Antiadhesive properties of biological surfaces are protective against stimulated granulocytes.

Despite the fact that a series of endogenous and exogenous inflammatory mediators are potent activators of circulating granulocytes, damage of vascular endothelium, a primary target tissue, is a rather unusual event in systemic inflammatory states. Since mediator-induced neutrophil hyperadhesiveness on plastic tissue culture dishes is invariably accompanied by intense release of lysosomal granule constituents and respiratory burst activation, thus representing a powerful model to investigate neutrophil cytotoxic states, comparative studies with neutrophils suspended in autologous plasma in the presence or absence of N-formyl-Met-Leu-Phe (2.5 microM), the most potent adhesion inducer, were performed on different biologic surfaces. On optimally adherent closed monolayers of cultured endothelial cells or fibroblasts we observed poor stimulation of adhesion as well as minimal granule release and hexose monophosphate pathway activation. Functional behavior of neutrophils on single molecular components of basal laminas such as fibronectin and collagen (type IV) coats was intermediate, with positive adhesion promotion but markedly reduced metabolic activation. When tested on endothelial cell-derived extracellular matrices, neutrophils again showed functional nonresponsiveness to N-formyl-Met-Leu-Phe. Scanning electron microscopy revealed an impressive congruency between the degree of cellular spreading and metabolic activation in the presence of N-formyl-Met-Leu-Phe, with maximally flattened neutrophils on plastic vs. nonspread, polarized cells on monolayers. Identical results were obtained by using other adhesion inducers such as complement-activated plasma or endotoxin. Lack of cell injury by N-formyl-Met-Leu-Phe-exposed neutrophils was corroborated by the absence of tracer release from [111In]tropolonate-labeled endothelium. These results indicate that biologic surfaces possess antiadhesive properties that protect them from cytotoxic damage by stimulated angry phagocytes.

Interleukin 1 and tumor necrosis factor stimulate human vascular endothelial cells to promote transendothelial neutrophil passage.

In an attempt to understand the regulatory mechanisms governing passage of neutrophils from the vascular bed to the interstitial tissue, we analyzed the effect of the pleiotropic monokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) on transendothelial neutrophil traffic. Short-time preincubation of human umbilical vein endothelial cell (HUVE) monolayers with IL-1 and TNF led to an impressive time- and dose-dependent increase of endothelial cell-associated neutrophils when working in a full plasma system on petri dishes. Electron microscopic analysis revealed junctional penetration of monolayers by neutrophils. More quantitatively, when using a monolayer-on-filter-system, priming led to a severalfold increase in complete layer passage occurring in the absence of an external chemotactic gradient. Direct comparison with an upside-down modification of the system together with data demonstrating the vectorial behavior of such migration revealed that IL-1-stimulated transendothelial neutrophil traffic is polarized. The described enhancement of neutrophil transendothelial passage was found to be a unique feature of IL-1/TNF-activated HUVE since HUVE-dependent transmigration potentiation was not observed as a consequence of mere neutrophil attachment to endothelial cells (e.g., induced by Fc-mediated adherence of PMN to HUVE). IL-1 acts selectively on endothelial cells as demonstrated by total inhibition of its effect by actinomycin D. Moreover, IL-1 does not induce HUVE monolayers to secrete a chemotaxin, and the neutrophil passage guiding principle is removable from the HUVE surface by short trypsin exposure. Congruent results were obtained with human adult arterial as well as saphenous vein endothelial cells. As shown by blockade of neutrophil migration with pertussis toxin, IL-1- and TNF-inducible transendothelial migration can be dissected into an initial anchoring step, which is succeeded by active neutrophil migration, possibly along a putative endothelial membrane-bound gradient.

Anti-Fas/APO-1 antibody-mediated apoptosis of cultured human glioma cells. Induction and modulation of sensitivity by cytokines.

Fas/APO-1 is a transmembrane protein of the nerve growth factor/TNF alpha receptor family which signals apoptotic cell death in susceptible target cells. We have investigated the susceptibility of seven human malignant glioma cell lines to Fas/APO-1-dependent apoptosis. Sensitivity to Fas/APO-1 antibody-mediated cell killing correlated with cell surface expression of Fas/APO-1. Expression of Fas/APO-1 as well as Fas/APO-1-dependent cytotoxicity were augmented by preexposure of human malignant glioma cells to IFN gamma and TNF alpha. Further, pretreatment with TGF beta 2, IL1 and IL8 enhanced Fas/APO-1 antibody-induced glioma cell apoptosis whereas other cytokines including TNF beta, IL6, macrophage colony-stimulating factor, IL10 and IL13 had no such effect. None of the human malignant glioma cell lines was susceptible to TNF alpha-induced cytotoxicity. Fas/APO-1 antibody-sensitive glioma cell lines (n = 5), but not Fas/APO-1 antibody-resistant glioma cell lines (n = 2), became sensitive to TNF alpha when co-treated with inhibitors of RNA and protein synthesis. Resistance of human glioma cells to Fas/APO-1 antibody-mediated apoptosis was mainly related to low level expression of Fas/APO-1 and appeared not to be linked to overexpression of the anti-apoptotic protooncogene, bcl-2. Given the resistance of human malignant glioma to surgery, irradiation, chemotherapy and immunotherapy, we propose that Fas/APO-1 may be a promising target for a novel locoregionary approach to human malignant glioma. This strategy gains support from the demonstration of Fas/APO-1 expression in ex vivo human malignant glioma specimens and from the absence of Fas/APO-1 in normal human brain parenchyma.

Imaging and therapy of small cell carcinoma xenografts using 131I-labeled monoclonal antibody SWA11.

The IgG2a monoclonal antibody SWA11 has been evaluated as a radioimmunotherapeutic agent for use in the treatment of small cell cancer of the lung. This antibody was initially selected for in vivo localization studies in a nude mouse model system because of its high affinity for the SW2 small cell cancer cell line in vitro. Following i.v. injection of 125I labeled antibody into nude mice bearing SW2 xenografts good selective accumulation was observed with 10.5% of injected material/g of tumor. The level remained constant from day 2 to day 4 following injection. At day 4 the tumor:blood ratio was 7:1 and tumor:liver, tumor:kidney, and tumor:lung ratios were 17:1, 24:1, and 12:1, respectively. Initial radioimmunotherapeutic studies performed on established small cell cancer of the lung xenografts have shown reduction in tumor burden following a single injection of 300 microCi of 131I labeled SWA11 with no evidence of regrowth up to day 34 postinjection. Histological evaluation of treated tumors revealed large areas of necrosis and extensive fibrosis. A few residual cells of tumor origin could be observed and these displayed atypical morphology. The clonogenic potential of such cells remains to be determined by long term observation.

Death ligand/receptor-independent caspase activation mediates drug-induced cytotoxic cell death in human malignant glioma cells.

Death ligand/receptor interactions and caspase activation mediate drug-induced apoptosis in certain cancer cells. The molecular mechanisms responsible for the chemoresistance of human malignant gliomas are largely unknown. Here, we report that malignant glioma cells co-express CD95 and CD95L without undergoing suicidal or fratricidal apoptosis. Glioma cells do not commit CD95/CD95L-dependent suicide or fratricide even when RNA and protein synthesis are inhibited. This is because ectopic expression of the viral caspase inhibitor, crm-A, or exposure to a neutralizing CD95L antibody, block apoptosis induced by exogenous CD95L but not cell death induced by cytotoxic concentrations of inhibitors of RNA and protein synthesis. Although some cytotoxic drugs enhance the expression of CD95 or CD95L, crm-A fails to block drug-induced cytotoxic and clonogenic cell death, suggesting that the drug-induced changes in CD95 and CD95L expression are epiphenomenal. There is also no difference in drug-induced apoptosis between crm-A-transfected and control cells as assessed by electron microscopy, in situ DNA end labeling and DNA fragmentation. Further, glioma cells selected for resistance to CD95L do not acquire cross-resistance to chemotherapy. However, the broad spectrum caspase inhibitor, ZVAD-fmk, inhibits drug-induced cytotoxic cell death, suggesting a role of crm-A-insensitive caspases in drug-induced apoptosis of glioma cells. Thus, drug resistance of malignant glioma cells may involve deficiencies in two interrelated pathways that mediate death in order tumor cell types: (i) death ligand/receptor signalling; and (ii) caspase activation.

Comparative studies of the preparation of immunoliposomes with the use of two bifunctional coupling agents and investigation of in vitro immunoliposome-target cell binding by cytofluorometry and electron microscopy.

The two coupling agents SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) and SATA (N-succinimidyl-S-acetylthioacetate) were compared in their efficiency and feasibility to couple monoclonal antibodies (Abs) via thioether linkage to liposomes functionalized by various lipophilic maleimide compounds like N-(3-maleimidopropionyl)-N2-palmitoyl-L-lysine methyl ester (MP-PL), N-(3-maleimidopropionyl)phosphatidylethanolamide (MP-PE), N6-(6-maleimidocaproyl)-N2-palmitoyl-L-lysine methyl ester (EMC-PL), and N-(6-maleimidocaproyl)phosphatidylethanolamine (EMC-PE). The composition of the liposomes was soy phosphatidylcholine (SPC), cholesterol, maleimide compounds and alpha-tocopherol (1:0.2:0.02:0.01, mol parts), plus N4-oleylcytosine arabinoside (NOAC) as cytostatic prodrug (0.2 mol parts) and a new, lipophilic and highly fluorescent dye N,N'-bis(1-hexylhfetyl)-3,4:9,10-perylenebis(dicarboximid ) (BHPD, 0.006 mol parts). From the maleimide derivatives MP-PL was the most effective in terms of preservation of the coupling activity in dependence of liposome storage. The coupling of the monoclonal A B8-24.3 (mouse IgG2b, MHC class I, anti H-2kb) and IB16-6 (rat IgG2a, anti B16 mouse melanoma) to the drug carrying liposomes was more effective and easier to accomplish with SATA as compared to SPDP. Coupling rates of 60-65% were obtained with SATA at molar ratios of 12 SATA:1 Ab:40 maleimide spacer groups on the surface of one liposome. The highest coupling rates with SPDP were obtained at the ratio of 24 SPDP:1 Ab:40 liposomal maleimide groups, with an Ab binding efficiency of only 20-25%. The optimal in vitro binding conditions to specific target cells (EL4 for B8-24.3-liposomes and B16-F10 for IB16-6-liposomes) were determined by cytofluorometric measurement of the liposomal BHPD fluorescence with SATA linked Abs. Optimal immunoliposome binding to specific epitopes on the target cells was achieved with 1-2 Ab molecules coupled to one liposome, with immunoliposome concentrations of 20-130 nM and with a small incubation volume of 0.3-0.4 ml. The specificity of the binding of B8-24.3-liposomes to EL4 target cells was visualized by scanning electron microscopy. Antibody mediated endocytic uptake of immunoliposomes could be demonstrated by transmission electron microscopy.

Pathways of tumour cell killing by activated macrophages: both tumour cell membrane and nucleus can be the primary target.

Plasma membrane and nucleus can be primary targets of tumour cell killing by activated macrophages (AMø). Necrotic-type cytotoxicity with loss of membrane integrity and cytoplasmic swelling was expressed by AMø from normal and from perforin-deficient mice, indicating that perforin was not involved. Incubation with AMø consistently triggered the release of thymidine from prelabelled targets, whereas chromatin condensation and small DNA fragments were only occasionally detected. It is shown by means of Pulsed-Field Gel Electrophoresis that DNA degradation in target cells is a slowly progressing process that may stop at any time, indicating that nuclear-type killing doesnot necessarily lead to the formation of low molecular weight fragments. Neither Fas nor the p55 tumour necrosis factor receptor appear to be involved in signalling nuclear-type killing. Accordingly, AMø do mediate membrane- and nuclear-type killing but the mechanisms differ from those identified in T cell cytotoxicity.

Extended therapeutic window for caspase inhibition and synergy with MK-801 in the treatment of cerebral histotoxic hypoxia.

In rats, striatal histotoxic hypoxic lesions produced by the mitochondrial toxin malonate resemble those of focal cerebral ischemia. Intrastriatal injections of malonate induced cleavage of caspase-2 beginning at 6 h, and caspase-3-like activity as identified by DEVD biotin affinity-labeling within 12 h. DEVD affinity-labeling was prevented and lesion volume reduced in transgenic mice overexpressing BCL-2 in neuronal cells. Intrastriatal injection of the tripeptide, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a caspase inhibitor, at 3 h, 6 h, or 9 h after malonate injections reduced the lesion volume produced by malonate. A combination of pretreatment with the NMDA antagonist, dizocilpine (MK-801), and delayed treatment with zVAD-fmk provided synergistic protection compared with either treatment alone and extended the therapeutic window for caspase inhibition to 12 h. Treatment with cycloheximide and zVAD-fmk, but not with MK-801, blocked the malonate-induced cleavage of caspase-2. NMDA injections alone resulted in a weak caspase-2 cleavage. These results suggest that malonate toxicity induces neuronal death by more than one pathway. They strongly implicate early excitotoxicity and delayed caspase activation in neuronal loss after focal ischemic lesions and offer a new strategy for the treatment of stroke.

A new look at the role of p53 in leukemia cell sensitivity to chemotherapy.

A gene encoding the p53 val135 mutant, which assumes mutant conformation at 38.5 degrees C and wild-type conformation at 32.5 degrees C, was introduced into p53-deficient K562 myeloid leukemia cells. Forced expression of wild-type, but not mutant, p53 resulted in growth arrest, accumulation of p21 and Bax proteins, and delayed cell death. Wild-type p53 enhanced the cytotoxic effects of some drugs and attenuated those of others. Compared with wild-type p53, mutant p53 induced much stronger sensitization to drug cytotoxicity. This occurred in the absence of effects on cell cycle progression or activation of several p53 target genes. Although both mutant and wild-type p53 induced changes of immunophenotype, no specific pattern of differentiation was associated with enhanced chemosensitivity. Thus, (1) induction of growth arrest and activation of p53 target genes such as p21 and bax are linked to the wild-type conformation of p53; (2) p53 induces immunophenotypic changes of myeloid leukemia cells suggestive of multidirectional differentiation in a conformation-dependent manner; and (3) (so-called) mutant p53 induces chemosensitization in the absence of effects on cell cycle progression, activation of bax, p21, gadd45 and mdm-2, or a specific pattern of differentiation; and (4) chemosensitization mediated by wild-type p53 may be masked by transcription-dependent induction of growth arrest.

PGI2 and PGE1 induce morphological alterations in human platelets similar to those of the initial phase of activation.

The effects of PGI2 and PGE1 on the ultrastructure of human platelets were studied by scanning (SEM) and transmission (TEM) electron microscopy in relation to the record of an optical aggregometer. Addition of PGI2 or PGE1 to citrated platelet-rich plasma (C-PRP) resulted in a permanent slight decrease in percent light transmission (%T) recorded by the aggregometer. SEM investigation of the platelets showed marginal pseudopods and occasional large stomata after application of prostaglandins. These alterations occurred within the initial 30 s and remained constant during the subsequent 20 min of incubation. TEM studies revealed morphological changes of alpha granules and moderately electron-dense material in the dilated profiles of the surface-connected canalicular system (SCCS). Addition of 10 microM ADP to C-PRP preincubated for 30 s with either 2 ng/ml (5 nmol/liter) PGI2 or 30 ng/ml (85 nmol/liter) PGE1 resulted in a further decrease of %T followed by a slight increase. The alterations of the aggregometer tracing were characterized in SEM by platelet shape change and the generation of primary aggregates. C-PRP samples preincubated with 3 and 9 ng/ml (8 nmol/liter and 24 nmol/liter) PGI2 or 40 and 120 ng/ml (113 nmol/liter and 338 nmol/liter) PGE1 did not produce additional changes in the aggregometer curves or in the ultrastructure of platelets in response to ADP. Our morphological study indicates that antiaggregatory prostaglandins induce an early phase of platelet activation but inhibit "shape change" and the formation of aggregates.

Comparison of FRTL-5 cell growth in vitro with that of xenotransplanted cells and the thyroid of the recipient mouse.

The present work was designed to compare in vitro cell growth kinetics with in vivo growth under conditions as similar as possible using labeling with [3H]thymidine. To this purpose, FRTL-5 cells were cultured as monolayers and as three-dimensional spheroids embedded in collagen gels and transplanted simultaneously into nude mice treated with perchlorate and a low iodine diet. The growth of the transplants was compared to that of the thyroids in host mice. In the intact thyroid, the fraction of [3H]thymidine-labeled follicular cells (FLC; 24-h labeling) increased sluggishly to a maximum of 10% after 3 weeks of goitrogen exposure, with a subsequent autoregulatory decrease to 3% at 7 weeks. A 4-fold higher FLC was found in six adenomas, indicating focal failure of growth-restraining mechanisms. In nonconfluent monolayer cultures the FLC was as high as 90%, even within large individual clusters where cells are in tight mutual contact. Solid, highly cellular grafts growing from transplanted monodispersed cells showed an average FLC of 20%, which is 5 times higher than the FLC in the identically stimulated mouse thyroid. In collagen-embedded cells, forming three-dimensional spheroids, the mean FLC decreased from 70% at 1 week in vitro (40% in vivo) to 20% at 3 weeks both in vitro and in vivo, suggesting effective auto-regulation of excessive growth in both conditions. However, these FLC were again much higher than the 3% FLC in simultaneously assessed host thyroids. The difference remained throughout the 45-day period studied. We conclude that FRTL-5 cells growing as monolayers and as three-dimensional spheroids in vitro or after xenotransplantation in vivo invariably show much higher proliferation rates under comparable environmental conditions than the normal follicular epithelium in the thyroids of host mice. The one exception is the confluent monolayer with near-zero growth, while densely packed three-dimensional transplants still grow intensively. Although growth-retarding cell to cell interactions are also clearly operative in growing FRTL-5 cells, they are less effective than those dampening the replication rate of the thyrocytes within the monolayer hull of normal follicles. A local failure of these mechanisms, allowing growth rates comparable to those of grafted FRTL-5 cells results in adenoma formation in normal thyroids. These observations call for caution in the transfer of in vitro growth studies with FRTL-5 cells to in vivo conditions prevailing in the normal thyroid.

Autonomous growth, but not autonomous function, in embryonic human thyroids: a clue to understanding autonomous goiter growth?

Thyroid glands from six 8- to 10-week-old fetuses obtained at the time of legal abortion were cryopreserved in liquid nitrogen and transplanted into nude nu/nu mice. Histological and autoradiographic studies of the grafts labeled with [3H]thymidine and [125I]iodine showed proliferation and functional differentiation of the fetal thyroid tissue. Despite T4-mediated suppression of host TSH secretion, up to 36% of the follicular cell nuclei incorporated the thymidine label, reflecting autonomous proliferation, while iodine organification was almost entirely obliterated. Methimazole-induced TSH hypersecretion readily stimulated both growth and function of the transplanted tissue. Thus, during early development, the human thyroid largely depends on TSH for function, but not for growth. Similar findings were obtained in newborn mice, in whom 58% of the thyroid follicular cells proliferated autonomously, i.e. in the absence of TSH. The number of autonomously proliferating cells gradually declined with increasing age to about 1% in 60-day-old animals and, as reported previously, in xenotransplanted normal human thyroid tissue, whereas the number of autonomously proliferating cells was previously found to be several times higher in xenotransplanted human multinodular goiters. We, therefore, hypothesize that the rapidly and autonomously replicating cells that initiate nodule formation in human multinodular goiters reflect the persistence in the adult gland of cells with fetal growth potential.

Preparation of basal cell membranes for scanning probe microscopy.

Scanning probe microscopy has the potential for investigating membranes in a physiological environment. We prepared with a lysis-squirting protocol basal cell membranes, that are suitable for scanning probe microscopy. Investigations using atomic force microscopy under liquid revealed cellular filaments which correlated perfectly with fluorescently stained actin filaments. Globular structures with a diameter as little as 10 nm could be resolved by stripping cytoplasmic components from the membranes. Therefore, cytoplasmic sides of supported basal cell membranes prove useful to gain high resolution with scanning probe microscopy in studies of plasma membrane associated structures and processes under buffer solution.

Modulation of the antilisterial activity of human blood-derived macrophages by activating and deactivating cytokines.

A concept of macrophage deactivation by hormones and cytokines that opposes activation was recently proposed. Deactivation of the antilisterial activity of macrophages by IL-4, IL-10, and TGF-beta, as well as by dexamethasone, was studied here. IL-4, IL-10, and dexamethasone, but not TGF-beta, caused a complete loss of the competence of human blood-derived macrophages infected with Listeria monocytogenes to control or eliminate ingested bacteria. IL-10 and, to a lesser degree, dexamethasone lessened in parallel the capacity of macrophages to secrete H2O2. The antilisterial activity of cells simultaneously exposed to deactivating agents could be significantly augmented by IFN-gamma. Likewise, TNF-alpha and to a limited degree GM-CSF increased the antilisterial activity of cells treated with IL-10 and dexamethasone but not that of cells treated with IL-4. Suppression of TNF-alpha secretion in response to Listeria by TGF-beta, IL-10, dexamethasone, or pentoxifylline did not closely parallel antilisterial activity. Studies by transmission electron microscopy and actin staining suggested that deactivation by IL-10, IL-4, and dexamethasone of human blood-derived macrophages resulted in intraphagosomal multiplication of Listeria followed only then by an escape of bacteria into the cytoplasm. The antibacterial competence of human macrophages is lessened by IL-4 and IL-10 and augmented by IFN-gamma, TNF-alpha, and GM-CSF. The success of human macrophages in controlling intracellular pathogens appears to depend on the balance of activating and deactivating mediators modulating their activity.

Carcinoembryonic antigen (CEA) presentation and specific T cell-priming by human dendritic cells transfected with CEA-mRNA.

The feasibility of dendritic cells (DC) for cancer immunotherapy after transfection by electroporation with mRNA encoding the human carcinoembryonic antigen (CEA) was investigated. Both, total RNA from the CEA(+) colon cancer cell line SW480 and mRNA transcribed in vitro from cDNA3.1-plasmids (pcDNA3.1+/-HisC) with a CEA-insert (ivt-CEA-mRNA, ivt-CEA/HisC-mRNA) were used. Labelled ivt-CEA-mRNA was detectable in DC by light and electron microscopy and by fluorescence-activated cell-sorting (FACS) even 15 min after electroporation. Four hours after transfection with ivt-CEA/HisC-mRNA, we detected specific expression of CEA and the histidine-tag by immunofluorescence microscopy and by FACS. CEA-specific T lymphocytes were successfully primed by transfected DC and were able to lyse CEA-expressing target cells, even from the CEA-expressing human colon adenocarcinoma cell line SW480. Thus, DC transfected by electroporation with CEA-mRNA are valuable tools for the immunotherapy of CEA(+) tumour entities.

A chemiluminescent assay for mycoplasmas in cell cultures.

A chemiluminescent assay for the detection of mycoplasma contamination of cell cultures is described. Cells (and supernatant) derived from mycoplasma-contaminated cultures stimulate a burst of luminol-dependent chemiluminescence in cell suspensions containing phagocytic effector cell types. The assay conditions for spleen cells, human and bovine polymorphonuclear leucocytes as the responder or indicator cells have been optimized. The chemiluminescent assay can be utilized for both monolayer and suspension cell cultures and is more sensitive than colony formation on agar plates and electron microscopy. Results are obtained within 3-5 h including the time required for the preparation of the indicator cells. CL can be measured in the tritium window of standard liquid scintillation spectrometers after switching off the coincidence circuit.

Evidence for an active type of cell death with ultrastructural features distinct from apoptosis: the effects of 3-acetylpyridine neurotoxicity.

3-Acetylpyridine is a niacinamide antagonist with potent neurotoxic properties in vitro and in vivo. 3-Acetylpyridine neurotoxicity was associated with positive DNA end-labelling and displayed features of active cell death without the ultrastructural changes of apoptotic cell death. After systemic administration in rats (70 mg/kg), we detected labelled nuclei in the inferior olive using in situ DNA end-labelling. However, the conventional chromatin stain did not show chromatin condensation or fragmentation and electron microscopy studies failed to reveal features of apoptosis. Although areas of condensed chromatin were present in some nuclei, cytoplasmic damage with extensive organelle swelling was the most prominent finding. In vitro, 3-acetylpyridine (0.1-1 mM) induced degeneration of cerebellar granule neurons in a concentration- and time-dependent manner. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) and the transcriptional inhibitor actinomycin D (10 microM) protected against 3-acetylpyridine toxicity. In contrast, neither the free radical scavenger alpha-phenyl-N-tertbutylnitron (100 microM), nor glutathione ethyl ester (10-100 microM), N-acetyl-cysteine (10-200 microM) or 3-aminobenzamide (0.1-4 mM), an inhibitor of poly(ADP-ribose) synthesis, were effective. 3-Acetylpyridine-induced neuronal death in vitro was associated with positive in situ DNA labelling. However, DNA fragmentation could not be demonstrated prior to neuronal cell loss and no DNA "laddering" was detected by DNA gel electrophoresis. Correspondingly, no apoptotic nuclei were revealed upon electron microscopy but organelle swelling and extensive vacuolization, changes similar to autophagocytosis. In conclusion, 3-acetylpyridine induces an active form of cell death that required de novo protein synthesis but is distinct from apoptosis. A loss of glutathione accompanies, but does not precede, cell death.

Blood platelets in cardiopulmonary bypass operations. Recovery occurs after initial stimulation, rather than continual activation.

The ultrastructure of blood platelets was related to platelet function and secretion products before, during, and after cardiopulmonary bypass. Circulating platelets from 15 patients undergoing aorta-coronary bypass operations were investigated at ten predetermined points of time by scanning and transmission electron microscopy. Simultaneously, platelet adenosine triphosphate, diphosphate, and serotonin, as well as plasma levels of platelet factor 4, beta-thromboglobulin, serotonin, thromboxane B2, lactic dehydrogenase, and free hemoglobin were measured. Moreover, platelet responsiveness toward adenosine diphosphate and collagen was determined by optical aggregometry. By scanning electron microscopy, the number of unactivated platelets dropped from 96% +/- 4% to 54% +/- 19% (p less than 0.05) 8 minutes after the onset of bypass. Simultaneously, the percentage of "shape changed" platelets significantly increased. No major release reaction was detected at this time. After the initial activation, platelet morphology began to recover although the bypass continued. During the late period of bypass, a highly significant correlation between increasing plasma levels of alpha-granule compounds (platelet factor 4 and beta-thromboglobulin) and lysis parameters (lactic dehydrogenase and free hemoglobin) was found. However, transmission electron microscopic analysis of the arterial filter and scanning electron microscopic findings of circulating platelets indicated that the release products in plasma were due not only to platelet lysis but also to a limited extent to secondary aggregation. In an inverse and probably causative manner, platelet morphology recovered, whereas the sensitivity of platelets to adenosine diphosphate and collagen decreased toward the end of bypass.

Preparation and in vivo testing of porous alumina ceramics for cell carrier applications.

Microporous alumina was used to develop implantable cell carriers shaped as a hollow-sphere with a central opening to allow ingrowth of vascularised tissues. The carriers were produced by suspending the ceramic raw materials in water, homogenising and dropping the resulting slurry onto a heated plate (hot plate moulding, HPM). Morphological characteristics of the cell carriers were investigated by SEM and optical microscopy. Produced carriers had an average diameter of 4.9 mm. The material was highly porous (56 +/- 8%). For in vivo testing the cell carriers were implanted into abdominal wall of Zur: SIV rats for up to 50 weeks and investigated by light microscopy, SEM and TEM. The surface of the hollow carriers was in close contact with unirritated muscle tissue; no inflammation or capsule formation was observed. Loose connective tissue had grown into the hollow cell carrier, and after prolonged implantation >20 weeks adipocytes were observed. The absence of scar tissue formation around the implant and the vitality within the cavity of the hollow carriers indicate that porous alumina may be used for cell transplantation devices.