Isolation and culture of hepatic stellate cells.

Hepatic stellate cells (HSCs) are routinely prepared by collagenase/pronase digestion of liver using a perfusion system and subsequent fractionation of the heterogeneous cell suspension on continuous density gradients made out of Nycodenz, metrizamide, stractan, or percoll. Because of their lipid content, stellate cells are the least dense fraction of the nonparenchymal cells, and during centrifugation they float effectively away from other hepatic cells resulting in preparations containing almost 80% stellate cells. The degree of purity can be increased by further enrichment of cells by methods like centrifugal elutriation or Scatter-activated cell sorting. We present a detailed protocol from our laboratory to obtain a high number of pure, viable, freshly isolated hepatic stellate cells from rat liver. This two-step protocol (collagenase/pronase digestion and Nycodenz gradient) yields a preparation of approx 4-5 x 107 cells enriched in 74% HSC having a viability of at least 76% as estimated by Trypan blue exclusion test. Further purification by centrifugal elutriation results in virtually pure HSC preparations ( >98%).

Evaluation of donor-specific transfusion sources: unique failure of bone marrow cells to induce prolonged skin allograft survival with anti-CD154 monoclonal antibody.

BACKGROUND: Treatment with anti-CD154 monoclonal antibody (mAb) plus a donor-specific transfusion (DST) of spleen cells prolongs skin allograft survival in mice through a mechanism involving deletion of host alloreactive CD8(+) T cells. It is unknown if other lymphohematopoietic cell populations can be used as a DST. METHODS: Murine recipients of allogeneic skin grafts on day 0 were either untreated or given a DST on day -7 plus 4 doses of anti-CD154 mAb on days -7, -4, 0, and +4. Deletion of CD8(+) alloreactive cells was measured using "synchimeric" CBA recipients, which circulate trace populations of TCR transgenic alloreactive CD8(+) T cells. RESULTS: Transfusion of splenocytes, thymocytes, lymph node cells, or buffy coat cells led to prolonged skin allograft survival in recipients treated with anti-CD154 mAb. In contrast, bone marrow DST failed to delete host alloreactive CD8(+) T cells and was associated with brief skin allograft survival. Transfusions consisting of bone marrow-derived dendritic cells or a mixture of splenocytes and bone marrow cells were also ineffective. CONCLUSIONS: Donor-specific transfusions of splenocytes, thymocytes, lymph node cells, or buffy coat cells can prolong skin allograft survival in recipients treated with costimulation blockade. Bone marrow cells fail to serve this function, in part by failing to delete host alloreactive CD8(+) T cells, and they may actively interfere with the function of a spleen cell DST. The data suggest that transplantation tolerance induction protocols that incorporate bone marrow cells to serve as a DST may not be effective.

Enrichment of unlabelled human Langerhans cells by modified discontinuous Ficoll-metrizoate density medium and following Langerhans cell culture.

Highly enriched and specifically unlabelled human Langerhans cells (LC) from epidermal cell suspensions (ECS) are indispensable for the intensive study of LC function. Discontinuous Ficoll-metrizoate density gradient centrifugation was found to be a satisfactory method of enriching LC from ECS. In contrast to a previous study, however, the majority of LC floated on Ficoll-metrizoate with a density of 1.057 g/cm3 instead of 1.068 g/cm3. The concentration of unlabelled LC can be enriched to as high as 92% from the original 1.8-3.1% LC in ECS. Approximately 40-50% of original LC can be harvested. The procedure was also simplified by using a Schick razor instead of a dermatome to obtain thin epidermal sheets (about 0.25 mm in thickness), omitting the density gradients of 1.089 and 1.10 g/cm3 and using the avidin-biotin complex method to identify LC. LC are non-proliferative cells in in vitro culture systems. The viability of LC dropped to 20-30% after 3 days in the culture medium. It was also observed that LC tended to attach to aggregated keratinocytes in the culture system.

Effect of thyroid hormones and other iodinated compounds on the transition of monocytes into veiled/dendritic cells: role of granulocyte-macrophage colony-stimulating factor, tumour-necrosis factor-alpha and interleukin-6.

Stimulation of human peripheral blood monocytes with the thyroid hormones tri-iodothyronine (T3) and thyroxine (T4) enhanced their ability to mature into cytologically and functionally characteristic veiled/dendritic cells. Veiled/dendritic cell transition induced by T3 and T4 was dependent on the production of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF alpha) and interleukin-6 (IL-6) in the culture, since the addition of antibodies specific for GM-CSF, TNF alpha and IL-6 to the culture system had blocking effects. The addition of antibodies to macrophage colony-stimulating factor and IL-1 had no effects. Contaminating T cells and B cells did not contribute to the transition of monocytes to veiled/dendritic cells, and it is therefore likely that the GM-CSF, TNF alpha and IL-6 produced in the culture system were derived from the monocytes themselves. Stimulation of the blood monocytes with an optimal concentration of metrizamide (14.5%), reverse T3 (rT3; 2 x 10(-10) M) or highly iodinated thyroglobulin (Tg; 2 x 10(-11) M) also resulted in an increased transition of monocytes to veiled/dendritic cells, but to a lesser extent in comparison with the thyroid hormones (T3, 31 +/- 6% and T4, 25 +/- 5% vs rT3, 22 +/- 8% and Tg with an iodination grade of 0.37%: 20 +/- 4% veiled/dendritic cells). Administration of anti-GM-CSF, anti-TNF alpha and anti-IL-6 to the culture system also had blocking effects on the transition from monocytes to veiled/dendritic cells induced by the iodinated compounds. The mechanisms by which such iodinated compounds act on the monocyte to veiled/dendritic cell transition can only be speculated on (interference H2O2-generating system?).

Evidence that swainsonine pretreatment of rats leads to the formation of autophagic vacuoles and endosomes with decreased capacity to mature to, or fuse with, active lysosomes.

The plant toxin swainsonine causes a variety of biochemical and morphological changes in animal tissues. In rat liver there is an extensive vacuolization which is not accompanied by an accumulation of oligosaccharide. In investigating this proliferation of autophagic vacuoles we have found that swainsonine administration leads to a shift in the density of liver lysosomes as indicated by the distribution of several lysosomal glycosidases in sucrose gradients. Whereas most of these activities are normally found in low density fractions, only a minor portion occurring in high density fractions, the reverse distribution is observed after the administration of microgram doses of swainsonine. Two promoters of the accumulation of autophagic vacuoles, vinblastine and chloroquine, caused the expected increase in very light vacuoles as measured by localization of two acid hydrolases. However, this effect of the two agents was blocked by swainsonine pretreatment. Moreover, swainsonine decreased the degradation of endocytosed asialofetuin and increased the retention of the glycoprotein in very light fractions. These results suggest that vesicle movement and/or fusion is inhibited by the pretreatment with the toxin. That the effects are mediated by a change in vacuolar membrane is suggested by the finding that lysosomes prepared from the livers of swainsonine-fed rats are much more fragile than control lysosomes, more so in metrizamide solutions than in sucrose solutions. The swainsonine may exert its effect through its known ability to alter the biosynthesis of complex glycoproteins, which are abundant and distinctive in lysosomal membranes.

A species/strain comparison of hepatic natural lymphocytotoxic activities in rats and mice.

The natural killer (NK) and natural cytotoxic (NC) cell activities in livers from certain rat and mouse strains were compared. This included the two rodent strains used in animal carcinogenicity bioassays, i.e. Fischer 344 and B6C3F1 mice. Sprague-Dawley and Fischer 344 rats exhibited high hepatic NK activity, which was greater than the levels seen in all of the five mouse strains studied. However, the hepatic NC activity in rats was comparable to the activities observed in C57BL and BALB/c mice. An inverse relationship was observed between the two tumoricidal activities in all but one of the mouse strains examined; that is (at 8 weeks of age), NK activity: C3H greater than B6C3F1 greater than CBA greater than BALB/c; NC activity: BALB/c much greater than CBA greater than B6C3F1 greater than C3H. The C57BL mouse strain was the only strain to express both activities at comparatively high levels. Female mice exhibited a similar profile of cytotoxic activities. Rats also possessed high activities of a presently ill-defined tumoricidal activity, this being the spontaneous P815 mastocytoma killing by unstimulated effector cells, over an 18 h period. Both adherent and nonadherent effector cells from rat livers, but only the nonadherent cell population isolated from male mouse livers, exhibited this activity which may represent a distinct hepato-specific population of natural lymphocytotoxic effector cells. The tumoricidal activities in liver-derived cells were greater than those of effector cells isolated from the spleen. The differences in natural immunity reported in this study may be related to the varying background incidences of hepatic tumors, i.e. the mouse strains susceptible to high background incidences of liver tumors have relatively low natural immunity, whereas the two mouse strains resistant to hepatic tumors possess high levels of at least one hepatic NLC activity. Similarly, rats have relatively low hepatic tumor rates and high levels of hepatic natural immunity.

A neurite-promoting factor from muscle supports the survival of cultured chicken spinal motor neurons.

During embryonic development, spinal motor neurons require muscle-derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle-derived neurite-promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6-day-old chicken embryos by a metrizamide step-gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%-6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutinin-horseradish peroxidase or the fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6-fold enriched in fraction I and the specific CAT activity was 4.4-fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle-derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high-affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle-derived NPF and laminin was used as the substrate. Muscle-derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite-promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons.

Uptake of antigen by afferent lymph dendritic cells mediated by antibody.

Dendritic cells isolated from sheep afferent lymph were examined for their ability to bind soluble protein and peptide antigens labeled with fluorescein both in in vitro assays and following intradermal injection of antigen in vivo. Analysis of dendritic cells by flow cytometry revealed weak direct binding of proteins and peptide antigens. However, the degree of uptake was greatly enhanced in the presence of specific antibody in vitro or if antigen was injected intradermally into antigen-primed sheep. About 60% of dendritic cells possessed the ability to take up antigen in both the in vitro and in vivo experiments. The uptake of antigen occurred very rapidly, reaching maximum values in terms of cell numbers and fluorescence intensity in less than 5 min in vitro and 20-40 min following in vivo challenge. Both sheep IgG subclasses could mediate this effect, but F(ab')2 fragments were ineffective. Procedures adopted to remove complement components from the in vitro test mixtures did not result in any reduction in the binding of antigen by dendritic cells. Two-color flow cytometry analysis of the dendritic cell population further showed that 43% of cells taking up the antigen/antibody complexes were CD1+, suggesting a relationship between these cells and Langerhans' cells or other dendritic cells in skin. The results, thus, indicate that approximately two thirds of sheep afferent lymph dendritic cells bind antigen/antibody complexes via an Fc receptor, a mechanism which could be important in the accentuated accessory function of these cells known to occur following secondary antigen challenge.

Local glucose utilization changes caused by subarachnoid contrast media in the rabbit.

The glucose metabolism effects of six hour exposures to subarachnoid injections of metrizamide, iohexol, iodixanol and control solutions were studied in vivo in 18 rabbits. The brain tissue uptake of intravenously injected 14C labelled deoxyglucose was measured using autoradiographic techniques. Metrizamide and iodixanol caused significant (p less than 0.05) decreases in deoxyglucose uptake in the outer cortical areas where the contrast medium concentrations were highest. Iohexol and the control CSF solution did not cause significant effects. The results appear to indicate that iohexol has less effect on brain tissue glucose metabolism than either metrizamide or the new non-ionic dimer iodixanol.

Accessory cells with a morphology and marker pattern of dendritic cells can be obtained from elutriator-purified blood monocyte fractions. An enhancing effect of metrizamide in this differentiation.

Human monocytic cell fractions obtained by counterflow elutriation centrifugation (with regard to specific monocyte/macrophage characteristics: 82-88% were positive for nonspecific esterase; 86-94% for CD14) were cultured (overnight, 37 degrees C) under nonadhering conditions (polypropylene tubes). Thirty to 40% of the cells were found to differentiate into large, monocytoid cells with a dendritic morphology. These cells expressed a marker of active dendritic cells RFD1 in 76-89% and were also positive for class II MHC antigens as identified by OKIa (95-97%). An exposure of the blood monocytic cells to metrizamide (30 min, 14.5%) prior to the overnight culture enhanced this differentiation, and 47-58% of cells with a dendritic morphology were found (of which 80-87% RFD1 positive, and 95-97% Class II MHC positive). The cultured cell populations containing the cells with the morphology and marker pattern typifying dendritic cells, appeared functionally more active as accessory populations when compared to freshly isolated blood monocytic cell fractions; the cultured cells had an enhanced stimulator capability in MLR, and cluster formation with lymphocytes was more prominent. At the same time, the cultured cell population showed a decreased bactericidal activity when compared to the freshly isolated monocytic populations, and in addition, all the cultured monocytoid cells had lost non-specific-esterase activity, while only approximately 10% of cells were still positive for the CD14 marker. When U937 cells were exposed to metrizamide (14.5% concentration, 30 min) and cultured under nonadhering conditions for 36 hours, similar changes were observed (30-45% became dendritic, 20% RFD1 positive).(ABSTRACT TRUNCATED AT 250 WORDS)

[Electronystagmographic evaluation of changes in vestibular reactions after administration of diverse substances into cerebral ventricles]. / Elektronistagmograficheskaia otsenka izmeneniia vestibuliarnykh reaktsii posle vvedeniia v zheludochki mozga razlichnykh veshchestv.

Vestibulo-oculomotor reflexes (nystagmus) were recorded by the method of electronystagmography in 33 neurosurgical patients before and after ventriculography. Cerebral ventricles were examined using water soluble compounds (conray, dimeriks, amipaque) in 18 patients or water soluble compounds combined with majodil emulsion in 15 patients. Ventriculography by means of water soluble compounds led to insignificant changes in nystagmic parameters while that by means of X-ray contrasting mixtures caused a frequent and noticeable enhancement of stem vestibular reactions as related to all nystagmic parameters and a significant increase of vestibulo-autonomic reactions.

Selective fractionation of hypoxic B16 melanoma cells by density gradient centrifugation.

Cells of the mouse B16 melanoma growing in monolayer culture and as tumors were fractionated by isopycnic density centrifugation in a linear-density (1.02-1.20 g/ml) metrizamide gradient. Cultured cells concentrated into one or two distinct bands, with densities of 1.02-1.04 g/ml and 1.06-1.10 g/ml, depending on growth conditions. Cells subjected to extreme hypoxia (less than 0.02% O2) banded predominantly at the lower density, and normally-oxygenated cells banded at the higher density. Fractionated tumor cells concentrated at both densities. Compared with cells at the higher density, lower-density cells incorporated more of the hypoxic cell radiosensitizer [14C]misonidazole and less [3H]thymidine in vivo, were less clonogenic but more resistant to X-irradiation in situ, and labeled to a lesser extent with intravenously-delivered Hoechst 33342 fluorochrome, a marker for cells proximal to tumor blood vessels. Lower-density tumor cells were, therefore, enriched in non-proliferating radioresistant hypoxic cells from tumor regions remote from blood vessels.

Effects of contrast media on neural tissue glucose uptake in vitro.

The authors previously showed that metrizamide causes an inhibition in CO2 production in rat neural tissue. The purposes of this work were to test if this inhibition was the result of a competitive inhibition of metrizamide with the D-glucose transport system and to test the effect of other contrast media. Deoxyglucose was used as a marker for glucose. The first cellular system using rat hippocampus slices was designed to examine the effect of 15 mM and 80 mM metrizamide on deoxyglucose uptake. The second cell-free system, using isolated rat brain synaptosomes, was designed to evaluate more accurately the mechanism and kinetics of metrizamide's inhibitory effect on the uptake of deoxyglucose and to compare metrizamide to other nonionic contrast media (iohexol, iopamidol, iotrol, and iodixanol). These experiments demonstrate that there is inhibition of D-glucose uptake only in hippocampus slices and that the inhibition is dependent on the concentration of metrizamide. This does not, however, appear to be a competitive inhibitory effect on the carrier such as that between D-glucose and 2-deoxy-D-glucose. In synaptosomes, none of the contrast media had a significant effect on the uptake of 2-deoxyglucose.

Diurnal variation of HMG-CoA reductase activity in rat liver peroxisomes.

The specific activity of hepatic microsomal and peroxisomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) was determined at different times during a 24 hour cycle from cholestyramine treated rats. The microsomal HMG-CoA reductase activity displayed a peak at D-6 (6th hour of the dark cycle) as previously reported, whereas, the peroxisomal HMG-CoA reductase activity was the highest at L-2 (2nd hour of the light cycle). Immunoblots of the peroxisomal HMG-CoA reductase suggest that the increase in enzyme activity at L-2 is due to changes in enzyme mass. The different cyclic variations observed in microsomal and peroxisomal HMG-CoA reductase activity may suggest different mechanisms of regulation.

Iopamidol and neural tissue metabolism. A comparative in vitro study.

Metrizamide was the first water-soluble contrast medium with a neurotoxicity low enough to allow it to be used routinely in the entire subarachnoid space. However, neurologic complications are still observed in some patients following the use of metrizamide. The cause of this toxicity has not been established, but existing evidence suggests an interference with glucose metabolism. In previous studies, a depression in CO2 production in neural tissue slices was demonstrated when isotonic metrizamide was added but not isotonic iohexol. In addition to iohexol, there is another new, nonionic, monomeric, water-soluble CM, iopamidol, soon to be released for clinical use in the United States. Iopamidol, like iohexol, has shown fewer adverse reactions and seems to be safer for myelography than metrizamide. Direct comparative studies of iopamidol and iohexol are sparse and the cause of their toxicity is not yet understood. This study was performed to determine the effect of iopamidol on neural tissue glucose metabolism as compared with the effects of iohexol and metrizamide. Metrizamide decreased CO2 production in neural tissue slices by 23%. Iopamidol and iohexol did not produce significant depression. Moreover, this model could not demonstrate any significant difference between iopamidol and iohexol in direct comparisons. The new monomeric contrast media, iopamidol and iohexol, thus do not appear to interfere with glucose metabolism. Adverse reactions to these new media are most likely caused by other mechanisms.

Effect of various contrast media on coagulation, fibrinolysis, and platelet function. An in vitro and in vivo study.

An in vitro and in vivo study of the effect of ionic and nonionic contrast media (CM) on coagulation and platelet function is reported. The methods employed were tests for extrinsic and intrinsic coagulation together with a fibrinolytic parameter and aggregation using ADP and collagen as inducers. The in vivo study utilized patients undergoing routine cerebral angiography. The in vitro results showed a modest influence of the nonionic CM in contrast to the ionic. The marked inhibitory effect of the latter was mainly caused by inherent toxicity, osmolality/ionic strength being of minor importance. The in vivo results showed a negligible influence of CM on systemic hemostatic parameters, but catheter-derived samples indicated desirability of premedication with ASA or heparin. The nonionic CM caused less discomfort than the ionic CM.