96-well Tissue Culture Research Articles

Aprotinin inhibits platelet adhesion to endothelial cells.

Studies were conducted to assess the effect of the serine protease inhibitor aprotinin on platelet adherence to both thrombin-stimulated and unstimulated human umbilical vein endothelial cells. Aprotinin treatment reduced significantly the adherence of platelets to endothelium pretreated or not with thrombin. In addition, aprotinin similarly reduced the adherence of platelets to plastic or collagen-coated tissue culture wells suggesting that the main site of action of the drug in this system is on the platelets. The role of endothelium-derived relaxing factor (EDRF; nitric oxide) in these platelet-endothelium reactions was investigated by prior incubation of both platelets and endothelial cells with NG-monomethyl-L-arginine (L-NMMA) which prevents the production of nitric oxide. The results demonstrated that nitric oxide was a significant inhibitor of the thrombin-induced platelet adherence in this assay system. Treatment with aprotinin in the presence or absence of L-NMMA reduced adherence of platelets to equivalent levels suggesting that aprotinin acts directly on the platelets via a mechanism that is EDRF-independent, to inhibit adherence.

Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes.

When mesenchyme from fetal mammary or salivary gland is implanted into adult mouse mammary gland, adjacent epithelium responds with intense hyperplasia. The hyperplastic cells are more vulnerable than are non-stimulated cells to transformation in vivo by a chemical carcinogen or by mammary tumor virus. This system provides a potentially useful model for determining how stroma contributes to mammary tumorigenesis. We have developed co-culture systems and used them to investigate in more detail the nature of the signal produced by the mesenchyme cells. Monolayers of mesenchyme cells were prepared on tissue-culture wells. The mesenchyme cells were trapped on the surface by a thin overlay of agarose. Primary mammary epithelial cells were cultured atop this barrier layer, either as organoids in collagen gels for assessment of anchorage-dependent growth, or as single-cell dispersions in soft agarose for assessment of anchorage-independent growth. Our procedures for assay of anchorage-independent growth allow us for the first time to detect and measure this transformation-defining characteristic in non-immortalized mammary epithelial cells in primary culture. Fetal mammary fat pad precursor tissue and fetal salivary mesenchyme both stimulated anchorage-dependent growth of mammary epithelium, with cell number increasing as much as fifteenfold during a 6-day culture period. These same fetal tissues also stimulated anchorage-independent growth of the mammary epithelial cells, with colony-forming efficiencies of up to 40% in co-cultures with salivary mesenchyme. No colonies formed in the absence of mesenchyme. Cells of colonies contained keratin, which indicates that the colonies grew from epithelial cells and not from a contaminant of another cell type. When co-cultured epithelial cells were subsequently re-cultured in the absence of mesenchyme, they lost their ability to grow independent of anchorage. No colonies grew in co-cultures with fetal cells from heart, kidney, or lung, which is consistent with the lack of stimulation by these tissues in the mammary gland in vivo. A tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA), also caused anchorage-independent growth of the dispersed mammary epithelial cells. Culture medium conditioned by primary or early-passage salivary mesenchyme cells was capable of stimulating growth under both anchorage-dependent and anchorage-independent conditions, confirming that these effects are mediated by a paracrine factor. The results indicate that stimulatory fetal mesenchymes produce soluble molecules that act analogously to transforming growth factors.

Plasminogen activator inhibitor-1 binds to fibrin and inhibits tissue-type plasminogen activator-mediated fibrin dissolution.

Plasminogen activator inhibitor-1 (PAI-1) accumulates within thrombi and forming whole blood clots. To explore this phenomenon at the molecular level, PAI-1 binding to fibrin was examined. The experiments were performed by adding 125I-PAI-1, which retains its complete tissue-type plasminogen (t-PA) inhibitory activity, to fibrin matrices formed in 2-cm2 tissue culture wells. Guanidine HCl-activated PAI-1 binding was reversible and was inhibited in the presence of excess, unlabeled PAI-1. Activated 125I-PAI-1 recognized 2 sites on fibrin: a very small number of high affinity sites (Kd less than 1 nM) and principally a large number of low affinity sites with an approximate Kd of 3.8 microM. Latent PAI-1 bound to fibrin at a site indistinguishable from the lower affinity site recognized by activated PAI-1. Fibrin, pretreated with activated PAI-1, was protected from t-PA-mediated plasmin degradation in a PAI-1 dose-responsive manner (IC50 = 12.3 nM). Clot protection correlated with partial occupancy of the low affinity PAI-1 binding site on fibrin and was due to the formation of sodium dodecyl sulfate-stable, PAI-1.t-PA complexes. Latent PAI-1 (27 nM) did not protect the fibrin from dissolution. The localization of PAI-1 to a thrombus by virtue of its fibrin binding potential could result in significant protection of the thrombus from the degradative effects of the fibrinolytic system.

Secretion and hormonal regulation of interleukin-6 production by mouse uterine stromal and polarized epithelial cells cultured in vitro.

Uterine stromal (USC) and uterine epithelial (UEC) cells were isolated from immature and mature mice to determine their ability to secrete interleukin-6 (IL-6) in response to ovarian steroids, IL-1 alpha, and soluble products produced by the heterologous cell type. In addition, the effect of IL-6 on embryo attachment and outgrowth in vitro was determined. UEC cultured on nitrocellulose filter inserts in a polarized manner secreted IL-6 with a 2.5- to 5-fold apical vs. basal preference, as determined by a B9 hybridoma cell proliferation assay and enzyme-linked immunosorbent assay. The hormonal status of animals at the time uteri were removed did not influence subsequent secretion of IL-6, as UEC isolated from immature, diestrous, and estrous stage mice exhibited both a similar amount and had a similar apical preference for secretion of IL-6. The addition of 17 beta-estradiol (E) to UEC cultures markedly inhibited total IL-6 secretion, but did not affect vectorial secretion. The inhibitory effect of E on IL-6 secretion by UEC was consistent with an apparent decrease in IL-6 transcript observed by a reverse transcriptase polymerase chain reaction assay. Other transcripts detected by this assay in UEC included IL-1 alpha, but not IL-1 beta or tumor necrosis factor-alpha. Secretion of IL-6 by UEC was not stimulated by IL-1 alpha, conditioned medium from USC, or coculture with USC. USC secreted IL-6, and while this also was inhibited by E, progesterone was more effective in this regard at physiological concentrations. In addition, there was a synergistic effect of E plus progesterone on inhibition of IL-6 secretion by USC. Secretion of IL-6 by USC was stimulated by IL-1 alpha, and coculture studies demonstrated the ability of UEC to stimulate a several-fold increase in IL-6 secretion by USC. The cytokine transcripts detected in USC cultures included IL-6 and IL-1 alpha, but not IL-1 beta. Transcripts for tumor necrosis factor-alpha were present in USC only after culture with IL-1 alpha. IL-6 added to blastocysts on laminin-coated tissue culture wells resulted in a transient inhibition of the rate of blastocyst attachment and, to a greater extent, an inhibition of the rate of embryo outgrowth. In addition, IL-6 inhibited the size of embryo outgrowths at 24 and 48 h of culture.(ABSTRACT TRUNCATED AT 400 WORDS)

Polar secretion of endothelin-1 by cultured endothelial cells.

The aim of this study was to determine the permeability of endothelial monolayers for endothelin-1 and a possible directionality of the endothelin-1 secretion process. Human umbilical vein endothelial cells were cultured on acellular amniotic membranes, dividing the tissue culture wells into an apical (luminal) and a basolateral (abluminal) compartment. Whereas in the absence of endothelial monolayers 44.9 +/- 2.3 and 43.5 +/- 2.0% of the unilaterally added endothelin-1 permeated from the apical to the basolateral side and from the basolateral to the apical side, respectively, only 6.5 +/- 0.6 and 6.6 +/- 0.4% diffused in the presence of endothelial cells. Analyzing endothelin-1 secretion, approximately 80% of the total amount of synthesized endothelin-1 was found in the basolateral compartment; thrombin (10 units/ml) stimulated the production of endothelin-1 approximately 2-fold, but did not change the relative distribution of endothelin-1 between the apical and basolateral compartments. In the presence of dexamethasone (10(-7) M), a decrease in the level of endothelin-1 was found in the apical compartment, whereas the total amount of endothelin-1 produced was not affected. Dexamethasone did not influence the permeability of human umbilical vein endothelial cell monolayers for endothelin-1. These results strongly support the hypothesis that endothelin-1 is a local paracrine regulator of vasotone.

A method for rapid screening of recombinant proteins for recognition by T lymphocytes.

A simple, cost-effective method is described that allows rapid screening of recombinant protein sequences for their ability to stimulate T cells. Individual microcultures of E. coli each expressing a gene product or peptide sequence fused to protein A are grown in 96-well plates. Following lysis of the bacteria, the fusion peptide is readily captured with immobilized immunoglobulin in tissue culture wells. No further purification is required. T lymphocytes plus appropriate antigen-presenting cells are added directly to the wells and assayed for proliferation. The DNA in bacteria from wells stimulating T cell proliferation is then sequenced. The technique allows rapid mapping of T cell epitopes by facilitating screening of truncation mutants without extensive purification. Described here is a further application of the technique to study monosubstituted analogues of a known T cell epitope.

Nephrotoxic antiserum identifies a beta 1-integrin on rat glomerular epithelial cells

A postulated mechanism of immune glomerular injury is a direct interaction between antibody and glomerular epithelial cell (GEC) surface antigens. To explore this hypothesis, we examined the interaction of the noncomplement-fixing gamma 2-subclass of sheep anti-rat nephrotoxic serum (NTS), which causes immediate complement- and neutrophil-independent proteinuria in vivo, with rat GECs in culture. Reactivity of NTS with GEC surface antigens was determined by positive immunofluorescence of GEC plasma membranes and by the ability of NTS-coated tissue culture wells to provide an adhesive substrate for GECs. NTS immunoprecipitated two proteins (135 and 118 kDa) from surface-labeled GECs. Proteins of similar molecular mass were precipitated by a polyclonal rabbit antibody that identifies the beta 1-integrin chain of the mouse fibronectin receptor (anti-FnR). In addition, NTS identified similarly sized bands on Western blot analysis of cell membranes from isolated rat glomeruli. Similar reactivity was eluted from the glomeruli of proteinuric rats injected with NTS. NTS significantly inhibited GEC adhesion to laminin, types I and IV collagen, and fibronectin and prevented GEC spreading on types I and IV collagen. Anti-FnR similarly inhibited GEC adhesion. Cell viability was not affected. These results show that NTS recognizes a pair of GEC surface proteins that have the characteristics of an alpha- and beta 1-integrin and, at low concentrations, disrupt cell-matrix interactions.

Regulation of human colon-carcinoma cell adhesion to extracellular matrix by transforming growth factor beta 1.

The regulatory effect of transforming growth factor beta 1 (TGF-beta 1) on the adhesion of human colon-carcinoma cells to the extracellular matrix (ECM) was investigated. ECMs used in this study included tissue-culture wells coated with fibronectin, laminin, collagen and BSA, as well as plastic wells. Three phenotypically different human colon-carcinoma cell lines (Moser, HCT116, and KM12SM) were used. The Moser cell line is moderately differentiated and, in terms of the diversity of responses elicited by TGF-beta 1, is the human colon-carcinoma cell line most responsive to TGF-beta 1 as reported to date. By comparison, the undifferentiated HCT116 and the highly metastatic KM12SM cells are unresponsive to this growth factor. We showed that TGF-beta 1 regulated the adhesion responses of all 3 cell lines. However, the response profiles as well as the endogenous adhesive properties of each cell line were quite different from those of the others. Endogenous Arg-Gly-Asp(RGD)-related receptors were present on the HCT116 but not on the other cells. The observed regulatory effect of TGF-beta 1 was contingent on the cell line, the type of ECM, and the growth-factor treatment protocol used. When cells were treated with TGF-beta 1 for 16 hr prior to exposure to ECM in a 4-hr adhesion assay, a significant increase in adhesion to fibronectin and collagen was observed for the Moser cells. For the identical experimental protocol, the KM12SM cells responded by increasing adhesion to fibronectin, while the HCT116 cells responded by decreasing adhesion to collagen. Kinetic analyses of TGF-beta 1 treatment showed that increased adhesion response to laminin was induced in the Moser cells after 2 hr of growth-factor treatment. This response declined rapidly upon further exposure of the cells to TGF-beta 1. Simultaneous exposure of cells to both TGF-beta 1 and ECM negated the adhesion responses described above. The up-modulation of adhesion to fibronectin, laminin and collagen by TGF-beta 1 was mediated through RGD-related integrin receptors. RGD-containing peptides effectively blocked the enhanced adhesion responses induced by TGF-beta 1.

Quantitative analysis of autocrine-regulated, matrix-induced, and tumor cell-stimulated endothelial cell migration using a silicon template compartmentalization technique

Autocrine-regulated, matrix-induced, and tumor cell-stimulated endothelial cell migration was quantitatively analyzed using a two-dimensional, two-compartment coculture system. Silicon templates were used to subdivide 35-mm tissue culture dishes into two separate compartments. Endothelial cells were grown to confluence in the inner compartment and released from growth arrest by removal of the silicon template. The distance of endothelial cell outgrowth from the monolayer was measured in 24-h intervals. Endothelial cells from different vascular beds migrated with different migration rates (large vessel endothelial cells > hemangioendothelioma cells > microvessel endothelial cells). Prior coating of tissue culture wells with fibronectin, type I collagen, or type IV collagen and increasing serum concentrations strongly enhanced endothelial cell migration. Seeding tumor cells into the outer compartment prior to removal of the silicon template permitted the direct coculture analysis of tumor cell-induced endothelial cell migration. Microvascular endothelial cell migration was stimulated in a tumor cell number-dependent fashion, whereas large vessel endothelial cells could not consistently be stimulated by coculture with tumor cells. It is concluded that silicon templates offer a useful approach for the quantitative study of migration of anchorage-dependent cells, permitting follow-up measurements over several days, the study of matrix effects, and the direct coculture analysis of cell migration.

Reduced tumor cell transfer with contact neodymium-yttrium-aluminium garnett laser scalpels.

The local recurrence of tumor growth after surgery is thought, in part, to be a consequence of seeding of tumor cells from the primary lesion. This study was directed at determining if the contact Nd:YAG laser provides any advantage over the use of traditional scalpel dissection for tumor resection. Five human tumor tissue culture cell lines were studied in this report. They included MCF-7 (breast), HeLa (cervical), SW-780 (bladder), HT29 (colon), and A549 (lung). Using a scalpel blade or contact laser scalpel (with or without laser energy), the ability to transfer viable cells from dense cell stocks to new tissue culture wells was tested. Using the A549 cell line, the extent that these instruments were able to "seed" tumor cells was also assessed in a soft agar, in vitro, "incision" model. Results from these studies suggest that the contact laser scalpel has a significantly lower potential to transmit tumor cells, when compared to traditional scalpel blades.

Signaling by lymphocyte function-associated antigen 1 (LFA-1) in B cells: enhanced antigen presentation after stimulation through LFA-1.

To examine the role of lymphocyte function-associated antigen 1 (LFA-1) expression on murine B cells as it pertains to their function in T cell activation, we carried out antigen-presentation assays in tissue culture wells coated with a purified, secreted form of the murine intercellular adhesion molecule 1 (ICAM-1). We observed a significant decrease in the concentration of antigen required to activate a T cell hybridoma and primary T cells in wells coated with ICAM-1. This effect was dependent on the amount of ICAM-1 used to coat the wells and was also observed in wells coated with anti-LFA-1-monoclonal antibodies and was blocked by soluble anti-LFA-1 antibodies. The effect on antigen dose was most pronounced in assays carried out with an ICAM-1-deficient mutant B lymphoma cell line, small resting primary B cells, and unfractionated primary B cells at low concentrations. No decrease in the antigen dose was observed if the B cells were chemically fixed or treated with ricin, or when antigen was presented by a HeLa cell line transfected with murine class II major histocompatibility complex (MHC) genes, indicating that the immobilized ICAM-1 was mediating its effect through B cell LFA-1, and that B cell protein synthesis was required. The enhancing effect was also observed if the B cells were prepulsed with antigen, indicating that improved uptake or processing of antigen, or increased class II MHC expression were unlikely mechanisms.

Specific monoclonal antibodies to Opisthorchis viverrini

A Balb/c mouse was immunized with a crude soluble antigen of Opisthorchis viverrini adult worms (OVAA) over a period of 7 months. Spleen cells from the immune mouse were fused with Sp2/0 myeloma cells. Among the 264 tissue culture wells containing the fused cells, cells of 96 wells (36%) produced antibodies to the immunizing agent. Antibodies produced by cells in several wells reacted with antigens from other species of parasite. Cells of 17 wells produced antibodies specific only to OVAA, thus cells from three representative wells were cloned by limiting dilution. Hybrids obtained produced antibodies which could be classified according to their tissue specificities into three groups. The first group of antibodies reacted strongly to the worm integument and weakly with the muscles while those belonging to the second group reacted only to muscles of the worms. The monoclonal antibodies of the third group gave a positive reaction to both muscles and tegument.

Surface contact modulation of inflammatory macrophage antibody dependent cytotoxicity and prostanoid release.

Adherence to extracellular matrix proteins modulates the functional and secretory activities of mononuclear phagocytes, although the mechanisms regulating these adherence-dependent changes are poorly understood. In this study, the ability of rat inflammatory peritoneal macrophages (PM) to adhere to an endothelial cell-derived extracellular matrix or a denatured collagen/fibronectin-coated surface and perform antibody dependent cell cytotoxicity (ADCC) and secrete reactive oxygen intermediates was compared with PM adherent to tissue culture plastic. Prostaglandin E2 (PGE2) and thromboxane B2 (TxB2), two major cyclooxygenase products released by inflammatory macrophages, were also measured by PM adherent to the protein coated surfaces. Rat exudate PM were equally adherent to tissue culture plastic or wells coated with either endothelial cell derived matrix or denatured collagen (gelatin)/fibronectin. PM adherent to a denatured collagen/fibronectin-coated wells demonstrated significantly less cytolytic activity (15 +/- 2% lysis) when compared with either tissue culture plastic adherent PM (43 +/- 7% lysis) or PM adherent to extracellular matrix (59 +/- 11% lysis). PM adherent to extracellular matrix released twofold more TxB2 than plastic adherent PM, while PM adherent to denatured collagen/fibronectin released 40% more PGE2 than cells adherent to tissue culture plastic or 80% more PGE2 than PM adherent to the extracellular matrix. PM adherent to denatured collagen/fibronectin release less superoxide anion (27 +/- .9 nmoles/10(6) PM) than PM adherent to either tissue culture plastic (43 +/- 1 nmoles/10(6) PM) or the extracellular matrix (60 +/- 0.5 nmoles/10(6) PM). Furthermore, incubation of plastic adherent PM with exogenous PGE2 reduced superoxide production in a dose-dependent manner. These results demonstrate that the inhibition of ADCC and secretion of reactive oxygen intermediates by PM adherent to a denatured collagen/fibronectin surface correlated with an increased release of the immunosuppressive prostanoid PGE2. Furthermore, the addition of exogenous PGE2 to plastic adherent PM reproduced the depression in ADCC and superoxide anion production observed by PM adherent to a denatured collagen/fibronectin surface. These studies suggest that the increased production and release of PGE2 by inflammatory macrophages adherent to a denatured collagen surface may act to suppress cytotoxic mechanisms and thereby constitutes part of an autocrine feedback mechanism regulating macrophage function during wound injury.

Proteolysis of gelatin-bound fibronectin by activated leukocytes: a role for leukocyte elastase.

Fragmentation of subendothelial matrix-bound fibronectin by proteases released from stimulated leukocytes has been implicated in lung vascular injury. We studied the degradation of fibronectin bound to denatured collagen by inflammatory polymorphonuclear leukocytes (PMNL). Tissue culture wells coated with denatured collagen (gelatin) were pretreated with 125I rat plasma fibronectin to allow for fibronectin binding prior to the addition of rat inflammatory PMNL. The release of both intact and fragmented fibronectin from the 125I-labelled artificial matrix was quantified following the addition of PMNL stimulated by the phagocytosis of opsonized zymosan as well as leukocyte elastase. Stimulated PMNL released three times more radiolabelled fibronectin from the denatured collagen surface during a 4 h incubation as compared with unstimulated PMNL. This pattern of 125I-fibronectin release could also be elicited by the addition of purified leukocyte elastase alone, in the absence of PMNL. The release of radiolabelled fibronectin by stimulated PMNL was blocked in a dose-dependent manner by the addition of both methoxysuccinyl-alanine-alanine-valine chloromethyl ketone (AAPVCK), a leukocyte elastase specific inhibitor as well as phenylmethylsulfonylfluoride (PMSF), a non-specific serine protease inhibitor. Western blot analysis coupled with autoradiography confirmed the presence of fibronectin fragments in the medium after addition of PMNL or leukocyte elastase. The large molecular weight fragments (60-200 kD) were not labelled, but the smaller molecular weight fragments (less than 45 kD), derived from the artificial matrix, were labelled. Thus, fibronectin complexed with denatured collagen is susceptible to proteolytic degradation by stimulated inflammatory PMNL. Such a process may have a role in the pathogenesis of acute vascular injury following microvascular margination of activated blood leukocytes.

Effect of tumor necrosis factor on the generation of chlorinated oxidants by adherent human neutrophils.

Human neutrophils adherent to simulated biologic surfaces undergo significant activation of the respiratory burst over prolonged periods of time in response to stimulation with the cytokines tumor necrosis factor-alpha (TNF alpha) or tumor necrosis factor-beta (TNF beta) or with the chemotactic peptide N-formyl-methionylleucylphenylalanine (FMLP). In this study, neutrophils were examined for their ability to generate the highly reactive and powerful oxidant hypochlorous acid (HOCl) and the longer-lived, less reactive endogenous nitrogen-chlorine (N-Cl) derivatives in response to these stimuli either alone or when exposed to recombinant human TNF alpha (rTNF alpha) or beta (rTNF beta) prior to addition of FMLP. Neutrophils adherent to fetal bovine serum-coated polystyrene tissue culture wells were able to generate only small quantities of HOCl when incubated with rTNF alpha, rTNF beta, or FMLP individually. However, when neutrophils were first incubated with either rTNF alpha or rTNF beta prior to addition of FMLP, there was a marked increase in HOCl generation. Neutrophils stimulated in such a manner consumed approximately 18% of the HOCl generated in the formation of N-Cl derivatives. Further scrutiny of the response to the combination of rTNF alpha and FMLP revealed that HOCl release was rapid, with 80% of total HOCl accumulation occurring within 15 min after FMLP addition. The amount of HOCl generated was dependent on the number of cells added and on the concentration of both rTNF alpha and FMLP. Comparison of HOCl generation with superoxide anion and myeloperoxidase release showed that the amount of HOCl generated was limited primarily by the amount of myeloperoxidase released rather than by the degree of respiratory burst activation. These results demonstrate that human neutrophils stimulated with FMLP after a brief incubation with rTNF alpha or rTNF beta can generate cytotoxic and microbicidal concentrations of chlorinated oxidants.

Effect of serum-free co-culture and synchrony of recipients on development of cultured sheep embryos to fetuses

The percentage of sheep embryos that continued to develop after collection and immediate transfer on d 2 after estrus was similar when phosphate-buffered saline with 10% fetal calf serum (PBSFCS, 45%), physiological saline (50%), or tissue culture medium 199 supplemented with 10% fetal calf serum (M199FCS, 47%) was used to flush embryos from oviducts. Co-culture of sheep embryos for 3 d with oviductal cells tended (P = .1) to reduce the percentage of embryos that developed to fetuses after transfer compared with those embryos transferred immediately. Tissue culture medium 199 supplemented with .3% BSA (M199BSA) was an adequate substitute for M199FCS for culture of sheep oviductal cells if tissue culture wells were pretreated with fibronectin. Estradiol in concentrations from 10 to 1,000 pg/ml and progesterone at concentrations of 1 or 10 ng/ml in M199BSA failed to stimulate embryo development during 3 d of co-culture beyond that seen in co-culture with M199FCS or M199BSA without added steroid. Transfer of sheep embyros co-cultured for 3 d in M199BSA or M199FCS to recipients synchronized with donors resulted in about 19% of the embryos developing to fetuses, whereas transfer to recipients that were in estrus 24 h after donors resulted in 33% of embryos developing to fetuses. The significant (P less than .05) improvement for delayed recipients may reflect the relatively lesser developmental rate of co-cultured embryos compared with that of embryos in vivo. Embryo development into fetuses was similar after co-culture in M199FCS or M199BSA co-cultures; therefore, serum is not required for the co-culture of sheep embryos.

Subpopulations of CD8+ cytotoxic T cell precursors collaborate in the absence of conventional CD4+ helper T cells.

Four different subpopulations (Ly6Cneg, Ly6Clow, Ly6Cint, and Ly6Chi) of CD8+ T cells were arbitrarily defined on the basis of differential expression of Ly6C Ag. By combining the processes of electronic cell sorting and automated cell deposition, small numbers of respective CD8+ T cell subpopulations were directly deposited into tissue culture wells in which mitogen-stimulated responses were studied. Anti-CD3-stimulated proliferation and IL-2 production were the strongest by Ly6Cneg/Ly6Clow T cells, moderate for Ly6Cint T cells, and highly deficient for Ly6Chi T cells. The level of IL-2 production for Ly6Cneg CD8+ T cells was comparable to that of conventional CD4+ Th cells. Allogeneic stimulator cells elicited a strong cytotoxic response by Ly6Cneg + low but not Ly6Chi CD8+ T cells in the absence of added lymphokines. When IL-2 was supplied in excess, anti-CD3 induced comparable levels of cell proliferation and cytotoxic activity in Ly6Cneg, Ly6Clow, Ly6Cint, and Ly6Chi CD8+ T cells whereas alloantigen stimulated an approximate fivefold higher cytotoxic response by Ly6Chi than Ly6Cneg + low CD8+ T cells. Stimulation of co-cultures of B10 (CD8b) Ly6Cneg + low and congenic B10.CD8a Ly6Chi CD8+ T cells in the absence of added lymphokines, followed by selective elimination of activated CD8.1+ (CD8.2+) T cells by anti-CD8.1 (anti-CD8.2) + C treatment, allowed the demonstration that help provided by Ly6Cneg + low T cells can be effectively used by both Ly6Cneg + low and Ly6Chi T cells in anti-CD3 and alloantigen induced proliferative and cytotoxic responses, respectively.

Effect of the Prostaglandin E1Analogue Misoprostol on the Carbon Tetrachloride-Induced Injury of Rat Liver Cells in Culture

Previous investigators have reported a protective effect of some prostaglandins and of the prostaglandin E2 analogue enprostil on carbon tetrachloride (CCl4)-induced injury of liver cells. In the present study liver cells were isolated from the rat liver by collagenase perfusion and suspended in F-10 medium, containing 20% foetal bovine serum, 1% gentamicin, and 1% glutamine. In the first study cells were cultured in T-flasks with 3 ml suspension of 6 x 10(6) cells/ml, and in the second study (extended dose response) cells were cultured in tissue culture wells with 0.5 ml cell suspension. Misoprostol was added to groups of cultures 15 min before CCl4, 2 microliters/ml, and the number of living cells was counted 45 min after the first addition. The number of living cells was compared with those of other groups with CCl4 only and control groups. In the first experiment misoprostol was given in doses of 200, 400, and 800 ng/ml medium and in the second experiment in 0.1, 1, 10, 100, and 1000 ng/ml medium. CCl4 is an agent well known to be toxic to liver cells, and in cultures to which only CCl4 was added, the number of living cells was significantly reduced compared with controls. When 0.1 ng misoprostol was added before CCl4, no significant difference in the number of living cells was shown compared with cultures with CCl4 only. On the other hand, misoprostol given in doses from 1 ng to 1000 ng before CCl4 resulted in a higher number of living cells, indicating a protective effect.

Natural products phytotoxicity A bioassay suitable for small quantities of slightly water-soluble compounds

A large variety of secondary metabolites that can inhibit germination and/or seedling growth are produced by plants in low quantities. The objective of this study was to develop a bioassay capable of reliably assessing reductions in germination percentage and seedling length of small-seeded plant species caused by exposure to minute quantities of these compounds. The germination and growth of alfalfa (Medicago saliva), annual ryegrass (Lolium multiflorum), and velvetleaf (Abutilon theophrasti) were evaluated against six known phytotoxins from five chemical classes; cinmethylin (a herbicidal cineole derivative) was selected as a comparison standard. Each phytotoxin, dissolved in a suitable organic solvent, was placed on water-agar in small tissue culture wells. After the solvent evaporated, imbibed seeds were placed on the agar; after three days, germination percentages and seedling lengths were measured. Compared to a commonly used filter paper procedure, this modified agar bioassay required smaller quantities of compound per seed for comparable bioassay results. This bioassay also readily permitted the measurement of seedling length, a more sensitive indicator of phytotoxicity than germination. Seedling length decreased sigmoidally as the toxin concentration increased logarithmically. Phytotoxicity was a function of both compound and plant species. Cinmethylin, a grass herbicide, reduced the length of annual ryegrass seedlings by 90-100%, whereas that of alfalfa and velvetleaf was inhibited slightly. The agar bioassay facilitated the rapid and reliable testing of slightly water-soluble compounds, requiring only minute quantities of each compound to give reproducible results.

Parainfluenza virus infection of cultured airway epithelial cells

Studies of the cellular effects of respiratory viruses have generally used cultures of non-airway (particularly renal) epithelial cells. This requires the assumption that, despite the marked differences between renal epithelium and airway epithelium, the virus-host cell interactions in cultures of renal epithelium will be relevant to those in airway epithelium. To study viral infection of airway epithelial cells, we removed the epithelial cells from ferret tracheas using 0.1% pronase solution, and plated them at a density of 5 × 10 5 cells/cm 2 in collagen-coated plastic tissue culture wells. Cultures grew to confluence after 5–7 days. Viral inocula, consisting of supernatants from parainfluenza type 1-infected rhesus monkey kidney cell monolayers, were added to the culture medium in a concentration 10 3 times that sufficient to produce infection in 50% of rhesus monkey kidney monolayers (TCID 50). Cytopathic changes, consisting of cellular elongation and detachment, became apparent after 3–6 days, at which time the medium contained 5 × 10 8 TCID 50/ml. The monolayer appeared to be uniformly infected as revealed by adsorption of guinea pig erythrocytes. Specific immunofluorescence revealed uniformly positive staining for parainfluenza type 1 antigens. The ability to infect pure cultures of airway epithelial cells with viruses will allow us to examine the effects of these viruses on epithelial cell function, and to study virus-host cell interactions in cell cultures derived from the natural host cell.