 2.54 MPa, 6.55 ± 1.67 MPa and 8.42 ± 2.79 MPa, respectively. Contamination by blood and blood-saliva significantly decreased the µSBS (p<0.001, p=0.029). Saliva contamination alone had no statistically significant effect on the µSBS (p=0.524). A statistically significant difference in the mode of failure was detected between the experimental groups (p=0.012). CONCLUSION: Saliva contamination has no influence on µSBS between RMGIC and RC when it is dried thoroughly, while blood and blood-saliva contamination reduced µSBS between RMGIC and RC even when dried thoroughly.

Dynamic load response of human dura mater at different velocities.

Despite of its assumed role to mitigate brain tissue response under dynamic loading conditions, the human dura mater is frequently neglected in computational and physical human head models. A reason for this is the lack of load-deformation data when the dura mater is loaded dynamically. To date, the biomechanical characterization of the human dura mater predominantly involved quasi-static testing setups. This study aimed to investigate the strain rate-dependent mechanical properties of the human dura mater comparing three different velocities of 0.3, 0.5 and 0.7 m/s. Samples were chosen in a perpendicular orientation to the visible main fiber direction on the samples' surface, which was mostly neglected in previous studies. The elastic modulus of dura mater significantly increased at higher velocities (5.16 [3.38; 7.27] MPa at 0.3 m/s versus 44.38 [35.30; 74.94] MPa at 0.7 m/s). Both the stretch at yield point λf (1.148 [1.137; 1.188] for 0.3 m/s, 1.062 [1.054; 1.066] for 0.5 m/s and 1.015 [1.012; 1.021] for 0.7 m/s) and stress at yield point σf of dura mater (519.14 [366.74; 707.99] kPa for 0.3 m/s versus 300.52 [245.31; 354.89] kPa at 0.7 m/s) significantly decreased with increasing velocities. Conclusively, increasing the load application velocity increases stiffness and decreases tensile strength as well as straining potential of human dura mater between 0.3 and 0.7 m/s. The elastic modulus of human dura mater should be adapted to the respective velocities in computational head impact simulations.

The relationship between the pH value of a hydration solution and the biomechanical properties of Crosado-embalmed human iliotibial bands.

Determining the biomechanical properties of human tissues commonly involves the immersion or spraying of the tissues to maintain them in a hydrated state. However, the influence of the pH value of these solutions on the biomechanical properties of the tissues is not well understood. This study investigated the effects of the pH value on the biomechanical properties of the collagen-rich human iliotibial band (ITB). A total of 124 samples were allocated to polyethylene glycol (PEG) solutions of pH values between 3 and 13 for 24 h, which is a frequently used immersion time prior to biomechanical tests. After this, the samples were biomechanically tested in a uniaxial tensile testing setup using an established testing routine. Similarly, 69 samples were allocated to pH groups of 6, 7 and 8 and biomechanically tested after 1, 2 and 3 weeks. The cross-sectional area of all samples was determined after immersion into the PEG solutions for the specified time frames. In the 24-h experiment, the elastic modulus (pH 12: p ≤ 0.045; pH 13: p ≤ 0.020) and the ultimate tensile strength (pH 12: p ≤ 0.031; pH 13: p ≤ 0.026) of the pH groups 12 and 13 were significantly lower and their cross-sectional areas were higher (pH 12: p ≤ 0.005; pH 13: p ≤ 0.003) compared to several groups of acidic to alkaline pH values. There was no difference in the maximum forces between the different groups within a 24-h immersion time (p > 0.999). In the 3-week-test, a decrease of the ultimate tensile strength was noted between the 24-h and 3 week values for the pH groups 7 (p = 0.034) and 8 (p = 0.029). It is concluded that pH-dependent tissue swelling influences the cross-sectional area-dependent biomechanical properties of the human ITB. Therefore, the pH value of storage and hydration solutions for the preparation of biomechanical tests should be recorded. From a biomechanical perspective, the collagen stability of the human ITB is largely unaltered in PEG solutions with pH values between 3 and 13 over 24 h.

Biomechanics of vascular areas of the human cranial dura mater.

Accurate biomechanical properties of the human cranial dura mater are paramount for computational head models, artificial graft developments and biomechanical basic research. Yet, it is unclear whether areas of the dura containing meningeal vessels biomechanically differ from avascular areas. Here, 244 dura mater samples with or without vessels from 32 cadavers were tested in a quasi-static uniaxial tensile testing setup. The thicknesses of the meningeal and periosteal dura in vascular and avascular areas were histologically investigated in 36 samples using van Gieson staining. The elastic modulus of 112 MPa from dura samples containing vessels running transversely was significantly lower than samples with vessels running longitudinally (151 MPa; p < 0.001). The ultimate tensile strength of dura samples with transversely running vessels (11.1 MPa) was significantly lower in comparison to both avascular samples (14.9 MPa; p < 0.001) and samples with a longitudinally running vessel (15.0 MPa; p < 0.001). The maximum force of dura samples with longitudinally running vessels was 37 N (p < 0.001), this was significantly higher compared to the other groups which were 23 N (p < 0.001). The meningeal and periosteal dura layer thicknesses were not statistically different in avascular areas (p > 0.222). However, around the vessels, the meningeal dura layer was significantly thicker compared to the periosteal layer (p ≤ 0.019). The sum of the meningeal and periosteal layers was similar between vascular and avascular areas (p ≥ 0.071). Vascular areas of the human cranial dura mater withstand the same forces as avascular areas when being stretched. When stretched along the vessel, the dura-vessel composite can withstand even higher tensile forces compared to avascular areas. Vascular areas of the cranial dura mater seem to be similar when compared to avascular areas making their separate simulation in computational models non-essential.

On the correlations of biomechanical properties of super-imposed temporal tissue layers and their age-, sex-, side- and post-mortem interval dependence.

Obtaining biomechanical properties of biological tissues for simulation purposes or graft developments is time and resource consuming. The number of samples required for biomechanical tests could be reduced if the load-deformation properties of a given tissue layer could be estimated from adjacent layers or if the biomechanical parameters were unaffected by age, bodyside, sex or post-mortem interval. This study investigates for the first time potential correlations of multiple super-imposed tissue layers using the temporal region of the human head as an area of broad interest in biomechanical modelling. Spearman correlations between biomechanical properties of the scalp, muscle fascia, muscle, bone and dura mater from up to 83 chemically unfixed cadavers were investigated. The association with age, sex and post-mortem interval was assessed. The results revealed sporadic correlations between the corresponding layers, such as the maximum force (r = 0.43) and ultimate tensile strength (r = 0.33) between scalp and muscle. Side- and age-dependence of the biomechanical properties were different between the tissue types. Strain at maximum force of fascia (r = -0.37) and elastic modulus of temporal muscle (r = 0.26) weakly correlated with post-mortem interval. Only strain at maximum force of scalp differed significantly between sexes. Uniaxial biomechanical properties of individual head tissue layers can thus not be estimated solely based on adjacent layers. Therefore, correlations between the tissues' biomechanical properties, anthropometric data and post-mortem interval need to be established independently for each layer. Sex seems not to be a relevant influencing factor for the passive tissue mechanics of the here investigated temporal head tissue layers.

Computed tomography osteoabsorptiometry-based investigation on subchondral bone plate alterations in sacroiliac joint dysfunction.

Sacroiliac joint dysfunction (SIJD) is an underappreciated source of back pain. Mineralization patterns of the sacroiliac (SIJ) subchondral bone plate (SCB) may reflect long-term adaptations to the loading of the joint. Mineralization densitograms of 27 SIJD patients and 39 controls, were obtained using CT osteoabsorptiometry. Hounsfield unit (HU) values of the SCB mineralization of superior, anterior and inferior regions on the iliac and sacral auricular surfaces were derived and statistically compared between SIJD-affected and control cohorts. Healthy controls showed higher HU values in the iliac; 868 ± 211 (superior), 825 ± 121 (anterior), 509 ± 114 (inferior), than in the sacral side; 541 ± 136 (superior), 618 ± 159 (anterior), 447 ± 91 (inferior), of all regions (p < 0.01). This was similar in SIJD; ilium 908 ± 170 (superior), 799 ± 166 (anterior), 560 ± 135 (inferior), sacrum 518 ± 150 (superior), 667 ± 151 (anterior), 524 ± 94 (inferior). In SIJD, no significant HU differences were found when comparing inferior sacral and iliac regions. Furthermore, HU values in the inferior sacral region were significantly higher when compared to the same region of the healthy controls (524 ± 94 vs. 447 ± 91, p < 0.01). Region mineralization correlated negatively with age (p < 0.01). SIJD-affected joints reflect a high mineralization of the sacral inferior region, suggesting increased SIJD-related mechanical stresses. Age-related SCB demineralization is present in all individuals, regardless of dysfunction.

On the morphological relations of the Achilles tendon and plantar fascia via the calcaneus: a cadaveric study.

Current treatments of plantar fasciitis are based on the premise that the Achilles tendon (AT) and plantar fascia (PF) are mechanically directly linked, which is an area of debate. The aim of this study was to assess the morphological relationship between the AT and PF. Nineteen cadaveric feet were x-ray imaged, serially sectioned and plastinated for digital image analyses. Measurements of the AT and PF thicknesses and cross-sectional areas (CSA) were performed at their calcaneal insertion. The fiber continuity was histologically assessed in representative subsamples. Strong correlations exist between the CSA of the AT and PF at calcaneal insertion and the CSA of PF's insertional length (r = 0.80), and between the CSAs of AT's and PF's insertional lengths. Further correlations were observed between AT and PF thicknesses (r = 0.62). This close morphological relationship could, however, not be confirmed through x-ray nor complete fiber continuity in histology. This study provides evidence for a morphometric relationship between the AT and PF, which suggests the presence of a functional relationship between these two structures following the biological key idea that the structure determines the function. The observed morphological correlations substantiate the existing mechanical link between the AT and PF via the posterior calcaneus and might explain why calf stretches are a successful treatment option for plantar heel pain.

Assessment of plantaris and peroneus tertius tendons as graft materials for ankle ligament reconstructions - A cadaveric biomechanical study.

Both the plantaris tendon and the peroneus tertius tendon are used as auto- and allogenous graft materials to reconstruct the ankle ligament complex. However, it is unclear to what extent these graft materials resemble the load-deformation behavior of the ankle ligaments. A total of 34 human ankle ligaments and 35 tendons were assessed mechanically deploying a quasi-static tensile testing setup. Tendons were significantly stiffer (median elastic moduli: plantaris tendon = 465.7 MPa, peroneus tertius tendon = 338.5 MPa, medial ligament = 61.4 MPa, lateral ligament = 49.3 MPa; p ≤ 0.035), but more distensible (median strain at maximum force: plantaris tendon = 15.1%, peroneus tertius tendon = 15.3%, medial ligament = 9.3%, lateral ligament = 9.6%; p ≤ 0.008) and mechanically tougher (median ultimate tensile strength: plantaris tendon = 51.0 MPa, peroneus tertius tendon = 40.5 MPa, medial ligament = 4.1 MPa, lateral ligament = 3.5 MPa; p ≤ 0.033) when compared to medial and lateral ankle ligaments. The lateral ligaments of the right ankle were significantly tougher compared to the left side (p = 0.015). The elastic modulus of the medial ligament (r = 0.489, p = 0.045) and the peroneus tertius tendon (r = 0.517, p = 0.014) yielded an age-dependent increase. Both tendons seem biomechanically suitable graft materials to replace the medial and lateral ankle ligaments during physiological loading. The age-dependent increase in tissue elastic properties of the medial vs. lateral ankle ligaments, and differences in ultimate tensile strength between the lateral ligaments left vs. right, may reflect the complex asymmetric loading behavior of both ankle ligaments.

Passive load-deformation properties of human temporal muscle.

The passive load-deformation properties of the human temporal muscle applicable to computer simulations of the human head or the comparison of the temporal muscle to other graft materials are unexplored to date and it is unclear, if these properties depend on age, sex, post-mortem interval or body side. Eighty-eight fresh temporal muscle samples from 69 human cadavers (age range 4 months - 94 years) were investigated in a quasi-static tensile setup. For comparative reasons, 20 age-matched human temporal muscle fascia and scalp samples were tested in the same manner as the temporal muscle. Human temporal muscle showed an elastic modulus of 1.58 ± 0.64 MPa, an ultimate tensile strength of 0.26 ± 0.11 MPa and a strain at maximum force of 26.21 ± 12.48%. These parameters were independent of sex (p > 0.88), side (p > 0.92) and post-mortem interval (p > 0.09). All passive load-deformation parameters of the human temporal muscle differed from temporal muscle fascia and scalp except for the strain at maximum force of the temporal muscle and scalp. Significantly different load-deformation properties of the human temporal muscle from temporal muscle fascia and scalp indicate the need for a separate simulation of these soft tissue layers in computational head models to reflect lifelike conditions. Contrary to other tissues such as scalp or temporal muscle fascia the biomechanical temporal muscle properties in head models may not require adjustments for sex, side and age based on the here-presented findings.

Mechanical properties of native and acellular temporal muscle fascia for surgical reconstruction and computational modelling purposes.

The temporal muscle fascia (TMF) is a widely used graft material and of interest for computational simulations of the temporomandibular joint as well as computational and physical human head models in general. However, reliable biomechanical properties of the TMF are lacking to date. This study provides tensile data of 52 TMFs at an age range of 18 to 94 years. It further investigates, if acellular fascia scaffolds differ from native counterparts in their biomechanical behaviour. Native TMF has a median elastic modulus of 26.2 MPa (acellular: 24.5 MPa), an ultimate tensile strength of 2.9 MPa (acellular: 2.1 MPa), a maximum force of 12.6 N (acellular: 9.9 N) and a strain at failure of 14.1% (acellular: 14.8%). No significant difference was found regarding the properties of native and acellular samples. Elastic modulus and the ultimate tensile strength increased with age but only in the acellular group (p < 0.01). Decorin and fibronectin seemed to be washed out by the acellularization procedure. The absence of cells in acellular TMF samples is not of biomechanical relevance compared to the native state. Acellular TMF is a biomechanically promising scaffold material for graft purposes, which can be retrieved easily due to its superficial location.

Suicide by the intraoral blast of firecrackers - experimental simulation using a skull simulant model.

Suicides committed by intraorally placed firecrackers are rare events. Given to the use of more powerful components such as flash powder recently, some firecrackers may cause massive life-threatening injuries in case of such misuse. Innocuous black powder firecrackers are subject to national explosives legislation and only have the potential to cause harmless injuries restricted to the soft tissue. We here report two cases of suicide committed by an intraoral placement of firecrackers, resulting in similar patterns of skull injury. As it was first unknown whether black powder firecrackers can potentially cause serious skull injury, we compared the potential of destruction using black powder and flash powder firecrackers in a standardized skull simulant model (Synbone, Malans, Switzerland). This was the first experiment to date simulating the impacts resulting from an intraoral burst in a skull simulant model. The intraoral burst of a "D-Böller" (an example of one of the most powerful black powder firecrackers in Germany) did not lead to any injuries of the osseous skull. In contrast, the "La Bomba" (an example of the weakest known flash powder firecrackers) caused complex fractures of both the viscero- and neurocranium. The results obtained from this experimental study indicate that black powder firecrackers are less likely to cause severe injuries as a consequence of intraoral explosions, whereas flash powder-based crackers may lead to massive life-threatening craniofacial destructions and potentially death.

Pulmonary artery perforation and coronary air embolism-two fatal outcomes in percutaneous left atrial appendage occlusion.

Percutaneous left atrial appendage (LAA) closure is a routinely performed method to reduce the risk of stroke in patients suffering from atrial fibrillation, when an oral anticoagulation is no longer indicated due to relevant bleeding complications. Currently, the Amplatzer Amulet and the Watchman system are two equally used systems. While there is an acute success rate of more than 95 per cent for this intervention, several minor and major complications such as pericardial effusions, air embolism, vascular lesions in proximity to the heart or even death can occur. Here, we report two cases of very rare fatal outcomes in percutaneous LAA occlusion. Eight hours after deployment of an Amplatzer Amulet a patient died, after the pulmonary trunk was perforated by a hook of the occluder device causing pericardial tamponade. In the second case during final radiological position control of the deployed Watchman occluder air was injected accidentally. The patient immediately died due to coronary air embolism. Forensic autopsies are necessary to solve the cause and manner of death, to evaluate and develop medical devices and to rule out medical malpractice. Thus, a close collaboration of legal medicine and the various cardiologic departments is proposed.

Circulating microparticles in patients with benign and malignant ovarian tumors.

BACKGROUND: Microparticles are known to be increased in various malignancies. In this prospective study, microparticle levels were evaluated in patients with benign and malignant ovarian lesions. PATIENTS AND METHODS: Microparticles from platelets/megakaryocytes, activated platelets and endothelial cells, tissue factor exposing microparticles and D-dimer values were examined in patients with newly diagnosed ovarian lesions before surgery, and were correlated with tumor histology. RESULTS: Higher counts of CD63-positive microparticles were detected in patients with ovarian cancer [mean=276×10(6) (range: 64-948)/l; n=12] as compared to patients with benign ovarian tumors [146×10(6) (45-390)/l; n=21; p=0.014]. D-dimer values were also increased in patients with cancer [860 (180-4500) ng/l versus 280 (170-2720) ng/l; p=0.001]. CONCLUSION: Elevated levels of CD63-positive microparticles and D-dimer reflect the procoagulant phenotype of these patients. However, for the discrimination between benign and malignant ovarian tumors, measuring preoperative levels of microparticles does not seem to be helpful.

Quantitative, phenotypic, and functional evaluation of basophils in myelodysplastic syndromes.

BACKGROUND: The myelodysplastic syndromes (MDS) are a group of clonal haematological disorders characterized by cytopenia(s), reduced differentiation-capacity of myeloid cells, and impaired leukocyte function. However, little is known so far about basophil granulocytes in MDS. DESIGN: We have compared the numbers, phenotype and function of basophils in MDS patients with those in healthy subjects. A total numer of 23 patients with MDS (refractory anaemia, n = 8; refractory anaemia with ringsideroblasts, n = 7; refractory anaemia with excess of blasts/refractory anaemia with excess of blasts in transformation, n = 8) and 20 healthy donors were included. RESULTS: The numbers of blood basophils in MDS patients (34.6 +/- 62.9 microL-1) was lower compared to healthy controls (58.6 +/- 64.9 microL-1). Correspondingly, whole blood histamine levels were lower in MDS patients (MDS 34.1 +/- 29.1 ng mL-1 vs. normal donors 72.0 +/- 36.9 ng mL-1). Like "normal" basophils, basophils in MDS expressed interleukin-3 receptor alpha (CD123), E-NPP3 (CD203c), CR1 (CD35), CR3 (CD11b), CR4 (CD11c), membrane co-factor protein (CD46), decay-accelerating factor (CD55) and membrane attack complex inhibitory factor (CD59), as well as receptors for C3a, C5a (CD88), and IgE. Recombinant human (rh) C5a and anti-IgE induced significant release of histamine from basophils in both groups of donors without significant differences between MDS and healthy controls. CONCLUSIONS: The absolute numbers of basophils in MDS patients are lower than in normal donors. However, basophils in MDS do not differ from their "normal counterparts" in terms of complement receptor expression, IgE-receptor expression, or functional responses to respective ligands.

Ex vivo generation of human anti-melanoma autologous cytolytic T cells by dentritic cell/melanoma cell hybridomas.

Due to their central role in controlling immunity, dendritic cells are logical targets for priming naive cytotoxic T lymphocytes against tumour cells. In a strictly autologous system, we fused dendritic cells with melanoma cells, both of which were derived from patients with metastatic malignant melanoma. Hybridomas were positive for major histocompatibility complex (MHC) class II, CD40, CD54, CD83, CD86, and the pro-inflammatory cytokine interleukin-12. Autologous T lymphocytes were co-incubated with hybridomas. After 6 days, in-vitro-primed T lymphocytes revealed a strong proliferation activity and released Th-1-associated, but not Th-2-associated, cytokines. Furthermore they showed effective anti-melanoma activity, resulting in death of 70 +/- 9% of autologous melanoma cells. After depletion of CD4+ cells from the mixed population of primed T lymphocytes, the remaining CD8+ cells were able to kill 63+/-8% of autologous melanoma cells. Following depletion of CD8+ cells, however, the cytotoxic capacity of the remaining T lymphocytes caused death in only 32+/-6% of autologous melanoma cells. Blocking of MHC class I, but not class II, molecules on hybridomas impaired T cell proliferation, secretion of Th-1-associated cytokines, as well as the cytotoxic activity of primed T cells. These findings strongly suggest that hybridomas deliver melanoma-associated antigens via MHC class I molecules to T lymphocytes, resulting in the generation of CD8+ cytotoxic T lymphocytes with effective anti-melanoma activity in vitro. The data may serve as a basis for the use of hybridomas in the immunotherapy of malignant melanoma in vivo.