In vitro efficacy of dicationic compounds and mefloquine enantiomers against Echinococcus multilocularis metacestodes

The current chemotherapy of alveolar echinococcosis (AE) is based on benzimidazoles such as albendazole and has been shown to be parasitostatic rather than parasiticidal, requiring lifelong duration. Thus, new and more efficient treatment options are urgently needed. By employing a recently validated assay based on the release of functional phosphoglucose isomerase (PGI) from dying parasites, the activities of 26 dicationic compounds and of the (+)- and (-)-erythro-enantiomers of mefloquine were investigated. Initial screening of compounds was performed at 40 muM, and those compounds exhibiting considerable antiparasitic activities were also assessed at lower concentrations. Of the dicationic drugs, DB1127 (a diguanidino compound) with activities comparable to nitazoxanide was further studied. The activity of DB1127 was dose dependent and led to severe structural alterations, as visualized by electron microscopy. The (+)- and (-)-erythro-enantiomers of mefloquine showed similar dose-dependent effects, although higher concentrations of these compounds than of DB1127 were required for metacestode damage. In conclusion, of the drugs investigated here, the diguanidino compound DB1127 represents the most promising compound for further study in appropriate in vivo models for Echinococcus multilocularis infection.

Pulmonary surfactant coating of multi-walled carbon nanotubes (MWCNTs) influences their oxidative and pro-inflammatory potential in vitro

Background Increasing concern has been expressed regarding the potential adverse health effects that may be associated with human exposure to inhaled multi-walled carbon nanotubes (MWCNTs). Thus it is imperative that an understanding as to the underlying mechanisms and the identification of the key factors involved in adverse effects are gained. In the alveoli, MWCNTs first interact with the pulmonary surfactant. At this interface, proteins and lipids of the pulmonary surfactant bind to MWCNTs, affecting their surface characteristics. Aim of the present study was to investigate if the pre-coating of MWCNTs with pulmonary surfactant has an influence on potential adverse effects, upon both (i) human monocyte derived macrophages (MDM) monocultures, and (ii) a sophisticated in vitro model of the human epithelial airway barrier. Both in vitro systems were exposed to MWCNTs either pre-coated with a porcine pulmonary surfactant (Curosurf) or not. The effect of MWCNTs surface charge was also investigated in terms of amino (−NH2) and carboxyl (−COOH) surface modifications. Results Pre-coating of MWCNTs with Curosurf affects their oxidative potential by increasing the reactive oxygen species levels and decreasing intracellular glutathione depletion in MDM as well as decreases the release of Tumour necrosis factor alpha (TNF-α). In addition, an induction of apoptosis was observed after exposure to Curosurf pre-coated MWCNTs. In triple cell-co cultures the release of Interleukin-8 (IL-8) was increased after exposure to Curosurf pre-coated MWCNTs. Effects of the MWCNTs functionalizations were minor in both MDM and triple cell co-cultures. Conclusions The present study clearly indicates that the pre-coating of MWCNTs with pulmonary surfactant more than the functionalization of the tubes is a key factor in determining their ability to cause oxidative stress, cytokine/chemokine release and apoptosis. Thus the coating of nano-objects with pulmonary surfactant should be considered for future lung in vitro risk assessment studies. Keywords: Multi-walled carbon nanotubes (MWCNTs); Pulmonary surfactant (Curosurf); Macrophages; Epithelial cells; Dendritic cells; Triple cell co-culture; Pro-inflammatory and oxidative reactions

Effect of a plasmaelectrolytic coating on the strength retention of in vivo and in vitro degraded magnesium implants

The strength decrease in magnesium implants was studied in vitro and in vivo, with and without a protective plasmaelectrolytic coating. In vivo, degradation was examined by implanting rectangular plates on top of the nasal bone of miniature pigs. The presence of gas pockets in the soft tissue surrounding the implants was evaluated with intermediate X-rays and computed X-ray tomography scans before euthanasia. After 12 and 24weeks of in vivo degradation, the large rectangular plates were removed and mechanically tested in three-point bending. In vitro, identical plates were immersed in simulated body fluid for 4, 8 and 12weeks. In vitro and in vivo results showed that onset of gas release can be delayed by the plasmaelectrolytic coating. Mass loss and strength retention during in vivo degradation is about four times slower than during in vitro degradation for the chosen test conditions. Despite the slow degradation of the investigated WE43 alloy, the occurrence of gas pockets could not be completely avoided. Nevertheless, uniformity of degradation and reliable strength retention make this alloy a prime candidate for the use of magnesium in cranio-maxillofacial surgery.

Aspiration toxicology of hydrocarbons and lamp oils studied by in vitro technology

Medical literature regularly reports on accidental poisoning in children after aspiration of combustibles such as lamp oils which usually contain hydrocarbons or rape methyl esters (RMEs). We aimed to analyze the toxic potential of alkanes and different combustible classes in vitro with regard to biologic responses and mechanisms mediating toxicity. Two different in vitro models were used, i.e. (i) a captive bubble surfactometer (CBS) to assess direct influence of combustibles on biophysical properties of surfactant film and (ii) cell cultures (BEAS-2B and R3/1 cells, primary macrophages, re-differentiated epithelia) closely mimicking the inner lung surface. Biological endpoints included cell viability, cytotoxicity and inflammatory mediator release. CBS measurements demonstrate that combustibles affect film dynamics, i.e. the surface tension/area characteristics during compression and expansion, in a dose and molecular chain length dependent manner. Cell culture results confirm the dose dependent toxicity. Generally, cytotoxicity and cytokine release are higher in short-chained alkanes and hydrocarbon-based combustibles than in long-chained substances, e.g. highest inducible cytotoxicity in BEAS-2B was for hexane 84.6%, decane 74% and hexadecane 30.8%. Effects of RME-based combustibles differed between the cell models. Our results confirm data from animal experiments and give new insights into the mechanisms underlying the adverse health effects observed.

Involvement of drug-specific T cells in acute drug-induced interstitial nephritis

Drug-induced interstitial nephritis can be caused by a plethora of drugs and is characterized by a sudden impairment of renal function, mild proteinuria, and sterile pyuria. For investigation of the possible pathomechanism of this disease, drug-specific T cells were analyzed, their function was characterized, and these in vitro findings were correlated to histopathologic changes that were observed in kidney biopsy specimens. Peripheral blood mononuclear cells from three patients showed a proliferative response to only one of the administered drugs, namely flucloxacillin, penicillin G, and disulfiram, respectively. The in vitro analysis of the flucloxacillin-reactive cells showed an oligoclonal immune response with an outgrowth of T cells bearing the T cell receptor Vbeta9 and Vbeta21.3. Moreover, flucloxacillin-specific T cell clones could be generated from peripheral blood, they expressed CD4 and the alphabeta-T cell receptor, and showed a heterogeneous cytokine secretion pattern with no clear commitment to either a Th1- or Th2-type response. The immunohistochemistry of kidney biopsies of these patients revealed cell infiltrations that consisted mostly of T cells (CD4+ and/or CD8+). An augmented presence of IL-5, eosinophils, neutrophils, CD68+ cells, and IL-12 was observed. In agreement with negative cytotoxicity assays, no cytotoxicity-related molecules such as Fas and perforin were detected by immunohistochemistry. The data indicate that drug-specific T cells are activated locally and orchestrate a local inflammation via secretion of various cytokines, the type of which depends on the cytokine pattern secreted and which probably is responsible for the renal damage.

Structure-activity relationships from in vitro efficacies of the thiazolide series against the intracellular apicomplexan protozoan Neospora caninum

Nitazoxanide (2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide; NTZ) represents the parent compound of a novel class of broad-spectrum anti-parasitic compounds named thiazolides. NTZ is active against a wide variety of intestinal and tissue-dwelling helminths, protozoa, enteric bacteria and a number of viruses infecting animals and humans. While potent, this poses a problem in practice, since this obvious non-selectivity can lead to undesired side effects in both humans and animals. In this study, we used real time PCR to determine the in vitro activities of 29 different thiazolides (NTZ-derivatives), which carry distinct modifications on both the thiazole- and the benzene moieties, against the tachyzoite stage of the intracellular protozoan Neospora caninum. The goal was to identify a highly active compound lacking the undesirable nitro group, which would have a more specific applicability, such as in food animals. By applying self-organizing molecular field analysis (SOMFA), these data were used to develop a predictive model for future drug design. SOMFA performs self-alignment of the molecules, and takes into account the steric and electrostatic properties, in order to determine 3D-quantitative structure activity relationship models. The best model was obtained by overlay of the thiazole moieties. Plotting of predicted versus experimentally determined activity produced an r2 value of 0.8052 and cross-validation using the "leave one out" methodology resulted in a q2 value of 0.7987. A master grid map showed that large steric groups at the R2 position, the nitrogen of the amide bond and position Y could greatly reduce activity, and the presence of large steric groups placed at positions X, R4 and surrounding the oxygen atom of the amide bond, may increase the activity of thiazolides against Neospora caninum tachyzoites. The model obtained here will be an important predictive tool for future development of this important class of drugs.

Tumor necrosis factor-alpha: alternative role as an inhibitor of osteoclast formation in vitro

TNFalpha is known to stimulate the development and activity of osteoclasts and of bone resorption. The cytokine was found to mediate bone loss in conjunction with inflammatory diseases such as rheumatoid arthritis or chronic aseptic inflammation induced by wear particles from implants and was suggested to be a prerequisite for the loss of bone mass under estrogen deficiency. In the present study, the regulation of osteoclastogenesis by TNFalpha was investigated in co-cultures of osteoblasts and bone marrow or spleen cells and in cultures of bone marrow and spleen cells grown with CSF-1 and RANKL. Low concentrations of TNFalpha (1 ng/ml) caused a >90% decrease in the number of osteoclasts in co-cultures, but did not affect the development of osteoclasts from bone marrow cells. In cultures with p55TNFR(-/-) osteoblasts and wt BMC, the inhibitory effect was abrogated and TNFalpha induced an increase in the number of osteoclasts in a dose-dependent manner. Osteoblasts were found to release the inhibitory factor(s) into the culture supernatant after simultaneous treatment with 1,25(OH)(2)D(3) and TNFalpha, this activity, but not its release, being resistant to treatment with anti-TNFalpha antibodies. Dexamethasone blocked the secretion of the TNFalpha-dependent inhibitor by osteoblasts, while stimulating the development of osteoclasts. The data suggest that the effects of TNFalpha on the differentiation of osteoclast lineage cells and on bone metabolism may be more complex than hitherto assumed and that these effects may play a role in vivo during therapies for inflammatory diseases.

Effect on infection resistance of a local antiseptic and antibiotic coating on osteosynthesis implants: an in vitro and in vivo study

The purpose of this study was to acquire information about the effect of an antibacterial and biodegradable poly-L-lactide (PLLA) coated titanium plate osteosynthesis on local infection resistance. For our in vitro and in vivo experiments, we used six-hole AO DC minifragment titanium plates. The implants were coated with biodegradable, semiamorphous PLLA (coating about 30 microm thick). This acted as a carrier substance to which either antibiotics or antiseptics were added. The antibiotic we applied was a combination of Rifampicin and fusidic acid; the antiseptic was a combination of Octenidin and Irgasan. This produced the following groups: Group I: six-hole AO DC minifragment titanium plate without PLLA; Group II: six-hole AO DC minifragment titanium plate with PLLA without antibiotics/antiseptics; Group III: six-hole AO DC minifragment titanium plate with PLLA + 3% Rifampicin and 7% fusidic acid; Group IV: six-hole AO DC minifragment titanium plate with PLLA + 2% Octenidin and 8% Irgasan. In vitro, we investigated the degradation and the release of the PLLA coating over a period of 6 weeks, the bactericidal efficacy of antibiotics/antiseptics after their release from the coating and the bacterial adhesion of Staphylococcus aureus to the implants. In vivo, we compared the infection rates in white New Zealand rabbits after titanium plate osteosynthesis of the tibia with or without antibacterial coating after local percutaneous bacterial inoculations at different concentrations (2 x 10(5)-2 x 10(8)): The plate, the contaminated soft tissues and the underlying bone were removed under sterile conditions after 28 days and quantitatively evaluated for bacterial growth. A stepwise experimental design with an "up-and-down" dosage technique was used to adjust the bacterial challenge in the area of the ID50 (50% infection dose). Statistical evaluation of the differences between the infection rates of both groups was performed using the two-sided Fisher exact test (p < 0.05). Over a period of 6 weeks, a continuous degradation of the PLLA coating of 13%, on average, was seen in vitro in 0.9% NaCl solution. The elution tests on titanium implants with antibiotic or antiseptic coatings produced average release values of 60% of the incorporated antibiotic or 62% of the incorporated antiseptic within the first 60 min. This was followed by a much slower, but nevertheless continuous, release of the incorporated antibiotic and antiseptic over days and weeks. At the end of the test period of 42 days, 20% of the incorporated antibiotic and 15% of the incorporated antiseptic had not yet been released from the coating. The antibacterial effect of the antibiotic/antiseptic is not lost by integrating it into the PLLA coating. The overall infection rate in the in vivo investigation was 50%. For Groups I and II the infection rate was both 83% (10 of 12 animals). In Groups III and IV with antibacterial coating, the infection rate was both 17% (2 of 12 animals). The ID50 in the antibacterial coated Groups III and IV was recorded as 1 x 10(8) CFU, whereas the ID50 values in the Groups I and II without antibacterial coating were a hundred times lower at 1 x 10(6) CFU, respectively. The difference between the groups with and without antibacterial coating was statistically significant (p = 0.033). Using an antibacterial biodegradable PLLA coating on titanium plates, a significant reduction of infection rate in an in vitro and in vivo investigation could be demonstrated. For the first time, to our knowledge, we were able to show, under standardized and reproducible conditions, that an antiseptic coating leads to the same reduction in infection rate as an antibiotic coating. Taking the problem of antibiotic-induced bacterial resistance into consideration, we thus regard the antiseptic coating, which shows the same level of effectiveness, as advantageous.

In vitro effects of bethanechol on specimens of intestinal smooth muscle obtained from the duodenum and jejunum of healthy dairy cows

OBJECTIVE: To describe the in vitro effects of bethanechol on contractility of smooth muscle preparations from the small intestines of healthy cows and define the muscarinic receptor subtypes involved in mediating contraction. SAMPLE POPULATION: Tissue samples from the duodenum and jejunum collected immediately after slaughter of 40 healthy cows. PROCEDURES: Cumulative concentration-response curves were determined for the muscarinic receptor agonist bethanechol with or without prior incubation with subtype-specific receptor antagonists in an organ bath. Effects of bethanechol and antagonists and the influence of intestinal location on basal tone, maximal amplitude (A(max)), and area under the curve (AUC) were evaluated. RESULTS: Bethanechol induced a significant, concentration-dependent increase in all preparations and variables. The effect of bethanechol was more pronounced in jejunal than in duodenal samples and in circular than in longitudinal preparations. Significant inhibition of the effects of bethanechol was observed after prior incubation with muscarinic receptor subtype M(3) antagonists (more commonly for basal tone than for A(max) and AUC). The M(2) receptor antagonists partly inhibited the response to bethanechol, especially for basal tone. The M(3) receptor antagonists were generally more potent than the M(2) receptor antagonists. In a protection experiment, an M(3) receptor antagonist was less potent than when used in combination with an M(2) receptor antagonist. Receptor antagonists for M(1) and M(4) did not affect contractility variables. CONCLUSIONS AND CLINICAL RELEVANCE: Bethanechol acting on muscarinic receptor sub-types M(2) and M(3) may be of clinical use as a prokinetic drug for motility disorders of the duodenum and jejunum in dairy cows.

Inhibition of hepatic tumor cell proliferation in vitro and tumor growth in vivo by taltobulin, a synthetic analogue of the tripeptide hemiasterlin

AIM: To investigate the inhibitory effects of taltobulin (HTI-286), a synthetic analogue of natural hemiasterlin derived from marine sponges, on hepatic tumor growth in vitro and in vivo. METHODS: The potential anti-proliferative effects of HTI-286 on different hepatic tumor cell lines in vitro and in vivo were examined. RESULTS: HTI-286 significantly inhibited proliferation of all three hepatic tumor cell lines (mean IC50 = 2 nmol/L +/- 1 nmol/L) in vitro. Interestingly, no decrease in viable primary human hepatocytes (PHH) was detected under HTI-286 exposure. Moreover, intravenous administration of HTI-286 significantly inhibited tumor growth in vivo (rat allograft model). CONCLUSION: HTI-286 might be considered a potent promising drug in treatment of liver malignancies. HTI-286 is currently undergoing clinical evaluation in cancer patients.

Dual in vitro perfusion of an isolated cotyledon as a model to study the implication of changes in the third trimester placenta on preeclampsia

In the current study perfusions of an isolated cotyledon of term placenta using standard medium were compared to medium containing xanthine plus xanthine oxidase (X+XO), which generates reactive oxygen species (ROS). A time-dependant increase in the levels of different cytokines (TNF-alpha, IL-1ss, IL-6, IL-8 and IL-10) was observed between 1 and 7h with more than 90% of the total recovered from the maternal compartment with no significant difference between the 2 groups. For 8-iso-PGF2alpha 90% of the total was found in the fetal compartment and a significantly higher total release was seen in the X+XO group. Microparticles (MPs) isolated from the maternal circuit were identified by flow cytometry as trophoblastic sheddings, whereas MPs from the fetal circuit were predominantly derived from endothelial cells. More than 90% of the total of MPs was found in the maternal circuit. The absolute amount of the total as well as the maternal fraction were significantly higher in the X+XO group. Immunohistochemistry (IHC) of the perfused tissue revealed staining for IL-1beta of villous stroma cells, which became clearly more pronounced in experiments with X+XO. Western blot of tissue homogenate revealed 2 isoforms of IL-1beta at 17 and 31kD. In X+XO experiments there was a tendency for increased expression of antioxidant enzymes in the tissue. Western blot of MPs from the maternal circuit showed increased expression of antioxidant enzymes in the X+XO group and for IL-1beta only the 17kD band was detected. In vitro reperfusion of human placental tissue results in mild tissue injury suggestive of oxidative stress. In view of the increased generation of ROS in perfused tissue with further increase under the influence of X+XO, the overall manifestation of oxidative stress remained rather mild. Preservation of antioxidant capacity of human placental tissue could be a sign of integrity of structure and function being maintained in vitro by dual perfusion of an isolated cotyledon. The observed changes resemble findings seen in placentae from preeclampsia.

In vitro efficacy of nitro- and bromo-thiazolyl-salicylamide compounds (thiazolides) against Besnoitia besnoiti infection in Vero cells

Nitazoxanide (NTZ) and its deacetylated metabolite tizoxanide (TIZ) exhibit considerable in vitro activity against Besnoitia besnoiti tachyzoites grown in Vero cells. Real-time-PCR was used to assess B. besnoiti tachyzoite adhesion, invasion, and intracellular proliferation in vitro. A number of NTZ-derivatives, including Rm4822 and Rm4803, were generated, in which the thiazole-ring-associated nitro-group was replaced by a bromo-moiety. We here show that replacement of the nitro-group on the thiazole ring with a bromo (as it occurs in Rm4822) does not impair the efficacy of the drug, but methylation of the salicylate ring at the ortho-position in a bromo-derivative (Rm4803) results in complete abrogation of the antiparasitic activity. Treatment of extracellular B. besnoiti tachyzoites with NTZ has an inhibitory effect on host cell invasion, while treatments with TIZ, Rm4822 do not. TEM demonstrates that the effects of Rm4822 treatment upon the parasites are similar to the damage induced by NTZ. This includes increased vacuolization of the parasite cytoplasm, and loss of the structural integrity of the parasitophorous vacuole and its membrane. Thus, Rm4822, due to the absence of a potentially mutagenic nitro-group, may represent an important potential addition to the anti-parasitic arsenal for food animal production, especially in cattle.

Intraindividual comparison of human radial and internal thoracic arteries in vitro and the effect of preoperative calcium blocker therapy

The patency rate of radial artery (RA) conduits is considerably lower than that of internal thoracic artery (ITA) grafts and the evidence suggests that this is due to a clinically suspected higher incidence of vasospasm. The aim of this study was, therefore, to compare intraindividually the pharmacological reactivity of RA with that of ITA. Both RA and ITA were taken from the same patients and investigated in parallel. Changes in tone were monitored isometrically on ring preparations from both arteries in organ baths with modified Krebs-Henseleit solution containing 1.25 mm calcium chloride at 1 g passive preload. In intraindividual comparisons maximal receptor-mediated contractile responses to noradrenaline and endothelin-1 and endothelium-dependent acetylcholine-induced relaxant responses revealed no differences between both arteries. By contrast, depolarization-induced contractions to potassium chloride (KCl) appeared to be significantly higher in RA than in ITA. Further analysis, however, revealed that this difference was due to preoperative calcium entry blocker (Ca(2+)eB) therapy. Compared with control tissues, maximal responses to KCl were significantly attenuated in the ITA but unchanged in RA when arteries were obtained from patients with preoperative Ca(2+)eB therapy. The present results suggested that functional responses to pharmacological stimuli of both RA and ITA were quite similar. Preoperative Ca(2+)eB therapy, however, attenuated markedly responses to KCl of the ITA leaving those of RA unchanged. These results, demonstrating a lower sensitivity to Ca(2+)eB of RA, therefore suggested that in addition to Ca(2+)eB other classes of drug may be more effective at reducing the propensity of RA conduits to vasospasm.

Design and in vitro characterization of highly sst2-selective somatostatin antagonists suitable for radiotargeting

Radiolabeled sst 2 and sst 3 antagonists are better candidates for tumor targeting than agonists with comparable binding characteristics (Ginj, M.; Zhang, H.; Waser, B.; Cescato, R.; Wild, D.; Erchegyi, J.; Rivier, J.; Mäcke, H. R.; Reubi, J. C. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16436-16441.). Because most of the neuroendocrine tumors express sst 2, we used the known antagonists acetyl- pNO 2Phe (2)- c[ dCys (3)-Tyr (7)- dTrp (8)-Lys (9)-Thr (10)-Cys (14)]- dTyr (15)-NH 2 ( 1) (Bass, R. T.; Buckwalter, B. L.; Patel, B. P.; Pausch, M. H.; Price, L. A.; Strnad, J.; Hadcock, J. R. Mol. Pharmacol. 1996, 50, 709-715. Bass, R. T.; Buckwalter, B. L.; Patel, B. P.; Pausch, M. H.; Price, L. A.; Strnad, J.; Hadcock, J. R. Mol. Pharmacol. 1997, 51, 170; Erratum.) and H-Cpa (2)- c[ dCys (3)-Tyr (7)- dTrp (8)-Lys (9)-Thr (10)-Cys (14)]-2Nal (15)-NH 2 ( 7) (Hocart, S. J.; Jain, R.; Murphy, W. A.; Taylor, J. E.; Coy, D. H. J. Med. Chem. 1999, 42, 1863-1871.) as leads for analogues with increased sst 2 binding affinity and selectivity. Among the 32 analogues reported here, DOTA- pNO 2Phe (2)- c[ dCys (3)-Tyr (7)- dAph (8)(Cbm)-Lys (9)-Thr (10)-Cys (14)- dTyr (15)-NH 2 ( 3) and DOTA-Cpa (2)- c[ dCys (3)-Aph (7)(Hor)- dAph (8)(Cbm)-Lys (9)-Thr (10)-Cys (14)]- dTyr (15)-NH 2 ( 31) had the highest sst 2 binding affinity and selectivity. All of the analogues tested kept their sst 2 antagonistic properties (i.e., did not affect calcium release in vitro and competitively antagonized the agonistic effect of [Tyr (3)]octreotide). Moreover, in an immunofluorescence-based internalization assay, the new analogues prevented sst 2 internalization induced by the sst 2 agonist [Tyr (3)]octreotide without being active by themselves. In conclusion, several analogues (in particular 3, 31, and 32) have outstanding sst 2 binding and functional antagonistic properties and, because of their DOTA moiety, are excellent candidates for in vivo targeting of sst 2-expressing cancers.

Switch from type II to I Fas/CD95 death signaling on in vitro culturing of primary hepatocytes

Fas/CD95-induced apoptosis of hepatocytes in vivo proceeds through the so-called type II pathway, requiring the proapoptotic BH3-only Bcl-2 family member Bid for mitochondrial death signaling. Consequently, Bid-deficient mice are protected from anti-Fas antibody injection induced fatal hepatitis. We report the unexpected finding that freshly isolated mouse hepatocytes, cultured on collagen or Matrigel, become independent of Bid for Fas-induced apoptosis, thereby switching death signaling from type II to type I. In such in vitro cultures, Fas ligand (FasL) activates caspase-3 without Bid cleavage, Bax/Bak activation or cytochrome c release, and neither Bid ablation nor Bcl-2 overexpression is protective. The type II to type I switch depends on extracellular matrix adhesion, as primary hepatocytes in suspension die in a Bid-dependent manner. Moreover, the switch is specific for FasL-induced apoptosis as collagen-plated Bid-deficient hepatocytes are protected from tumor necrosis factor alpha/actinomycin D (TNFalpha/ActD)-induced apoptosis. Conclusion: Our data suggest a selective crosstalk between extracellular matrix and Fas-mediated signaling that favors mitochondria-independent type I apoptosis induction.