Acta Histochem Suppl, 1985, 31, 37 - 46 The molecular mechanisms of the distinct calcium-dependent aggregation systems in marine sponges and corals; Muller WE et al.; During the last 15 years we have developed two biological systems, with whom it was possible to study the Ca++-dependent and the Ca++-independent adhesion on cellular level . In contrast to cells from other multicellular organisms, cells from the marine sponge Geodia cydonium are provided with Ca++-dependent adhesion mechanisms only . Two different mechanisms have been discovered by us, which were termed primary aggregation and secondary aggregation . In previous reports, we described that two macromolecules (aggregation factor {sAF} and aggregation receptor {AR} are involved in the secondary aggregation of sponge cells . The sAF was bound to a high-molecular-weight particle and was termed aggregation complex . The aggregation complex was shown to consist of two further functional subunits: UDP-glucuronosyltransferase and UDP-beta-D-galactosyltransferase . The AR with a molecular weight of approximately 17,000 was found to be a glycoprotein with D-glucuronic acid as the terminal sugar moiety . Data are presented from in vitro and in vivo experiments with the Geodia system, indicating that cell aggregation and cell separation are controlled first by alteration of the binding capacity of the aggregation receptor and second by an additional molecule (anti-aggregation receptor), which can decrease the interaction between the aggregation factor and the aggregation receptor . Recently we succeeded in the identification and isolation of the primary aggregation factor (pAF) from the same sponge species . This pAF is a glycoprotein that is firmly associated with the cell membrane . The Mr of the native pAF was 36,000; under denatured conditions three protein species were identified in the pAF preparation . We hypothesize that in contrast to the secondary aggregation, the initial aggregation of Geodia cells is mediated by the one-component system, the bivalent and bifunctional pAF . We were also able to dissociate the coral Eunicella cavolinii into single cells . These cells readily formed aggregates of a size of 2,100 micron during incubation in roller tubes: no aggregate formation was observed in non-rotating petri dishes . The formation of aggregates was not influenced by Ca++, urea or trypsin; it was also independent on temperature (4 degrees C to 30 degrees C) and pH (5.5-9.0) . The intercellular material of the gorgonian contains a galactose-specific lectin, as determined by double diffusion experiments and haemagglutination inhibition experiments using a series of galacto-glycoconjugates . This lectin converted the aggregation-susceptible cells to aggregation-deficient cells.(ABSTRACT TRUNCATED AT 400 WORDS) J Immunol Methods, 1984 Dec 14, 75(1), 129 - 40 Characterisation of the under-agarose method for quantifying migration of highly purified human monocytes; Al-Sumidaie AM et al.; Optimal conditions governing migration towards chemotactic agent (MTCA) and random migration of purified human monocytes under agarose have been determined . These include an incubation period of 20 h, use of blood group AB pooled human serum, 0.75% agarose type I, pH between 6.8 and 7.2 and performance in 35 mm Falcon plastic petri dishes . A discontinuous Percoll density gradient was used to give monocyte purity of 83 +/- 10% and viability 97-99% . The use of highly purified monocytes was found to be essential, as increasing lymphocyte: monocyte ratio caused inhibition of migration. Cell Mol Neurobiol, 1984 Dec, 4(4), 385 - 96 Dendritic membrane from insect olfactory hairs: isolation method and electron microscopic observations; Klein U et al.; Sensory hairs from antennae of male saturniid moths (Antheraea polyphemus) were separated while deep-frozen by shaking antennal branches with glass beads . The hairs were collected through their differential adhesion to the surface of a petri dish . The yield, determined by the length of the isolated hair fragments, was about 38% of the estimated total hair length per antenna . The dendritic membrane was separated from the hair fragments by centrifugation through Sephadex and further purified by ultracentrifugation in sucrose buffers . Transmission electron microscopy was used to monitor the steps of the hair and membrane isolation and to investigate the membrane pellet . Some membrane vesicles bound cationized ferritin, thus indicating a negatively charged cell surface coat . Negatively stained membrane vesicles exhibited a pattern of repetitive substructures irregularly distributed over the vesicle surface . The units had a diameter of about 3 nm and a maximal density of 30,000/micron2. Acta Physiol Scand, 1984 Dec, 122(4), 607 - 13 Preparation of cells from pig gastric mucosa . Isolation of parietal cells by isopycnic centrifugation on linear density gradients of Percoll; Mardh S et al.; Cells were isolated from pig gastric mucosa by a combination of mechanical and enzymatic treatment . Isopycnic centrifugation on linear density gradients of Percoll provided a simple and rapid procedure for obtaining highly enriched parietal cells and chief cells . Their densities were 1.06 and 1.10 g/ml, respectively . Isolated mucosal cells attached to Petri dishes coated with fibronectin or collagen . Both parietal cells and chief cells adhered more readily to fibronectin than collagen . Mucosal cells and cells from the Percoll gradients were maintained for up to one week as primary cell cultures . The ability of free parietal cells to produce acid was tested by the 14C-aminopyrine accumulation technique . Maximal accumulation was 2.5 pmol aminopyrine per 10(4) parietal cells after incubation for 45 min at 10(-4) M histamine . The EC50 for histamine was 5 X 10(-6) M . The accumulation of aminopyrine at 10(-6) M carbachol and 10(-7) M pentagastrin were for both secretagogues about 0.9 pmol per 10(4) parietal cells. Cancer Res, 1984 Dec, 44(12 Pt 1), 5605 - 8 Growth enhancement of N-nitrosomethylurea-induced rat mammary tumor cells in soft agar by estrogen or prolactin; Arafah BM et al.; Cells obtained from N-nitrosomethylurea-induced rat mammary tumors were grown in vitro using the soft-agar clonogenic assay technique . Their growth was studied in regular media containing serum as well as in media lacking serum, but to which insulin was added . Deletion of serum from the media resulted in a mean decrease of 49% in the number of colonies formed in vitro in 13 of 18 tumors and was without effect in the remaining 5 tumors . The addition of either 17 beta-estradiol (10(-8) M) or ovine prolactin (OPRL, 100 ng/ml) to the defined media resulted in an increase in the number of colonies formed in 12 of 18 tumors . The mean numbers of colonies per Petri dish in 17 beta-estradiol- and OPRL-treated Petri dishes were 95 +/- 5.4 (S.E.) and 92 +/- 6.2% of the values seen in serum-containing media . Simultaneous addition of both hormones to the defined media resulted in a significant increase in the number of colonies formed which was greater than that seen when either hormone was added separately . Of four tumors where neither hormone influenced colony formation, the addition of both 17 beta-estradiol and OPRL resulted in an increase in the number of colonies formed in three tumors . We conclude that the N-nitrosomethylurea-induced mammary tumors can be grown in soft agar using defined media and that their growth can be enhanced by either 17 beta-estradiol or OPRL . These hormones have a synergistic effect on the growth of some of these tumors in vitro . These data are consistent with the known in vivo effects of these hormones on the N-nitrosomethylurea-induced rat mammary tumors. Rev Esp Fisiol, 1984 Dec, 40(4), 463 - 7 Positive selection by "panning" of B lymphocytes using unfractionated anti-immunoglobulin antiserum; Rojo JM; A simple method allowing a positive selection of mouse B lymphocytes by "panning" has been achieved using rabbit anti-mouse immunoglobulin antisera without further purification . Plastic petri dishes were coated with the gamma-globulin fraction of normal mouse serum and then incubated with excess rabbit anti-mouse gamma globulin antiserum . These plates were then used for the selection of B lymphocytes by incubation of spleen cells in the plates . The adherent cells wee above 95% B lymphocytes as judged by immunofluorescence staining, proliferated in response to bacterial lipopolysaccharide and were not stimulated by concanavalin A. Cytometry, 1984 Nov, 5(6), 572 - 9 Preparative dual-beam sorting of the human Y chromosome and in situ hybridization of cloned DNA probes; Cremer C et al.; Bivariate Hoechst/chromomycin flow karyotypes for chromosomes from a Chinese hamster-human hybrid cell line (CH-Y-VII) were established that have retained a human Y chromosome . These bivariate flow karyotypes showed the human Y chromosome to be completely separated from the peaks for the Chinese hamster chromosomes . In preparative dual-beam sorting experiments, 3 X 10(6) chromosomes were sorted from the Y peak into frozen petri dishes . An examination of Q-banded samples of sorted chromosomes revealed that 82% +/- 5% of them were human Y chromosomes . The DNA from the sorted chromosomes (approximately 250 ng) was isolated and used to establish a genomic library (vector lambda gt WES . lambda B) . Three clones (YACG 45, 52, 54) of this library containing inserts of repetitive human DNA were used for chromosomal localization by means of in situ hybridization to metaphase spreads of male human lymphocytes and of CH-Y-VII cells . In all three cases, a significant binding to the human Y chromosome was observed . A more detailed study of the chromosomal distribution of sequences homologous to the insert of YACG 45 suggested the existence of minor binding sites on several human autosomes . Southern blot analysis revealed the existence of other human specific sequences without Y chromosome specificity. Arch Toxicol, 1984 Nov, 56(1), 29 - 32 Cell survival after the combined action of manganese (MnCl2) and X-rays in synchronized Chinese hamster cells; Skreb Y et al.; The interactions between the effects of manganese chloride and X-rays were studied in synchronized populations of V79 Chinese hamster fibroblasts . The cells were selected by shaking off asynchronous cultures for detachment of mitotic cells which were plated in petri dishes and exposed to various treatments . Irradiation was carried out with a Philips RT-100 X-ray unit . A final concentration of 0.25 mM MnCl2 was used . The main parameter was the colony forming ability of the surviving cell fraction . When MnCl2 was administered over 1 h, its toxicity was low regardless of the phase of the cell cycle . Administered separately, 2 Gy irradiation produced only a slight decrease in survival, less marked in the S phase . However, the two agents together induced a synergistic inhibition of the surviving fraction in the S phase when the metal was given immediately after irradiation . If manganese was administered 3 h after irradiation the two inhibitory effects apparently remained only additive . It seems that MnCl2 can impair some repair processes starting immediately after irradiation. Exp Cell Res, 1984 Oct, 154(2), 421 - 31 Adhesive and migratory behavior of normal and sulfate-deficient sea urchin cells in vitro; Venkatasubramanian K et al.; Dissociated cells from different stage embryos of the sea urchin Lytechinus pictus were compared in their adhesion to various substrates . Micromeres from 16-cell stage embryos bind to tissue culture and Petri dishes but not to Petri dishes coated with human plasma fibronectin . Other cell types did not adhere to any of the substrates tested . By hatched blastula stage, about 28% of the cells adhered to fibronectin as well as to tissue culture dishes . By the mesenchyme blastula stage, there was a further increase in the proportion of cells adhering to these substrates . At no stage did cells adhere to native rat tail collagen . Primary mesenchymal cells were isolated by their selective adhesion to tissue culture dishes in the presence of horse serum . These cells were then examined for their migratory capacity . Cell spreading and migration followed adhesion and occurred on fibronectin but not on the other substrates tested . Based on analysis of video tapes, greater than 60% of these cells moved faster than 1 micron/min . On the other hand, cells from sulfate-deprived embryos, in which primary mesenchyme migration is blocked in situ, failed to spread and migrated little on the same substratum . This defect was reversed by a 6 h pretreatment of the cells in normal sea water . Thus, the in vitro migratory behavior parallels that observed in vivo . These results support the hypothesis that the primary mesenchymal cells produce a sulfate-dependent component that is required for cell spreading and migration. J Immunol Methods, 1984 Sep 4, 72(2), 489 - 96 Reverse enzyme-linked immunospot assay (RELISPOT) for the detection of cells secreting immunoreactive substances; Czerkinsky CC et al.; A reverse modification of the recently described enzyme-linked immunospot assay (ELISPOT), based on localized enzyme-substrate reactions in gel, is described for the enumeration of antigen-secreting cells using petri dishes coated with specific antibodies . As a model the detection of mouse and human immunoglobulin-secreting cells has been evaluated . Simple and sensitive, this new method, termed RELISPOT, can be adapted for the quantitation of secreted antigen thus providing additional information on the metabolic state of the population of cells tested. Biull Eksp Biol Med, 1984 Sep, 98(9), 375 - 7 {Detection and isolation of antibody-forming clones by means of local cytolysis in gel}; Eraizer TL et al.; A method is suggested combining the screening and cloning of hybridomas producing cytotoxic antibodies . The method is based on the Erne local hemolysis principles . The cells from the preformed hybridoma line NATF 9.9 secreted monoclonal antibodies (McAb) against murine T lymphocyte differentiation antigen Lyt-3.2 . A mixture of hybridoma cells and target cells was attached to the plastic Petri dish surface pretreated with Poly-L-lysine and covered with agarose . McAb production by hybridoma cells was elicited by complement-dependent lysis of target cells . The lysis was detected by incorporation in dead cells of the fluorescent dye ethidium bromide . Subsequent studies made it possible to evaluate the clonogenic capacity of McAb production and isolate active clones. Cell Immunol, 1984 Aug, 87(1), 65 - 72 Regulation of NK cell activity by prostaglandin E2: the role of T cells; Zielinski CC et al.; The influence of T cells on the production of prostaglandins (PGE2) and on PGE2-mediated regulation of natural killer (NK) activity was studied . Supernatants from peripheral blood mononuclear cells (PBMC) and from PBMC depleted of T cells ((PBMC)-T), both of which had been incubated in plastic petri dishes overnight, contained similar amounts of PGE2, as detected by radioimmunoassay and by their potential to inhibit NK activity of peripheral blood mononuclear cells in a 51Cr release assay with K 562 cells as the target population . However, the NK activity of PBMC was inhibited significantly more strongly (P less than 0.005) by PGE2-containing supernatants than was the NK activity of (PBMC)-T . In further assays, in which synthetic PGE2 in concentrations of 10(-4) and 10(-5)M was added, a significant inhibition of NK activity was observed in PBMC populations (P less than 0.05), but not in (PBMC)-T . Thus, T cells did not seem to be involved in the control of PGE2 production, but their presence was necessary for PGE2-mediated inhibition of NK activity. Radiat Res, 1984 Aug, 99(2), 228 - 48 Evidence that a second event in X-ray-induced oncogenic transformation in vitro occurs during cellular proliferation; Kennedy AR et al.; It has previously been hypothesized that radiation transformation in vitro is a two-step process; the first step is a frequent alteration occurring among a large fraction of the irradiated cells, while the second step, malignant transformation, is a rare event occurring with an approximate frequency of 10(-6) among the progeny of the irradiated cells . Data are reported here on the distributions of transformed-cell clone sizes in irradiated cultures reseeded at various times post-treatment . The results suggest that the second event in transformation occurs randomly during the growth of irradiated cultures of C3H 10T1/2 cells to confluence . When the same number of irradiated C3H 10T1/2 cells were seeded into petri dishes of different sizes {35 mm (8 cm2), 60 mm (21 cm2), 100 mm (55 cm2), 150 mm (145 cm2)}, the number of foci which arose per dish was dependent on the final cell numbers at confluence in the various dish sizes, such that the number of foci/cm2 was constant . When irradiated cells and parental C3H 10T1/2 cells were mixed in different proportions at low density, the number of foci which ultimately arose was a function of the number of progeny of irradiated cells present in the culture at confluence . The results presented here confirm previous studies and give further evidence that the radiation-induced malignant transformation of cells occurs in an indirect, multistage fashion. Int J Cancer, 1984 Jul 15, 34(1), 49 - 56 The isolation and characterization of colorectal epithelial cell lines at different stages in malignant transformation from familial polyposis coli patients; Paraskeva C et al.; The genetic disease familial polyposis coli (hereditary adenomatosis of the colon and rectum) provides an excellent model for the study of tumour progression in the large bowel . We have isolated and characterized four epithelial cell lines from colorectal tumours from polyposis coli patients . These cell lines are grown on collagen-coated Petri dishes in the presence of mouse 3T3 feeder cells in medium containing 20% foetal bovine serum . Of these cell lines three were isolated from premalignant adenomas and one from an adenocarcinoma . All four lines have a characteristic cuboidal epithelial morphology, and their epithelial origin was confirmed by positive staining with a monoclonal antibody which reacts specifically with the keratin filaments of simple epithelia . The adenoma-derived lines display ultrastructural features characteristic of colonic epithelium including desmosomes, microvilli and mucin droplets . One of the adenoma-derived cell lines, designated PC/AA, has retained differentiated functions in culture, namely mucin production, after 21 in vitro passages . PC/AA has a karyotype of 46, XY with no detectable chromosome rearrangements . The adenoma-derived lines could be passaged from clumps of cells but not from single cells even in the presence of 3T3 feeder cells . The carcinoma-derived line, designated PC/JW, could however grow from single cells in the presence of a feeder layer . The one premalignant adenoma-derived line tested so far, PC/AA, did not produce tumours in athymic nude mice . In contrast, the carcinoma-derived line, PC/JW was tumorigenic in athymic nude mice . PC/JW produced moderately well-differentiated tumours which were histologically similar to the adenocarcinoma from which the cell line was isolated . PC/JW has a near-diploid chromosome number with an isochromosome (1q), an isochromosome (14q) and an (Xp; 17q) translocation . Unidentified marker chromosomes were present in a few cells . The features at present which distinguish the carcinoma-derived line from the adenoma-derived lines are tumorigenicity, growth from single cells and chromosomal abnormalities . The isolation and characterization of differentiating human epithelial cell lines at different stages in malignant transformation provide an opportunity to examine the cellular and molecular mechanisms controlling tumour progression in the large intestine, and to obtain an insight into the multistep process of human epithelial carcinogenesis. Int J Cancer, 1984 Jul 15, 34(1), 11 - 20 Antigenic heterogeneity of clones and subclones from human melanoma cell lines demonstrated by a panel of monoclonal antibodies and flow microfluorometry analysis; Cillo C et al.; Cells from two melanoma cell lines, Me43 and GLL-19, were cloned in methylcellulose cultures and 20 randomly selected colonies from each line were picked up by micromanipulation, expanded in liquid cultures, and considered as clones of the original cell lines . The antigenic cell surface phenotype of these clones defined by panel of 12 monoclonal antibodies (MAb) was analyzed by flow microfluorometry (FMF) using a fluorescence-activated cell sorter (FACS II) and compared with the known stable phenotype of the parent cell line . The antibody panel consisted of eight MAb against melanoma-associated antigens, two MAb against monomorphic determinants of HLA-DR (la) and HLA-ABC, respectively, one MAb against the common acute lymphoblastic leukemia antigen (CALLA) and one MAb against carcinoembryonic antigen used as control . A remarkable heterogeneity in terms of qualitative and quantitative expression of the cell surface antigens studied was observed among and within the different clones . The single-cell origin of the clones was assessed by comparing the clonogenic cell frequency, determined by limiting dilutions in microculture plates, with the cloning efficiency observed in Petri dishes . Both techniques using methylcellulose medium gave the same percentages of growing colonies . Cells from four Me43 clones were recloned in methylcellulose and the phenotype of five randomly selected subclones from each clone was analysed using the same panel of monoclonal antibodies . Each subclone also displayed heterogeneity with individual phenotypes different from that of the original clone and from the parental Me43 cell line . The antigen expression by individual cells in situ within clones was analyzed on frozen sections from colonies using the same panel of MAb and a biotin-avidin immunoperoxidase method . The results confirmed the marked heterogeneity of antigen expression within and among colonies, as indicated by the FMF analysis. Blut, 1984 Jul, 49(1), 13 - 7 A method of test substance removal in agar colony assays using glass capillaries; Kastner M et al.; A technique has been developed to remove test substances, after defined incubation periods, from clonogenic in vitro assays using agar-containing glass capillaries . Following removal from the capillaries, the entire agar gels were washed in petri dishes and redrawn into new capillaries . Using 8 radioactive biochemicals of molecular masses ranging from 150 to 1300 dalton the kinetics of diffusion between 1 and 20 min were determined . Using a wash solution-to-assay volume ratio of 20:1, a single washing for 10 min yielded between 90% and 99% removal by diffusion of test substances . By incorporating myelopoietic stem cells it was demonstrated that the cells to be assayed can be quantitatively transferred, without loss or stress, out of and back into capillaries . Thus the reversibility of test substance action can examined under defined conditions avoiding technical problems of previous methods. Dev Biol, 1984 Jul, 104(1), 86 - 96 Recognition of extracellular matrix components by neonatal and adult cardiac myocytes; Borg TK et al.; Recognition of extracellular matrix (ECM) components by isolated cardiac myocytes from neonatal (4-5 days postpartum) and adult rats was determined by measuring cell attachment to substrates made of ECM components . The substrates were petri dishes coated with either fibronectin, laminin, native monomers of collagen types I, II, III, IV, and V, denatured collagen, or gels containing reconstituted collagen fibers . Adult myocytes attached efficiently to laminin and type IV collagen, weakly to fibronectin, but not at all to the other types of collagen . Neonatal myocytes attached well to all types of collagen and to fibronectin and laminin . Antibodies raised against surface membranes of neonatal myocytes, adult myocytes, or adult hepatocytes were assayed for their ability to inhibit cell attachment to the various ECM substrates . Antibodies against the surface of neonatal myocytes as well as antibodies against the hepatocyte cell surface inhibited the attachment of neonatal myocytes and hepatocytes to collagen but not to fibronectin . Antibodies against the adult myocyte cell surface did not inhibit the attachment of neonatal myocytes or hepatocytes to ECM components . These results indicate the presence of binding molecules on the surface of neonatal myocytes that are involved in the recognition of collagen at a time when collagen is being secreted and formed into a three-dimensional network that attaches to the cell surface of the myocytes . This recognition and adhesion to collagen occurs by a mechanism independent of fibronectin . The binding molecules for collagen could not be detected on normal adult myocytes isolated at a time when the formation of the collagen network has already been completed. Cancer Res, 1984 Jul, 44(7), 3040 - 50 Actin cytoskeletal organization loss in the benign-to-malignant tumor transition in cultured human colonic epithelial cells; Friedman E et al.; The colonic epithelium in vivo is a highly indented sheet one cell thick . Culture methods have been developed to allow the normal cellular migration of the cells comprising this sheet to flatten it into a patch on the surface of a Petri dish {Friedman, E . A., Higgins, P.J., Lipkin , M., Shinya , H., and Gelb , A.M., In Vitro (Rockville), 17: 632-644, 1981} . Actin cytoskeletal organization was analyzed in such epithelial "patches" derived from several human colonic adenocarcinomas and their precursors, adenomas (benign tumors) . The actin cytoskeleton was visualized by fluorescence microscopy after the fixed, permeabilized cells were stained with rhodamine-conjugated phalloidin . This drug has a very high affinity for actin filaments and a much lower affinity for monomeric actin . Actin organization was scored from 0 (no cables) to 5 points (extensive intercellular cable network) . The phalloidin-stained actin found in seven adenocarcinomas had a predominantly granular fluorescence pattern with very little cable organization, scoring an average of 0.9 +/- 0.8 (S . D.) . Three established cell lines derived from human colon carcinomas contained no cables by this analysis, scoring 0.0 +/- 0.0 . In marked contrast, all 12 of the cultured adenomas had extensive actin cable networks, scoring an average of 4.3 +/- 0.4 . There was no statistical difference between adenomas of differing histopathology class and malignant potential . However, cytoskeletons of plasminogen-activator-secreting "late-stage" preneoplastic cells from adenomas became disorganized by exposure to 12-O-tetradecanoyl-phorbol-13-acetate or another tumor promoter, teleocidin B . They scored, respectively, average actin organization values of 0.0 +/- 0.0 and 0.4 +/- 0.6 . In contrast, nonplasminogen -activator- secreting "early-stage" preneoplastic cells from less advanced benign tumors were unaffected by 12-O-tetradecanoyl-phorbol-13- acetate or teleocidin B and retained extensive actin organization . Most, if not all, adenocarcinomas arise from preexisting preneoplastic adenomatous cells . Thus, loss of actin organization appears to mark the transition of noninvasive benign colonic tumors to invasive malignant tumors in humans . This transition is mimicked in vitro by exposure of certain "late-stage" preneoplastic cells to a tumor promoter which induces secretion of a plasminogen activator. Pflugers Arch, 1984 Jul, 401(3), 239 - 45 Electrophysiological study of single Leydig cells freshly isolated from rat testis . I . Technical approach and recordings of the membrane potential in standard solution; Joffre M et al.; Single Leydig cells were isolated from rat testis by a collagenase digestion procedure and purified through a 21,000 g self generated densities gradient of 35% Percoll . A method including collagen and fibronectin was proposed to attach freshly prepared Leydig cells to the bottom of plastic Petri dishes . Four hours after the isolation of the cells, it was simultaneously possible to determine their membrane potential by a standard electrophysiological technique using intracellular microelectrodes and to judge cellular integrity by direct microscopic observations . In standard Earle's solution, changes of membrane potentials appeared to be biphasic . On 198 impaled cells, 18 +/- 1 S after the impalement was effective, the membrane potential reached a most negative value (MP1) (-37.6 +/- 0.7 mV), followed by a gradual depolarization to a steady state (MP2) (-25.1 +/- 0.6 mV) which remained constant for a few minutes . In standard Earle's solution, the membrane resistance was low or decreasing towards the most negative potential, then it increased towards the steady potential . At this state, the average value of the cell input resistance was 65.9 +/- 6.0 M omega (n = 16) . No action potential was observed either in standard Earle's solution or under a depolarizing current state . It was concluded that the electrophysiological characteristics of the Leydig cell are similar to those of fibroblasts and macrophages, three types of cells with the same mesenchymal origin, present in the interstitial tissue of the rat testis . But the resting potential of the Leydig cell is higher and this secreting cell does not elicit hyperpolarizing oscillations at the steady state, under mechanical or electrical stimuli. In Vitro, 1984 Jul, 20(7), 585 - 96 Solute concentration effects on the expression of cellular heterogeneity of anchorage-independent growth among spontaneously transformed BALB/c3T3 cells; Rubin H et al.; Clones were derived in culture from a tumor initiated by spontaneously transformed 3T3 cells and tested for their colony-forming efficiency in agar (CFEag) . Incubation of petri dish cultures was done in subsaturation humidity to minimize mold contamination . There was great variation in CFEag between clones but also, under certain conditions, within clones . The most prominent condition that generated phenotypic diversity in CFEag was partial evaporation of the medium, which may occur during the protracted development of a mass population from a single cell . Evaporation was disproportionately great in 35-mm dishes and peripheral wells of multiwell plates . If the supraphysiological solute concentration resulting from evaporation was greater than 133% of normal, there was progressive suppression of cell growth in the succeeding transfer in agar or on plastic, even if isotonic medium was substituted 1 d before transfer . The effect of supraphysiological concentrations of all the solutes of the medium could be reproduced by simply increasing the NaCl concentration . Damaged cells were restored to their full growth potential after 3 d in isotonic medium . When nontransformed cells were chronically exposed to increased salt, irreversible increases in 2-deoxyglucose uptake were produced . With continued exposure of these cells to high salt, they became morphologically transformed, produced colonies in agar with high efficiency, and formed sarcomas when inoculated into nude mice. Cell Biol Int Rep, 1984 Jul, 8(7), 539 - 49 Role of substrata in determining the growth topology of transformed and nontransformed cells in culture; Reuveny S et al.; Cylindrical DEAE cellulose anion exchangers (DE-53), generally used for chromatography, were found suitable as a substratum for cultivating cells . Embryonic avian and mammalian cells cultured on DE-53 microcarriers (MC) grow in multilayers, while the same embryonic cells when transformed by avian sarcoma virus (ASV) grow in monolayers . These patterns of cell growth differ from those of normal and transformed cells grown on conventional glass or plastic Petri dishes, or on beaded MC . Cells derived from established cell lines such as BHK, HeLa, L-929, MDCK, and VERO grow in monolayer on these MC . A human adenocarcinoma cell line is the only exception growing in a multilayer form . These results indicate that the ability of cultured cells to grow in multilayers, is determined not only by their state of transformation but also by the properties of the support on which they are cultured. J Immunol Methods, 1984 Jun 8, 71(1), 37 - 42 Enumeration of rat allospecific antibody-producing cells by a solid-phase antibody-forming cell focus (AFCF) assay; Majoor GD; Individual rat cells producing antibody to rat MHC class I alloantigens ( RT1A ) and other erythrocyte (E)-associated alloantigens can be enumerated by a solid-phase antibody-forming cell focus ( AFCF ) assay . Cells are incubated in solid agarose medium in petri dishes coated with affinity-purified anti-immunoglobulin antibodies and antibody produced by any cell is captured by these anti-immunoglobulin antibodies . Foci of antibody to E alloantigens produced by single cells become visible upon subsequent addition of the appropriate target E . Execution of the assay in solid agarose medium facilitates handling of the petri dishes and reduces target background binding compared to performance of the assay in liquid medium. J Immunol, 1984 Jun, 132(6), 2813 - 9 Suppression of Con A mitogen-induced proliferation of normal spleen cells by macrophages from chickens with hereditary muscular dystrophy; Kline K et al.; Spleen cells from chickens with hereditary muscular dystrophy (MD) give low blastogenic responses to the T cell mitogen concanavalin A (Con A) while exhibiting normal mitogen stimulated blastogenic responses to the T cell mitogen phytohemagglutinin (PHA) . The addition of MD spleen cells to normal spleen cells caused a marked suppression of the Con A response of the normal cells while not affecting the PHA response of the normal cells . The suppressive activity by the MD spleen cells requires viable cells and is contact mediated . The suppressive activity is attributed to the presence in MD spleens of a population of suppressor cells with characteristics typical of macrophages . The suppressor cell activity was not removable by complement-mediated lysis using anti-T or anti-B sera, but it was reversible by treatment with carrageenan or carbonyl iron magnet, by passage through a Sephadex G-10 column, and by adherence to plastic petri dishes or glass beads . MD spleen cells depleted of the suppressor cell population remained unable to respond to Con A. Endocrinology, 1984 Jun, 114(6), 2060 - 7 Mammotroph autoregulation: uptake of secreted prolactin and inhibition of secretion; Kadowaki J et al.; A dissociated preparation of normal adult rat pituitary cells has been used to study PRL autoregulation at the level of the mammotroph . Female rat pituitary cells previously cultured for 48 h on polylysine-coated petri dishes were washed to remove serum and accumulated PRL and then incubated in fresh medium in the absence or presence of increasing concentrations of rat PRL . Accurate balance sheets, allowing for degradation and nonspecific adsorption of PRL, showed exogenous PRL to regulate the amount of PRL released by the cells . That this regulation was partly produced by uptake of secreted PRL from the medium was demonstrated by supplementing the medium with {125I}iodo-rat PRL . Inhibition of secretion also played a role and was implied by experiments showing that ease of reversal of the inhibition was inversely proportional to the density of cell culture, which was itself proportional to the amount of PRL in the medium and the duration of autoregulation . These results indicate that normal adult rat pituitary cells in primary culture are capable of regulating the amount of PRL in their external milieu and that uptake of already secreted PRL is an important component of the regulatory mechanism. Southeast Asian J Trop Med Public Health, 1984 Jun, 15(2), 148 - 54 Experimental infection of Oncomelania quadrasi with Paragonimus ohirai; Kawashima K et al.; Two hundred Oncomelania quadrasi collected from Leyte, Philippines were exposed to infection with Paragonimus ohirai, a rodent type lung fluke . In a group, each snail was exposed individually to 10 miracidia hatched from eggs which were brought from Japan to the Philippines . In another group, 100 snails were placed in a Petri dish and P . ohirai miracidia were added to provide 10 per snail . The observations were made each successive week after exposure . All the snails examined were positive for the larvae of P . ohirai . Nine-ten weeks after exposure, many cercariae were recognized . It was proved that O . quadrasi is highly susceptible to P . ohirai. Ann Trop Med Parasitol, 1984 Jun, 78(3), 307 - 18 Oviposition by African malaria vector mosquitoes . II . Effects of site tone, water type and conspecific immatures on target selection by freshwater Anopheles gambiae Giles, sensu lato; McCrae AW; Females of Anopheles gambiae s . lat., most of which would have been A . gambiae s . str., were collected from houses in coastal Kenya and tested for their oviposition preferences using Petri dishes in large laboratory cages with lighting equivalent to weak moonlight . Significantly more eggs were laid overnight in water over black than over paler tones, and this difference increased as contrast with the surrounding floor was increased . Direct observation revealed that over white targets, females oviposited from a settled posture, whereas over black targets they did so from flight . The influence on this behaviour of target darkness (tone) overrode that of cage size or target size . In tests which yielded markedly fewer eggs in sea water than in tap water, no significant difference was detected when cage floors were either black or white, although a black floor might have resulted in significantly greater discrimination against sea water had more tests been conducted . All further testing was done over black cage floors . Turbid water from a natural development site received more eggs than distilled, tap or swamp water, even though the turbid water appeared paler than the others . The females did not discriminate between rearing water and tap water, or tap water with and without pupae, but the presence of larvae was repellent . Turbid water from a development site thus seemed to possess an arrestant property which overrode selection favouring darker targets, and which was not derived from prior presence of conspecific immatures . It is suggested that for A . gambiae, oviposition from a settled posture is a response to sub-optimal stimuli, possibly indicating conditions under which oviposition would not occur in nature, and hence why cage experiments using white targets have in the past yielded confusing results. Int J Cell Cloning, 1984 May, 2(3), 142 - 60 Evaluation of an automated image analysis system for counting human tumor colonies; Salmon SE et al.; The Omnicon FAS II image analysis system was applied to counting tumor colonies grown in a soft agar human tumor clonogenic assay with a detailed protocol designed to assess the instrument's sensitivity, specificity, precision, and accuracy . Comparisons of technician and instrument counts were done on a blinded basis . Sensitivity studies (which used metal microspheres) yielded a correlation coefficient (r) of 0.999 between technicians and the counter . A field-by-field analysis of the instrument's specificity for identifying individual objects correctly as tumor colonies rather than artifacts (as identified by the technician) was excellent (r = 0.95) . In the precision studies (determined with repeated automated counting of the same samples for five days), the median coefficient of variation was less than 7% . Accuracy was evaluated on cultures of fresh biopsies from 30 human cancers obtained for drug sensitivity testing as well as on a series of tumor cell lines . The correlation between the mean number of colonies counted by the technicians and by the colony counter was greater than 0.91 . Similar comparisons of mean percent survival of tumor colony-forming cells after drug exposure between technician and machine were also quite acceptable (r = 0.85) . We conclude that the colony counter provided sufficient reliability to be applied to counting human tumor colonies grown in vitro . In addition, the colony counter performed the Petri dish counts ten times faster than experienced technicians and without associated operator fatigue. Metabolism, 1984 May, 33(5), 447 - 53 Dispersed adult rat pancreatic islet cells in culture: A, B, and D cell function; Weir GC et al.; The availability of suitably characterized dispersed islet cell preparations may assist in studies of islet function . Since freshly dispersed adult rat islet cells failed to respond appropriately to secretagogues (no alteration in insulin, glucagon, or somatostatin release after glucose change; modest response to IBMX), these cells were established in primary monolayer culture . We then tested the hypothesis that islet function is at least partially determined by islet structure . B cells which had attached to Petri dishes during a culture period of four days were well preserved at the ultrastructural level, with mitochondria clustered at the cell face attached to the Petri dish and secretory granules concentrated towards the portion of the cell facing the medium . Since it was not possible to estimate cellular hormone content or hormone release as a function of the number of specific types of cells, fractional rates of release and hormone content ratios were compared with those for intact islets maintained in culture in parallel . Whereas the ratio of somatostatin:insulin content was similar for islets and cells (approximately 0.7:100), the dispersed cell population appeared depleted in glucagon (glucagon:insulin ratios being 17:100 for islets and 4:100 for cells) reflecting either degranulation or relative loss of A cells . In contrast to the lack of responsiveness seen with freshly dispersed islet cells, the cultured cells released insulin in response to glucose and glucose plus IBMX in a fashion comparable to that seen with cultured islets . Proinsulin biosynthesis (incorporation of {3H} leucine) was higher in cultured cells than islets . Somatostatin release was lower from dispersed cells than from islets while the opposite was true for glucagon.(ABSTRACT TRUNCATED AT 250 WORDS) Bull Soc Pathol Exot Filiales, 1984 May-Jun, 77(3), 401 - 6 {A case of mixed otomyiasis in Ivory Coast}; Fakry K et al.; Authors relate the first case of otomyiasis in Ivory Coast due to Chrysomyia bezziana and Sarcophaga sp . They insist on scarcity of such a localisation . Egg-laying and development of larvae in tympanum case was favoured by a preexisting cholesteatoma . Precise entomological diagnosis needs incubation of the larvae in Petri dishes. Mutat Res, 1984 Apr, 130(2), 127 - 37 Induction of mutations by chemical agents at the hypoxanthine-guanine phosphoribosyl transferase locus in human epithelial teratoma cells; Huberman E et al.; Induction of 6-thioguanine (TG) resistance by chemical mutagens was examined in a line of cells derived from a human epithelial teratocarcinoma cell clone . The cells, designated as P3 cells, have a stable diploid karyotype with 46(XX) chromosomes, including a translocation between chromosomes 15 and 20 . Efficient recovery of TG-resistant mutants induced by the direct-acting mutagens: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10 -tetrahydrobenzo{a}pyrene (BPDE); and benzo{a}pyrene (B{a}P); activated in a cell-mediated assay, required an expression time of 7 days and a saturation density of 2 X 10(4) cells/60-mm petri dish . The TG-resistant mutant cells induced by MNNG and BPDE maintained their resistant phenotype 4-6 weeks after isolation . This mutant phenotype was associated with a more than 10-fold reduction in hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity relative to that of the parental P3 cell line, which was shown to catalyze the formation of 4.6 pmoles inosine-5'-monophosphate (IMP)/min/microgram protein . Induction of TG resistance was also observed in P3 cells cocultivated in a cell-mediated assay with human breast carcinoma cells, which are capable of polycyclic aromatic hydrocarbon (PAH) metabolism, after treatment with the carcinogenic PAHs: B{a}P, chrysene, 7,12-dimethylbenz{a}anthracene (DMBA), and 3-methylcholanthrene (MCA) . The degree of mutant induction in this assay was related to the carcinogenic potency of these PAHs in experimental animals . The most potent mutagen was DMBA, followed in decreasing order by MCA, B{a}P, and chrysene . DMBA, at 0.4 microM, increased the frequency of mutants for TG resistance from 2 for the control to about 200 TG-resistant mutants/10(6) colony-forming cells (CFC) . Benzo{e}pyrene (B{e}P) and pyrene, which are not carcinogenic, were not effective in the assay . None of the PAHs was mutagenic in the P3 cells cultivated in the absence of the PAH-metabolizing cells . These results indicate that the P3 cells can be useful for the study of mutagenesis at the HGPRT locus by direct-acting chemical mutagens, as well as by chemicals activated in a cell-mediated assay. Physiol Behav, 1984 Apr, 32(4), 565 - 73 An empirical study of the structure of the patrol/marking motivational system in the rat; Lee S et al.; The existence and structure of an hypothesized motivational system of patrol/marking was supported and elucidated by a behavioral study on untrained highly inbred laboratory rats . One rat (the "runner") was placed into a test chamber containing a wire-mesh running wheel flanked by two chambers, one of which contained another rat (the "target") . Four conditions of runners (socially-isolated males, socially-housed males, non-estrous females, and estrous females) were exposed to four types of targets (socially-housed males, non-estrous females, estrous females, and blank targets consisting of any empty target chamber) . Also placed in the chamber was a Petri dish containing scent-markings of the target rat . The experiment was designed in a counterbalanced way with 10 replications and repeated two times in two separate years . As predicted from the hypothesis, scent-marking, sniffing the dish, locomotion (number of wheel revolutions), and approach (differential running towards the target) were all correlated with each other and varied in the same way as a function of the hormonal and experiential condition of the runner and the type of target . They were interpreted to reflect motor patterns of a single unitary patrol/marking motivational system . Grooming, on the other hand, did not correlate with the other behaviors and facial gland secretion was, therefore, rejected as a motor pattern of patrol/marking. Scand J Work Environ Health, 1984 Apr, 10(2), 109 - 13 Sampling of high concentrations of airborne fungi; Blomquist G et al.; The paper discusses a method developed for determining airborne fungi particles in environments highly contaminated with mold fungi . The collection of airborne fungi was performed with two slit samplers . These sampling devices were found to give the highest values out of the three different types tested simultaneously . After spores had been collected with the slit samplers, the collection medium, an agar gel, was removed from the petri dishes and homogenized in a sterile 0.9% sodium chloride solution . The homogenate was diluted stepwise and spread on agar plates prior to the cultivation and the determination of viable counts . When the homogenization procedure was tested on samples collected from three different work environments, no increase or decrease in the number of colony-forming units could be detected . Storage of the homogenate at 2 degrees C over 9 d did not increase the number of viable fungal colonies. Exp Mol Pathol, 1984 Apr, 40(2), 223 - 34 Epidermis promotion of collagenase in hypertrophic scar organ culture; Ehrlich HP et al.; The pathophysiological biology of human hypertrophic scar was examined in a long-term organ culture system . Fresh full-thickness, thin slices of scar were placed in petri dishes . Tissue was successfully maintained for 2 weeks in an environment made up of CMRL-1066 medium, fetal bovine serum, insulin, and hydrocortisone under an environment of 40% O2, 5% CO2, and 55% N2 at 37 degrees C on a rocking platform . Histologically the explants were viable and remained differentiated . The omission of hydrocortisone caused localized destruction of the connective tissue matrix under the epidermal layer . Transplanting the epidermis to the adipose surface of an explant before culturing in hydrocortisone-free medium, produced localized connective tissue matrix destruction only within the deep dermal layer . Removal of the epidermis before culturing in hydrocortisone-free medium produced no localized connective tissue matrix destruction . Culture medium from intact explants maintained in hydrocortisone-free media had higher levels of latent collagenase activity compared to epidermal free explants and intact explants in hydrocortisone-containing medium . This hypertrophic scar latent collagenase had a molecular weight estimated to be 33,000 by molecular-sieve chromatography . The active form of the enzyme had a molecular weight estimated to be 26,000 . When examined by gel electrophoresis, activated collagenase cleaved types I and III native collagens, producing TC-A peptide fragments of alpha chains . Type V collagen was not cleaved by this enzyme . Metal chelators such as 1,10-phenanthroline blocked enzymatic activity . Serine and sulfhydryl proteolytic inhibitors showed no effects . Intact hypertrophic scar has the capacity to produce collagenase which appears responsible for the destruction of the connective tissue matrix of the scar . The production of hypertrophic scar collagenase is somehow controlled by the epidermis. EMBO J, 1984 Mar, 3(3), 637 - 44 Synthesis of a testosterone-dependent secretory protein by rat seminal vesicle-derived cell lines; Tajana GF et al.; Primary cell cultures were established from explants of rat seminal vesicle . The establishment of primary cell cultures required, among other factors, the presence of testosterone . Two cell populations were detected in such primary cultures: fibroblast-like cells and epithelial-like cells; the latter encompassed a subtype of small cells and a subtype of large squamous cells (most likely the result of a degenerative process acting upon the former) . Histochemical, as well as electron-microscopical observations, indicated the presence of a persistent secretory activity in the small epithelial cells; fibroblast and large squamous epithelial cells were inactive in this respect . Staining of the cells with a peroxidase-conjugated antibody and analysis of the proteins produced in the presence of labelled methionine, showed that one of the major rat seminal vesicle secretory proteins, namely RSV-IV, was also produced . Conditions which favoured the growth of epithelial cells, rather than of fibroblasts, were determined . The use of nearly homogeneous cell populations and the use of collagen-coated Petri dishes, allowed the cloning of two independently obtained permanent cell lines, namely SVC-1 and SVC-2 . The in vitro growth rate of both cell lines was modulated by the amount of testosterone in the medium . Both cell lines were able to synthesize a significant amount of RSV-IV protein under testosterone control. Clin Immunol Immunopathol, 1984 Mar, 30(3), 430 - 6 Deficient DR antigen expression on human cord blood monocytes: reversal with lymphokines; Stiehm ER et al.; Expression of DR antigen on cord blood (neonatal) human monocytes using complement-mediated cytotoxicity with anti-DR alloantisera and fluorescent-activated cell sorting (FACS) utilizing a battery of anti-DR mouse monoclonal antibodies was assessed . Forty preparations of neonatal cord blood monocytes were purified by adherence and elution from plastic petri dishes: lymphocyte contamination was less than 5% as indicated by esterase and peroxidase stains and cell sizing . By cytotoxicity tests 22 +/- 5.5% (SD) of neonatal monocytes expressed DR compared to its expression on 78.6 +/- 3.1% of adult monocytes . By FACS analysis, the frequency of DR expression on neonatal monocytes was 19-33% compared to 71-82% for adult monocytes . Incubation of neonatal monocytes with concanavalin A (Con A) or phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cell culture supernatants (lymphokine) or recombinant interferon-alpha (IFN-alpha) increased the expression of DR antigens in a dose- and time-dependent manner . A Con A-supplemented culture supernatant of unstimulated peripheral blood mononuclear cells had no effect on DR expression . Neonatal monocytes that were pretreated with anti-DR and complement in order to remove DR-positive cells were induced to express DR antigen after 2 days in culture with lymphokine . Thus DR-negative neonatal monocytes can be induced to express DR antigen . These results suggest a correctable maturational deficiency of neonatal monocytes . The inducibility of DR antigen expression by lymphokines and recombinant IFN-alpha suggests that they play an important role in the regulation of immune responses. In Vitro, 1984 Feb, 20(2), 95 - 102 Human bronchial explants in long-term culture: establishing a baseline for secretion; Snyder CE et al.; A serum-free culture medium was employed to evaluate both qualitative and quantitative aspects of secreted proteins derived from human bronchial explants over a period of 26 to 50 d . It was found that protein and hexose were secreted at a reasonably constant rate during this period . Viability of explants was assessed by histological examination, attachment to scored grids of the petri dish, incorporation of labeled precursors into tissue proteins, and by fluorographic analysis of the sodium dodecyl sulfate-polyacrylamide gel pattern of the secreted material . This culture system is useful as a model for the study of secretory products and processes and how they are affected by various stimuli. Radiat Res, 1984 Feb, 97(2), 434 - 42 A thin-film culturing technique allowing rapid gas-liquid equilibration (6 sec) with no toxicity to mammalian cells; Koch CJ; A method is described for inoculating mammalian cells onto the central area of glass petri dishes . The medium depth above the cells is only 100 microns for an added medium volume of 1 ml and increases linearly and rapidly with additional medium . The theoretical time constant for equilibration of the medium with the gas is related to the square of the medium depth . The experimental time constant was measured in two different ways for large and small medium depths, giving excellent agreement with the theoretical values . Although the time constant is only 6 sec for the case of 1 ml of added medium, there is no drying out of the medium or toxicity to the cells because of a large reservoir of medium in the meniscus at the periphery of the dish. J Clin Lab Immunol, 1984 Feb, 13(2), 97 - 100 Direct depletion and purification of monoclonal antibody defined cells from unfractionated human mononuclear leukocytes using antibody coated polystyrene Petri dishes; Cooper KD et al.; Human peripheral blood mononuclear leukocytes were depleted or enriched in various monoclonal antibody-defined subsets using a simplification of the indirect " panning " technique . Unfractionated mononuclear leukocytes (MNL) were sensitized with appropriate dilutions of monoclonal antibodies to human Lyt3 , OKT4 and OKT8 antigens, and to a monocyte-myeloid line antigen . The sensitized cells were then placed on polystyrene Petri dishes coated with goat antimouse IgG to obtain a population of cells depleted and a population of cells enriched in each cell type . Direct separation of surface immunoglobulin-bearing cells was similarly achieved by coating the Petri dish with goat antihuman immunoglobulins . Results showed that MNL could be depleted to greater than 95% purity with these methods and that positively selected adherent cells could be enriched to at least 85% purity . The relative proportions of MNL subpopulations in various sorted cell populations is reported . Cells obtained by panning are functionally intact . As compared to complement lysis both marker positive and marker negative cells can be obtained and the technique requires less technical expertise than using fluorescence activated cell sorting . The modification reported here is simpler and less time consuming for human T cell subpopulation separations than previously reported panning methods since an initial sheep erythrocyte rosetting step was not used. Immunology, 1984 Feb, 51(2), 275 - 85 Lymphocyte chemotaxis in inflammation . VII . Isolation and purification of chemotactic factors for T lymphocytes from PPD-induced delayed hypersensitivity skin reaction site in the guinea-pig; Shimokawa Y et al.; Four types of lymphocyte chemotactic factor (LCF-a, -b, -c and -d) could be isolated from extract of 24-hr-old delayed-type hypersensitivity (DTH) skin reaction sites induced with purified protein derivative (PPD) in guinea-pigs by gel filtration on Sephadex G-100 followed by chromatography with DEAE-Sephadex . Partially purified LCF-b was thought to be a heat-stable protein with a molecular weight (mol . wt.) of about 14,000 . LCF-c separated from LCF-d by chromatography with DEAE-Sephadex was highly purified by chromatography with CM-Sephadex, immunoadsorbent chromatography coupled with anti-IgG antibody, and chromatofocusing in that order . It was considered to be a heat-labile protein with a mol . wt . of about 160,000 and with pI of 8.1 +/- 0.2 . LCF-d first separated from LCF-c was also highly purified by chromatography with CM-Sephadex followed by preparative isotachophoresis . The factor was considered to be a heat-labile protein with a mol . wt . of approximately 300,000 and with pI of 6.2 +/- 0.2 . These factors were similarly active for non-adherent cells (mostly T cells) but not for cells (mostly B cells) adherent to anti-IgG antibody-coated petri-dishes . Since LCF-a was active for B cells as described earlier, it is thus suggested that LCF-b, LCF-c and LCF-d may be important for T cell migration in the DTH site to PPD. J Protozool, 1984 Feb, 31(1), 186 - 7 Development of second generation merozoites of Leucocytozoon caulleryi in vitro; Isobe T et al.; Sporozoites of Leucocytozoon caulleryi were inoculated into specific-pathogen-free (SPF) chickens intravenously . Fourteen days after inoculation, the infected blood, parasitized with second generation merozoites, was collected from the chickens . The blood was then suspended in SPF chicken serum or RPMI-1640 culture medium supplemented with 20% SPF chicken serum in petri dishes, which were cultured at 37 degrees C or 41 degrees C in a humidified atmosphere of 5% CO2 . Second generation merozoites in parasitized, immature erythrocytes developed continuously through several substages and finally became micro- and macro-gametocytes after 6 days of incubation. Z Parasitenkd, 1984, 70(3), 297 - 302 Further studies on in vitro pairing of Echinostoma revolutum (Trematoda) adults; Fried B et al.; Various factors that influence in vitro pairing of Echinostoma revolutum adults were studied . More worms paired in Locke's, Ringer's, and 0.85% NaCl solutions than in the defined medium NCTC 135 . Also, more pairing occurred at 39 or 43 degrees C than at 35 degrees C . Echinostomes placed 1, 3, or 5 cm apart in 5.5 cm diameter Petri dishes as well as those placed 2, 4, or 6 cm apart in 8.5 cm diameter Petri dishes paired; however, worms placed 8 cm apart in the latter dishes did not . Individuals maintained in Locke's solution at 20 or 39 degrees C for 1 h after having been removed from the chicks paired less frequently than those used for experiments immediately after their removal from chicks . Worms showed more pairing in fresh Locke's solution than in this solution preconditioned by excretory-secretory products of E . revolutum . Scanning electron microscopy revealed no differences between the tegument of fresh worms and of those maintained under experimental conditions for 2 h at 39 degrees C. Immunol Lett, 1984, 8(6), 295 - 9 Complement activation according to the alternate pathway by glass and plastic surfaces and its role in neutrophil adhesion; Hed J et al.; C3-deposition, generated by complement activation according to the alternate pathway, was detected on borosilicate glass slides and polystyrene Petri dishes . The C3-depositions grew peripherally until the entire surface was covered . The deposits were also visualized with scanning electron microscopy and could not be washed away with low-pH (3.5) or high-pH (9.6) buffers . No consumption of complement function was detected indicating a phenomenon restricted to the glass and plastic surfaces . The C3-deposits could mediate an adherence of human neutrophils. J Orthop Res, 1984, 2(1), 15 - 22 In vitro growth of bovine articular cartilage chondrocytes in various capacitively coupled electrical fields; Brighton CT et al.; Isolated articular cartilage chondrocytes from 1- to 3-week-old male Holstein calf knee joints were formed into pellets containing 4 X 10(6) isolated cells and were grown in tissue culture medium (minimum essential medium/NCTC 135) containing either 1 or 10% newborn calf serum (NBCS) in plastic Petri dishes in 5% CO2 and air at 37 degrees C in saturation humidity . On the 4th postisolation day either {35S}sulfate or {3H}thymidine was added to the medium, and the pellets were exposed for 24 h to capacitively coupled electrical fields (10, 100, 250, and 1,000 V peak-to-peak, 60 kHz, sine wave signals) . The pellets were then harvested, dialyzed, hydrolyzed, and assayed for DNA, protein, {35S}sulfate incorporation, and {3H}thymidine incorporation . Results indicated that at 250 V peak-to-peak there was a statistically significant increase in {35S}sulfate in 1% NBCS and a statistically significant increase in {3H}thymidine in 10% NBCS . At potentials above or below 250 V no changes were noted . Thus, articular cartilage chondrocytes grown in pellet form can be stimulated to increase glycosaminoglycan synthesis or to increase cell proliferation by an appropriate capacitively coupled electrical field . The importance of the serum concentration in the medium in evaluation of biosynthesis in vitro is noted. J Toxicol Environ Health, 1984, 13(2-3), 397 - 411 In vitro systems for exposure of lung cells to NO2 and O3; Rasmussen RE; In vitro studies of the effects of NO2 and O3 require development of methods for separation and culture of those lung cells that experience in vivo exposure, and also the design and construction of systems for controlled exposure of the cells to known concentrations of the gases . Separation of lung cell types has been accomplished by enzymatic dispersal of lung tissue and centrifugation of the mixed cells on media of various densities in order to separate the cells on the basis of buoyant density or sedimentation rate . The application of centrifugal elutriation has enabled separation of type II alveolar cells and Clara cells with a high degree of purity . Alveolar macrophages and endothelial cells have also been obtained in good yield . Exposure of cultured cells to test atmospheres requires precise control of pollutant levels, close contact of cells and gas without an intervening layer of medium, capability for prolonged exposure, and maintenance of sterile conditions, so that recovered cells can be cultured further or studied for other biological activity . Systems which meet these criteria include roller bottle cultures, petri dish cultures on rocker platforms, cell cultures on cellulose filters fed by perfusion of medium from the side opposite the cells, and cells grown in dishes with gas-permeable film bottoms . Systems that rely on solution of the gases in the overlaying medium do not resemble exposure conditions in vivo, and may not be suitable for studying effects of the poorly soluble oxidant gases . The cell exposure systems have not been used extensively for studies of the effects of pollutants on freshly isolated specific lung cell types . Such studies should be encouraged. J Steroid Biochem, 1984 Jan, 20(1), 449 - 54 Vasopressin: a potent growth factor in adrenal glomerulosa cells in culture; Payet N et al.; Previous studies have shown that vasopressin stimulates the mitotic activity in adrenal zona glomerulosa cells in intact as well as in hypophysectomized rats . (Payet and Isler, Cell and Tissue Res . 172, 1976; Payet and Lehoux, J . steroid Biochem . 12, 1980) . We now report that this effect is direct and specific, since vasopressin stimulates the mitotic activity of rat adrenal zona glomerulosa cells in primary cultures . These cells were prepared by dissociation with collagenase in the culture medium MEM-d-Valine . Isolated cells were placed in 3.5 diameter petri dishes in MEM-d-valine medium containing 15% fetal calf serum and antibiotics for two days and 5% fetal calf serum for subsequent cultures . The medium was changed at 24 hr intervals . The hormones were added 3 days after the culture was started . The mitogenic effect of vasopressin was found to be dependent both on time and hormone concentrations . Vasopressin (10(-11) M) stimulated thymidine incorporation 4.8 +/- 0.6-fold after 2 days of treatment and 5.3 +/- 1.6-fold after 8 days . When ACTH (10(-11) M) was added together with vasopressin (10(-11) M) the mitogenic effect was enhanced at 6.5 +/- 1.9-fold after 2 days and 12.9 +/- 6.9-fold after 8 days of treatment . The aldosterone and corticosterone outputs were also stimulated by the combined presence of vasopressin and ACTH in the incubation medium; a maximal effect was observed between 6 and 8 days of treatment . Vasopressin (10(-11) M) + ACTH (10(-11) M) stimulated the aldosterone output 7-fold and that of corticosterone by 18-fold . When added alone, vasopressin, as well as ACTH alone had only a small effect on the aldosterone output . However, ACTH alone stimulated the corticosterone output 10-fold . In conclusion, vasopressin is an important and specific growth factor of the adrenal zona glomerulosa cells . In addition, together with ACTH vasopressin stimulates the aldosterone and corticosterone output both in vivo and in vitro in primary cell cultures. Biol Cell, 1984, 50(1), 1 - 7 Sodium butyrate-induced adhesion in mastocytoma cells; Matsuhisa T et al.; Sodium butyrate induced adhesion of cultured mastocytoma p-815 cells to the surface of a standard tissue culture grade petri dish . The ratio of the number of adherent cells to that of total cells (adherent plus floating cells) was dependent on the serum concentration and on the dose of sodium butyrate . During approximately the first 6 hr after the addition of sodium butyrate, no cells adhered . The optimum conditions for adhesion were provided by 2 mM sodium butyrate and 15% fetal calf serum, 44 hr after addition of this compound . Morphologically, adherent cells consisted of spindle-shaped and round cells: the latter clustered to the former . Low concentrations of actinomycin D (0.005 microgram/mL) and of cycloheximide (0.5 microgram/mL) inhibited cell adhesion . Adherent cells were easily detached by 0.25% trypsin-0.02% EDTA but not by EDTA alone . Adherent mastocytoma cells which were cultured in the presence of 2 mM sodium butyrate, re-adhered to the surface of the dish . The ratio of adhesion in the second dish, however, was very low (35% after 2 hr incubation) . Radioactive iodinated surface proteins of butyrate-treated adherent cells showed two new bands (70,000 and 92,000 D) which were not detected in control cells, but there was no difference in the extent of labeling of high molecular weight protein (250,000 D) between butyrate-treated and control cells. J Biol Response Mod, 1984, 3(2), 219 - 25 Prostaglandin-related and adherent cell suppressor system in apparently cured Hodgkin's disease patients; Haim N et al.; Lymphocyte proliferation (LP) by phytohemagglutinin (PHA) and two suppressor systems of mononuclear cells was investigated in 22 patients with Hodgkin's disease (HD) who were apparently cured at the time of study . The results were compared with those obtained in healthy controls and in a group of patients with inoperable carcinoma of the bronchus . The prostaglandin-related suppressor cell system ( PGSS ) was tested by adding 1 microgram/ml indomethacin to the mononuclear leukocyte cultures . The adherent cell suppressor system ( ACSS ) was tested by removal of the adherent cells on plastic petri dishes . The PGSS index of suppression was defined as the ratio of LP by PHA with indomethacin to LP without indomethacin . The ACSS index was defined as the ratio of the nonadherent cell LP to the total LP . The mean PGSS index in HD patients (1.1 +/- 0.25) was significantly higher (p less than 0.01) than in healthy controls (0.94 +/- 0.2) . The ACSS index in HD patients (1.08 +/- 0.47) was also higher (p less than 0.02) than in controls . The LP by PHA value in HD patients (23,908 +/- 14,770 cpm) was significantly lower (p less than 0.001) than in controls (47,151 +/- 17,706 cpm) . ACSS values in bronchogenic cancer patients were lower than in HD patients . The LP by PHA value was significantly lower in HD patients compared with bronchogenic carcinoma patients (p less than 0.02) . HD patients who had a PGSS index of 1.3 or higher had a significantly lower LP value than those with a PGSS index lower than 1.3.(ABSTRACT TRUNCATED AT 250 WORDS) J Neurosci Res, 1984, 11(2), 131 - 44 Characterization of a subset of oligodendrocytes separated on the basis of selective adherence properties; Szuchet S et al.; A subset of oligodendrocytes (B3,f) was isolated by taking advantage of selective cell-substratum interaction . B3,f cells were characterized morphologically, biochemically, and immunocytochemically . Oligodendrocytes were isolated from 4-to-6-month-old lamb brains by a modified version of our published procedure {Szuchet et al, J Neurosci Methods 3:7-19, 1980} . Freshly isolated cells from band III were plated on plastic culture plates at a concentration of 2 X 10(6) cells/ml . Approximately 40% of the cells attached to the plate under these conditions . The remaining cells formed small floating clusters . We refer to the latter as B3,f oligodendrocytes . After 4 to 5 days, the supernatant containing B3,f cells was removed and centrifuged, and the pellet was resuspended in culture medium and replated on polylysine-coated petri dishes . B3,f oligodendrocytes attached to this surface and extended an intricate network of processes . The purity of the cultures, judged by the number of cells staining with a monoclonal antibody against galactocerebroside was 98-99% . This high degree of cell homogeneity was maintained throughout the life of the cultures . B3,f cells appeared to be highly differentiated and remained so in vitro . This is surmised by the expression of oligodendrocytic characteristic functions such as high levels of CNPase activity typically, 5 microM/min/mgP; high incorporation of H2 35SO4 into sulfatides, an overall lipid metabolism that mimics events associated with myelinogenesis {Szuchet et al, PNAS 80:7019-7023, 1983}; the presence, detected immunocytochemically, of myelin-associated glycoprotein and myelin basic proteins . It is concluded that this culture system offers an opportunity for studying the biology of interfascicular oligodendrocytes and their interaction with neurons and/or astrocytes . It also should open up a way of examining the relevance of oligodendrocyte polymorphism. Tissue Cell, 1984, 16(2), 157 - 66 Effect of epidermal growth factor on cultured adult rat hepatocytes; Jansing R et al.; When adult rat hepatocytes were cultured in plastic Petri dishes in a medium containing insulin and glucagon, supplementation with epidermal growth factor (EGF) had a pronounced effect on their viability, morphology, and biochemical integrity . Transmission and scanning electron microscopic studies showed that after 1 week cells denied EGF accumulated numerous non-electron-dense bodies and filamentous whorls, had irregular nuclei, and exhibited atypical cell surfaces . In contrast, cells grown for 2-3 weeks in the presence of EGF had well-preserved cellular organelles and remained as an epithelial-like monolayer . After 3 weeks EGF-exposed cultures were still inducible for liver-specific tyrosine aminotransferase, and both rat albumin and rat transferrin were recoverable from the culture medium . Virtually no viable cells were present at 3 weeks in EGF-deprived cultures. Cell Immunol, 1983 Dec, 82(2), 316 - 25 Enhanced natural killer (NK) cell activity and NK-sensitive thymic cells in murine muscular dystrophy; Semple JW et al.; Studies have shown that there is an abnormality in the thymus of dystrophic mice with respect to age-dependent thymus weight changes and altered morphology (T . DeKretser and B . Livett, Nature (London) 263, 682, 1976) . Recently, others have shown that natural killer (NK) cells can lyse cells of a large, immature, rapidly dividing cell subpopulation within the thymus of normal young (3 weeks of age) mice (M . Hansson, K . Karre, R . Kiessling, J . Roder, B . Anderson, and P . Hayry, J . Immunol . 123, 765, 1979) . The NK susceptibility of dystrophic mouse thymocytes as targets was therefore studied . Spleen cells from normal (+/+) and dystrophic (dy2J/dy2J) male C57BL/6J mice 8-10 weeks old were passed over nylon wool and the nonadherent cells were incubated with 51Cr-labeled YAC-1 lymphoma target cells or thymocytes in a 51Cr-release assay . Spleen cells from dystrophic mice killed twofold more YAC-1 target cells than did spleen cells from normal mice . Thymocytes from 3- to 4-week-old dystrophic mice were three to four times more susceptible to NK lysis by dystrophic mouse spleen cells as compared with normal mouse spleen cells . Spleen cells from dystrophic mice had the same NK activity against dystrophic and normal mouse thymocytes as targets . Normal mouse spleen cells killed three- to fourfold more dystrophic mouse thymocytes than that of normal mouse thymocytes as targets . Target cell-binding studies revealed that conjugate-forming cells from nylon nonadherent dystrophic mouse spleen cells were found to be two- to fourfold greater than for normal mouse spleen cells using YAC-1 tumor cells as targets . The number of lymphocytes bound per YAC-1 target cell ranged from 2 to 5 for dystrophic mouse spleen cells as compared with 1 to 2 for the normal control group . Using both normal and dystrophic mouse thymocytes as targets, the conjugate-forming cells from dystrophic mouse spleen cells were also found to be twofold greater than in the normal control group . Cold target inhibition studies revealed that the natural killing of dystrophic mouse thymocytes was due to a YAC-1-reactive NK cell . Effector cell depletion studies using monoclonal anti-Thy-1.2 plus complement treatment and plastic petri dish adherence also revealed that the natural killing of dystrophic mouse thymocytes was not due to either T lymphocytes or macrophages . Taken together, these results show an increase in NK-sensitive thymocyte targets in dystrophic mice, in combination with an increase in splenic NK activity. Toxicol Appl Pharmacol, 1983 Dec, 71(3), 330 - 41 Hamster hepatocytes in culture as a model for acetaminophen toxicity studies with inhibitors of drug metabolism; Harman AW et al.; A hamster hepatocyte system was developed for use in studying the toxicity of acetaminophen (APAP) . The cells were isolated and placed in culture conditions in petri dishes containing a film of collagen . Hepatocytes, after attachment to collagen, were exposed for various periods of time to different concentrations of APAP . Hepatocytes exposed to APAP exhibited concentration- and time-dependent GSH depletion followed by cytoplasmic enzyme leakage and an increase in malondialdehyde content (TBA-reactive material) . These effects were reduced by the drug metabolism inhibitors metyrapone, piperonyl butoxide, and dithiocarb . Removal of APAP and its unbound metabolites from cells prior to 1.5 hr followed by culture in drug-free medium resulted in no observable damage to the cells over a 24-hr period . Removal of drug after longer exposure times followed by culture in fresh medium resulted in eventual cell damage . This finding showed that deleterious changes caused by APAP occurred over a 1.5-hr period after which eventual hepatocyte damage could not be reversed by removal of the drug . Further experiments showed that metyrapone and dithiocarb had some protective effect when added after APAP had been completely removed from damaged cells . This result indicates that these agents have a protective action separate from, and in addition to, their ability to inhibit APAP oxidation via cytochromes P-450. Cell Immunol, 1983 Dec, 82(2), 422 - 5 The passive transfer of severe allergic neuritis in Lewis rats with lymphoid cells preincubated with P2 protein; Szymanska I et al.; The passive transfer of both clinical signs and histologic lesions characteristic of allergic neuritis was successfully performed in Lewis rats using pooled spleen and lymph node cells, or T lymphocytes therefrom, if first preincubated in petri dishes with P2 protein for 72 hr . For passive transfer, cells were taken from donors 8-16 days after sensitization with P2 protein or myelin in Freund's complete adjuvant, and administered via the tail vein; clinical signs appeared 12-13 days later . This study supports the importance of cell-mediated immunity in EAN and the antigenic role of the P2 protein. J Exp Med, 1983 Dec 1, 158(6), 1912 - 23 Acceptor-suppressor T cell hybridoma with a receptor recognizing antigen-specific suppressor factor; Takei I et al.; An acceptor hybridoma with a receptor that recognizes the keyhole limpet hemocyanin (KLH)-specific suppressor T cell factor (KLH-TsF) was established after the fusion of C57BL/6 splenic T cells enriched with KLH-coated petri dishes . The cloned hybridoma (34S-281) could be specifically activated by stimulation with the conventional KLH-TsF or monoclonal KLH-TsF from three different hybridomas in the absence of the relevant antigen (KLH) and it started to produce another factor that suppresses the antibody response against DNP-KLH in a KLH-specific fashion . The KLH specificity of the TsF was required for activation . The new factor was found not to bind the KLH but to be absorbed with the KLH-TsF-producing hybridoma . It is thus strongly suggested that the acceptor site has a complementary structure (antiidiotype) for the KLH-TsF . Moreover, the idiotypic determinant on KLH-TsF was found to have a structure similar to that on some of the anti-KLH antibodies, since the acceptor hybridoma was specifically killed by the conventional anti-KLH antibodies and complement . Drawing on the above results, the idiotype-antiidiotype network in the conventional antigen system is discussed. J Immunol Methods, 1983 Nov 25, 64(3), 345 - 51 Maintenance of granuloma macrophages in serum-free medium; Fauve RM et al.; Granulomas were induced by injecting polyacrylamide beads into subcutaneous pouches created by divulsion of the dorsal skin of mice . More than 10(7) phagocytic cells (60% macrophages, 40% polymorphonuclear cells) could be recovered from this granuloma . The separation of the phagocytic cells can be achieved either following sedimentation in Percoll or following the incubation of cells on plastic petri dishes . Phagocytosis of zymosan was observed in macrophages maintained in vitro for 1 month in Eagle's serum-free medium. Exp Cell Res, 1983 Nov, 149(1), 69 - 84 Interaction of bovine epithelial lens (BEL) cells with extracellular matrix (ECM) and eye-derived growth factor (EDGF) . I . Effects on short-term adhesiveness and on long-term organization of the culture; Tassin J et al.; A growth factor (EDGF) derived from the retina controls the proliferation and shape of adult bovine epithelial lens (BEL) cells in vitro as well as extracellular matrix (ECM) assembly . In order to analyse this mechanism and the specificity of the interactions between BEL cells and the extracellular matrix we have investigated the adhesion and growth of BEL cells on various substrata (fibronectin, laminin, ECM) . BEL cells treated with EDGF adhered more slowly to plastic Petri dishes than untreated cells, in part due to EDGF inhibition of fibronectin deposition . The untreated BEL cells spread less well on ECM or laminin than on fibronectin-coated plastic . The preferential adhesiveness of BEL cells on fibronectin vs laminin was confirmed by attachment experiments performed on replicas of SDS-PAGE of these proteins . However, in long-term cultures, 8 days after seeding, BEL cells were very differently arranged on plastic or on ECM . ECM by itself did not increase the proliferation rate but helped to restore an organized cell monolayer . BEL cells stimulated to grow on ECM by treatment with EDGF exhibited at least transiently contact inhibition producing a perfectly organized epithelium similar to the one observed in vivo . These results suggest specific interactions between ECM or ECM components with BEL cell that restrain excessive cell spreading and restore an original polarized phenotype of the cells seen in vivo. J Immunol, 1983 Nov, 131(5), 2254 - 7 Relationship of germinal centers in lymphoid tissue to immunologic memory . VI . Transfer of B cell memory with lymph node cells fractionated according to their receptors for peanut agglutinin; Coico RF et al.; We isolated germinal center B cells by exploiting their high affinity for peanut agglutinin (PNA) . The PNA+ and PNA- B cells, fractionated by panning on PNA-coated petri dishes, were examined for their ability to transfer memory responses to irradiated recipients at various times after priming . With such fractionated B cells from lymph nodes taken at the peak of germinal center formation, the largest response was obtained in recipients of the PNA+ B cell population . At 4 to 5 wk after priming, and 10 days after challenge with an unrelated antigen, memory responses were approximately equal in recipients of PNA+ or PNA- B cells . At 14 wk after priming, memory responses were found only in recipients of the PNA- B cell population . Memory B cells from the spleen, taken from mice primed in the footpad 8 wk earlier, were also PNA- . Finally, we show that boosting with a TNP-conjugate in the footpad, 6 mo after priming in the same footpad, induced the reappearance of marked memory responsiveness in the PNA+ B cell fraction of the draining node. Antonie Van Leeuwenhoek, 1983 Nov, 49(4-5), 387 - 97 Factors affecting the enumeration of coliphages in sewage and sewage-polluted waters; Havelaar AH et al.; The count of coliphages in naturally polluted waters was found to be dependent on many experimental factors . If Escherichia coli C was used as a host strain, consistently higher counts were obtained than with other strains (B,K-12-derivatives) . This could be explained partly by the absence of a restriction system in C . A nutrient medium (modified Scholtens' agar, MSA) was developed with optimal concentrations of calcium- and magnesium-ions . MSA was compared with other media used for phage work and was found to give higher counts than all but one medium, Phage Assay Agar (PAA), which performed equally . If plating was done in a single agar layer in a large-size Petri-dish, higher counts were found than with the well-known double-agar-layer method. Antiviral Res, 1983 Nov, 3(4), 235 - 9 A novel method for detecting the antiviral activity of flavans in their vapour phase; Bauer DJ et al.; This report describes a new method for detecting and measuring the antiviral activity of volatile compounds . This method, the vapour phase test, is a modification of the conventional plaque reduction test, in that the compounds instead of being incorporated in the overlay medium were deposited on the inner surface of the Petri dish lid . During the incubation period, the compounds (if volatile) permeated the overlay medium before exerting their antiviral effect . Compounds which have or may come to clinical trial against rhinovirus infections were compared by normal plaque reduction assays and by means of this new technique . Flavans were shown able to exert their antiviral activity in the vapour phase and thus display an advantage over non-volatile compounds . The report also briefly shows how the technique was used to evaluate the structure-activity relationships in a series of analogous compounds. FEBS Lett, 1983 Oct 31, 163(1), 54 - 6 Electrofusion of myeloma cells on the single cell level . Fusion under sterile conditions without proteolytic enzyme treatment; Vienken J et al.; A technique is presented which allows electrofusion of single cells under sterile conditions . The electrofusion chamber is placed in a Petri dish . Before a droplet of the fusion medium is pipetted between the electrodes, the chamber is completely covered with vaseline, which prevents the fusion medium evaporating . Additionally, the fusion chamber is treated with solutions containing poly(L)-lysine and pronase which results in a decreased movement of the cells on the glass between the electrodes and which allows electrofusion without any proteolytic pretreatment. J Immunol Methods, 1983 Oct 28, 63(3), 329 - 36 Purification of human basophils by negative selection; Landry FJ et al.; Enriched populations of human basophils were prepared using a combination of negative selection techniques . First, a 2-step discontinuous gradient was made using 65% and 55% isotonic Percoll . Purification of plasma basophils by centrifugation through the gradient increased basophils from 0.96 +/- 0.55% to 14.6 +/- 7.9% (n = 9) . In 5 other experiments a second step was added in which contaminating mononuclear cells were removed using a panning technique . In this technique, Percoll separated cells were treated with anti-T-cell antibodies . Contaminating T-cells were selectively removed by adherence to a petri dish coated with affinity purified goat anti-mouse IgG . Basophil purity was increased to 34 +/- 15% using this step . These experiments demonstrated that negative selection techniques can yield an increase in basophil purity 50-100-fold . The purified basophils contained the expected quantity of histamine (1.36-1.54 pg/basophil) and released the same amount of histamine in response response to different concentrations of goat anti-human IgE Fc as did unpurified cells. Cancer, 1983 Oct 15, 52(8), 1378 - 84 A case of T-cell chronic lymphocytic leukemia (T-CLL) expressing a peculiar phenotype (E+, OKM1+, Leu 1+, OKT3- and IgG EA-); Tagawa S et al.; A case of chronic leukemia is reported . Malignant cells had the morphology of lymphocytes and an affinity for skin . Cytochemically, they were peroxidase negative, and NaF-resistant alpha-naphthyl esterase positive . The peripheral mononuclear cells formed E-rosettes and reacted with Leu 1 and OKM1 . They were unreactive with OKT3, OKT4, OKT6, OKT8, and OKIa1 . The cells did not possess surface IG, C3b receptors, or IgG Fc receptors (EAIgG) . They responded weakly to phytohemagglutinin, and did not respond to concanavalin A . The cells did not adhere to Petri dishes or phagocytose latex beads . It is concluded that it was a case of OKT3-, OKM1+, EAIgG- T-cell chronic lymphocytic leukemia . This suggests that OKT3 does not recognize all peripheral T-cells, and that OKM1 is not specific for the monocyte-myeloid lineage . These cells may be useful in the characterization of E+, OKM1+ subset of peripheral mononuclear cells. Diabetes, 1983 Oct, 32(10), 915 - 20 Insulin secretion by fetal human pancreatic islets of Langerhans in prolonged organ culture; Mandel TE et al.; Fetal human pancreata, obtained from legally-induced abortions, were placed in organ culture to study their capacity to produce insulin over periods in vitro of between 18 and 40 days . Specimens obtained by hysterotomy usually showed increasing insulin secretion as measured by the insulin content of the media at each twice-weekly medium change . In most instances, relatively little insulin was produced during the first 7-10 days, but thereafter insulin secretion rapidly increased . In contrast, most specimens from prostaglandin-induced abortions showed high levels of insulin in the medium early in the culture period but little thereafter, indicating rapid release of insulin from damaged tissue . In some instances, after early insulin release by damaged tissue, some recovery of islet function occurred and insulin secretion again increased . The presence of differentiated endocrine cells was confirmed histologically . Tissue from each fetus was placed in a number of petri dishes, and for each individual fetus a qualitatively similar pattern of secretion was noted in each dish . These data suggest that representative and nondestructive monitoring of islet function is possible and may be important before such tissue is considered for use in islet transplantation in insulin-dependent diabetes. Int J Cancer, 1983 Sep 15, 32(3), 385 - 91 Studies of the mechanisms for the induction of in vivo tumor immunity . VII . Development of specific antitumor immunity in progressors and regressors; Ting CC et al.; The present study was aimed at comparing the development of specific antitumor immunity between hosts with progressively growing tumors and hosts with regressing tumors . The experiments were performed with a Friend virus-induced leukemia FBL-3 in syngeneic C57BL/6 mice . The specific antitumor immunity was determined by in vitro cell-mediated cytotoxicity assay and in vivo tumor neutralization test . Both the systemic immunity (demonstrated in spleen) and immunity developed at tumor site were examined . For progressors, the tumor site was in the peritoneal cavity . For regressors, it was in a subcutaneous site of both flanks . Testing by the cell-mediated cytotoxicity assay showed that immune hosts and regressors had higher levels of systemic immunity than the progressors . However, when lymphocytes isolated from tumor sites were assayed, it was found that there was no remarkable difference between lymphocytes from progressor tumors (PTL) and lymphocytes from regressor tumors (RTL) . Both lymphocyte populations were similar in profile analysis; they were characterized as T cells and possessed the same antigenic specificity . Nevertheless, when in vivo tumor transplantation experiments were performed, RTL were found to give protection against FBL-3 challenge whereas PTL consistently failed to do so . On cytomorphological examination, the PTL were seen to contain large amounts of macrophages . The presence of macrophages in PTL appeared to have an inverse relationship to the in vivo protective effect . After removal of macrophages from PTL by Petri dish adherence, the nonadherent PTL were found to give in vivo protection . Furthermore, thymocytes from progressors and macrophages isolated from the progressor tumors were found to suppress the in vivo T-cell-mediated immunity . These findings demonstrated that suppressor T cells and suppressor macrophages were present in tumor-bearing hosts . These suppressor cells could interfere with the function of immune T cells at the efferent arm of the immune response. Biochem J, 1983 Sep 15, 214(3), 871 - 7 Activity of lysosomal cysteine proteinase during differentiation of rat skeletal muscle; Kirschke H et al.; Cysteine-proteinase activities were measured in extracts of pre- and post-fusion populations of rat myogenic line L6 cells and in extracts of whole rat muscle . Activities of cathepsins B, L and H were compared . The substrates used included Z-Phe-Arg-NMec (cathepsins B and L), Z-Arg-Arg-NMec (cathepsin B), and Arg-NMec (cathepsin H) (where Z = benzyloxycarbonyl, and NMec = 4-methyl-7-coumarylamide); the enzyme activities were more specifically differentiated by appropriate concentrations of the inhibitors Z-Phe-Phe-CHN2 (CHN2 = diazomethane), bestatin and E-64 {L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane} . These experiments have demonstrated the feasibility of determining the cysteine-proteinase activities of myoblasts from a single (60 mm-diameter) Petri dish, with enzyme concentrations in the range of 5-20 ng/ml . Specific activities of the enzymes in L6 cells increased 2-20-fold after fusion . Concentrations of cysteine proteinases in extracts from cultured myoblasts were two orders of magnitude greater than those in muscle-tissue extracts . Cultured-cell extracts contained endogenous inhibitor(s) to purified rat cathepsins B, L and H. J Cell Physiol, 1983 Sep, 116(3), 345 - 51 Fibroblast contraction of collagen lattices in vitro: inhibition by chronic inflammatory cell mediators; Ehrlich HP et al.; Fibroblast-populated collagen lattices (FPCL), prepared in petri dishes with serum-containing culture medium and incubated at 37 degrees C, undergo progressive and symmetric contraction (reduction in size) over a period of days . The in vitro contraction process requires viable cells with intact cytoskeletal elements, is associated with cell elongation, and is believed to represent a fibroblast function which also occurs in vivo during wound healing and tissue fibrosis . We report that soluble mediators elaborated by chronic inflammatory cells cultured in vitro, when added to FPCL, inhibit lattice contraction . Granulomas, isolated from the liver of Schistosoma mansoni-infected mice, secrete a factor(s) with an estimated molecular weight between 13,700 and 43,000 daltons (gel filtration: Sephadex G-200) and pI = 6 (preparative isoelectrofocusing in granular gel) which inhibits lattice contraction but is not toxic to fibroblasts . Supernatants (cell-free conditioned culture medium) of cultured macrophages isolated from these granulomas also contain this activity . The contraction inhibitory activity present in granuloma culture supernatants is abrogated by the addition of indomethacin to the lattices, while the addition of prostaglandin E2 (PGE2) alone to lattices inhibits contraction . Furthermore, culture supernatants interfere with fibroblast elongation in lattices . We propose that the ability of fibroblasts to contract collagen lattices in vitro and a fibrotic mass in vivo may be regulated by soluble products of chronic inflammatory cells, including macrophages . This process may be mediated by fibroblast-derived prostaglandins which alter cytoskeletal functions and has implications for understanding regulation of tissue fibrogenesis in a variety of diseases. Sem Hop, 1983 Sep 1, 59(29-30), 2113 - 7 {Fungal flora in a hospital environment}; Mallea M et al.; The airborne fungal flora was studied in different departments of a hospital by using a slit sampler (Casella, London) where the measured volume of air is impacted to the surface of culture medium contained in Petri dishes . Colonies were counted after 3 days incubation at 25 degrees C and 37 degrees C . Many species of hyphomycetes were identified . The main fungi isolated were Cladosporium sp (especially C . cladosporioides), Penicillium sp., Alternaria alternata, Aspergillus sp . (A . flavus, A . niger, A . fumigatus) . The results show that the variability of the spore number is related to the ventilation . The number of spores found indoors seemed proportional to those present outdoors in naturally ventilated rooms . The results are quite different in the air-conditioned wards . Also, there might be a correlation between the counts and the activity and occupation of wards. Eur J Immunol, 1983 Sep, 13(9), 711 - 9 Idiotypic and fine specificity analysis of a (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific suppressor T cell hybridoma at the level of cell surface structures, isolated receptor material and functional suppressor factor; Suzuki G et al.; The (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific T suppressor cell hybridoma 7C3-13 was established by fusing splenic B10.BR T cells enriched on NP-coated petri dishes with the AKR thymoma BW5147 . 7C3-13 was selected by anti-NPb idiotypic and anti-I-Jk antibodies in microcytotoxicity tests . The hybridoma expressed H-2k, I-Jk, Qa-1, Thy-1.1 as well as idiotypic (binding site-related) and framework Ig VH determinants, while it was negative for I-A, I-E/C, Thy-1.2, Lyt-1, Lyt-2 and Ig constant region determinants . Hapten-binding receptor material could be isolated from 7C3-13 cells on NP-coupled nylon nets and functionally active T suppressor factor (TsF) could be extracted from the hybridoma . Both types of soluble molecules express NPb idiotype, but the TsF carries I-J determinants in addition while the isolated receptors do not . The molecular weight of the isolated receptor material is 80 000, that of the TsF activity is 27 000 and 57 000-64 000, respectively . We thus were able to show that NP-binding molecules can be obtained in the form of cellular surface receptors, isolated receptor material and extracted TsF from one and the same, monoclonal, cell source. Exp Hematol, 1983 Sep, 11(8), 709 - 13 The clotting and staining of methylcellulose cultures of human hemopoietic cells; Chan P et al.; We describe a technique for clotting, fixing and staining methylcellulose cultures of human hemopoietic progenitor cells . The entire cellular population of a 1-ml culture in a 35-mm plastic petri dish can be recovered and preserved permanently with this method . The technique also provides the opportunity for distinguishing between pure erythroid and mixed colonies, for examining cellular morphology without tediously picking individual colonies, and for terminating cultures on the appropriate day without the necessity of scoring on that same day. Experientia, 1983 Aug 15, 39(8), 859 - 60 Pheromone-induced aggregation of ixodid ticks before host contact; Leahy M et al.; The presence of a pre-feeding aggregation pheromone was demonstrated in the species Dermacentor variabilis, Dermacentor andersoni, Dermacentor parumapertus, Amblyomma americanum and Haemaphysalis leporispalustris by assay within a petri dish . However, Amblyomma maculatum and Amblyomma cajennense did not aggregate in the sector containing discs of presumed pheromone within the hour period . D . andersoni and A . americanum recognized each other's pheromone and A . americanum recognized that of H . leporispalustris . Preliminary experiments with guanine and hemin as possible aggregating factors have thus far given inconsistent results. Mutat Res, 1983 Aug, 110(2), 401 - 12 Influence of confluent holding time on UV light mutagenesis in human diploid fibroblasts; Grosovsky AJ et al.; We have investigated the induction of mutants resistant to 6-thioguanine (6TG) following 254 nm ultraviolet light exposure of density-inhibited cultures of human diploid fibroblasts . Phenotypic expression of 6TG resistance was maximal within 9 days and remained stable through 19 days after irradiation . In reconstruction studies, complete recovery of 6TG-resistant mutants occurred at cell densities of up to 35 000 cells per 100-mm petri dish . The induced mutation frequency increased linearly with dose over the range of 3-9 J/m2; the D0 of the survival curve was 4.2 J/m2 . Delaying subculture to low density for 1.5-24 h after irradiation produced unexpected alterations in induced mutation frequencies . An increase in UV-induced mutations of approximately 3-fold was observed in cultures maintained in confluence for 3 h . This trend was reversed with longer holding times: the mutation frequency declined sharply in cultures held for 6 h as compared to the 3-h value, and thereafter showed a steady and gradual diminution to background levels . These data suggest that the repair of potentially mutagenic damage is a complex phenomenon which can lead to an increase or decrease in mutation frequency as a function of holding time . Although the decline in mutation frequency observed following longer holding intervals is consistent with the notion of an error-free process, we hypothesize that the increased mutation frequency produced by a short holding period reflects the existence of a cell-mediated process which enhances the mutagenic potential of at least some UV-induced DNA photoproducts. J Appl Physiol, 1983 Aug, 55(2), 343 - 52 Oxygen toxicity in cultured aortic endothelium: selenium-induced partial protective effect; Housset B et al.; The time course of biochemical changes related to cell loss and damage during exposure to 95% O2 {DNA and protein content of dishes, lactate dehydrogenase (LDH) release} was studied in postconfluent endothelial cells isolated from pig aorta, cultured in standard medium and in medium supplemented with 2 X 10(-7) M selenomethionine (Se-Met) . A fourfold increase in glutathione peroxidase (G-Px) was the only major enzymatic Se-related effect under both normoxic and hyperoxic conditions, the other antioxidant enzymes being little or not at all affected by this treatment . The addition of Se-Met had a clearcut protective action against the cytotoxic effect of O2 as shown by measurements of DNA and protein content of Petri dishes and of LDH release . On the other hand, the most sensitive O2-related effect, namely the decrease in {3H}thymidine incorporation into DNA, was not affected by Se-Met addition . These experiments suggest that some of the O2-related toxic effects (but not the inhibition of DNA synthesis) could be mediated by lipid peroxides, since they were, at least partly, prevented by a Se-Met-induced increase in G-Px activity. J Immunol, 1983 Aug, 131(2), 572 - 80 Phenotypic and functional characterization of murine B lymphocyte precursors isolated from fetal and adult tissues; Landreth KS et al.; Murine fetal liver and adult bone marrow cells identified by monoclonal 14.8 antibody were enriched on antibody-coated polystyrene petri dishes . Cell surface immunoglobulin (sig)-bearing cells were depleted before this enrichment procedure, and the resulting preparations of 14.8+, slg- cells were characterized as to morphology, immunoglobulin gene expression, and functional potential in vivo and in vitro . All cells with detectable mu chains of IgM in the cytoplasm (cmu) were found to be included in the 14.8+ population . The enriched cells did not contain significant numbers of committed granulocyte-macrophage progenitor cells or putative hemopoietic stem cells . Selected cells from 16-day fetal liver were large, a majority of the cells had a lobulated rather than a spherical nuclear outline, and less than 1% had detectable cmu . Enriched cells from 19-day fetal liver were on the average smaller than those from 16-day-gestation liver and had a more typical lymphoid morphology; 30% were cmu+ . Adult bone marrow 14.8+, slg- cells were similar to 19-day fetal liver cells in morphology, and approximately half were cmu+ . These selected precursor cells retained the capacity to mature in vivo and in vitro . Fetal and adult 14.8+, slg- cells were efficient in generating newly formed B cells in vivo, and this maturation step appeared to be dependent on the presence of microenvironmental accessory cells . However, the ability of positively selected cells to mature in vitro was markedly decreased, and this potential was not rescued by providing known sources of accessory cells . Possible reasons for this difference are considered . This technique for positively selecting cells has allowed us to directly compare for the first time B cell precursors from fetal and adult tissues and will be invaluable for resolution of the cell compartments in the differentiation of B lymphocyte precursors, in the study of accessory cells known to facilitate this process, in the definition of humoral factors which may act on pre-B cells, in the study of immunoglobulin gene rearrangements which take place during normal differentiation, and for further comparative studies of fetal and adult lymphopoiesis. J Virol Methods, 1983 Aug, 7(2), 93 - 102 Optimal conditions for titration of SV40 by the plaque assay method; Fendrick JL et al.; The parameters of the Simian Virus 40 (SV40) plaque assay on African green monkey kidney cells were optimized for reproducibility and maximum plaquing efficiency . Plaques were visible as early as 8 days postinfection; maximum titers were obtained with a 10- to 11-day incubation period . Titers read 12-16 days postinfection were not significantly higher than those observed after 10-11 days . Adsorption volumes greater than 0.1 ml/60 mm Petri dish decreased plaque forming units (PFUs) detected . Times greater than 60 min for adsorption of virus to the cell monolayer did not significantly increase the titer; adsorption times less than 60 min resulted in decreased titers . Under standard conditions, 3 ml of overlay medium containing 0.8% agar was applied following virus adsorption and again on days 5 and 10 . Concentrations of fetal calf serum (FCS) in the overlay medium of 2.5 to 7.5% gave equal plaque formation . FCS concentrations of 1 and 10% resulted in slightly decreased and increased plaquing efficiencies respectively . Of the reagents tested, agar or agarose containing overlay media produced plaques of maximum number and size . An overlay of methyl cellulose resulted in the same number of plaques, but their size was reduced by approximately 70% relative to those observed in agar; thus longer incubation times were required . Gum tragacanth overlay medium was actually inhibitory to plaque development . DEAE-dextran, dextran sulfate, or DMSO added to agar overlay medium did not enhance plaque number or size, nor did they shorten the incubation period required for their detection. J Clin Microbiol, 1983 Aug, 18(2), 296 - 9 Microassay for Sindbis virus and interferon activity; Lloyd RE et al.; A simple and rapid microplaque assay for Sindbis virus was developed which uses microtiter plates and overlay medium consisting of methylcellulose and specific antibody to Sindbis virus . Discrete plaque formation was consistently observed on baby hamster kidney (BHK-15) cells within 24 h . The assay was reproducible, quantitative, and had about the same sensitivity as the agar overlay technique on chicken embryo cells in 35-mm petri dishes . The microplaque assay could be accurately applied to measuring interferon activity, particularly at low interferon levels . Overall, the microassay methods described here for assay of Sindbis virus yields and interferon activity retain the accuracy of chicken cell macroplaque assays while offering greater simplicity, rapidity, and economy of materials . This assay is also potentially applicable for use with other togaviruses. Biochem Biophys Res Commun, 1983 Jul 29, 114(2), 663 - 9 Homokaryon production by electrofusion: a convenient way to produce a large number of viable mammalian fused cells; Claude B et al.; A convenient technique to obtain homokaryons is described that provides large amounts of fused mammalian cells . Chinese hamster ovary cells grown in monolayers on a Petri dish are submitted to square wave electric pulses . Viability of cells is observed not to be affected by this electric treatment . The yield of fusion is strongly dependent on the strength of the field (KV/cm range) and on the duration of the pulse (microsecond range) . The yield is not improved by accumulation of pulses . Yields up to 80% are obtained and under our experimental conditions 200 000 cells are fused per assay. J Invest Dermatol, 1983 Jul, 81(1 Suppl), 127s - 31s Selective enrichment of human epidermal cell subpopulations using monoclonal antibodies; Morhenn VB et al.; In studying the mechanisms that regulate the growth and differentiation of the human epidermis, it would be helpful to obtain relatively pure populations of the different epidermal cell types . We have used a solid-phase immunoabsorption method termed "panning" to positively select two types of epidermal cells: Langerhans cells and the keratinocytes found in the basal cell layer (basal cells) . To attach basal cells to a goat anti-mouse IgG-coated plastic surface, we used murine monoclonal antibodies (VM-1 or VM-2), which were recently produced in our laboratory and bind specifically to antigens on human basal cells . Using antibodies VM-1 or VM-2, we panned for basal cells and obtained a yield of about 40 percent (an enrichment of about 2.5-fold) . The cells enriched for basal cells demonstrated much better growth and DNA synthesis than did the cell fraction depleted of basal cells . For positive selection of Langerhans cells, we used OKT6, a murine monoclonal antibody that binds specifically to Langerhans cells in the epidermis . We determined that of those cells preincubated with OKT6 and adherent to an antibody-coated petri dish surface, about 70 percent demonstrated OKT6 binding by fluorescence microscopy . This represents a 15- to 20-fold enrichment for Langerhans cells . The nonadherent cell fraction contained less than 1 percent OKT6-positive cells . Ultrastructural studies showed that the cells thus separated were Langerhans cells . The OKT6-positive but not the OKT6-negative cells were capable of stimulating allogeneic lymphocytes in the skin-cell-lymphocyte reaction . Thus the panning technique is an effective method for obtaining greatly enriched subpopulations of viable epidermal cells. Mol Cell Biol, 1983 Jul, 3(7), 1310 - 6 Immunological selection of variant mouse lymphoid cells with altered glucocorticoid responsiveness; Danielsen M et al.; We have devised an immunological procedure to separate cells on the basis of expression of mouse mammary tumor virus (MMTV) gene products . Plastic petri dishes coated with specific antibodies against MMTV proteins bind cells with an efficiency that correlates with the level of MMTV gene expression . Glucocorticoid-sensitive mouse thymoma cell line W7 was infected with MMTV . Clones from the infected population retain the relatively slow cytolytic glucocorticoid response and, in addition, exhibit a rapid induction of MMTV-specific RNA and proteins . By combining our immunological selection with the selection for resistance to hormone-mediated cytolysis, we have isolated variant cells which are resistant to the cytotoxic effect of glucocorticoids but which retain the induction of viral gene products and must therefore have a functional glucocorticoid receptor protein. J Clin Invest, 1983 Jul, 72(1), 113 - 21 Role of cell surface contact in the kinetics of superoxide production by granulocytes; Dahinden CA et al.; The complement-derived anaphylatoxin C5a and a putative analogue of bacterial chemotactic factor (N-formyl-methionyl-leucyl-phenylalanyl {fMLP}), as well as bacterial lipid A, all stimulate human granulocyte (PMN) adhesiveness and superoxide (O-2) production in a concentration-dependent manner . Since attachment of particulate matter to the PMN membrane is an early event in the triggering of respiratory burst of these cells, we further examined how adherence might modulate the release of O-2 induced by soluble mediators of inflammation . We found that both the quantity and kinetics of O-2 production depend on prior attachment of the cells to a surface . In stirred suspensions of PMN, fMLP induces only a short burst (2.5 min) of O-2 release associated with reversible PMN aggregation . The magnitude, but not the time course, of both these responses depend on the fMLP concentration . Unlike the short respiratory response of cells in suspension, PMN allowed to settle onto stationary petri dishes, then overlaid with fMLP, rapidly spread and attach to the surface where they remain and release O-2 throughout the 60-min test period . Prolonged O-2 release also follows fMLP stimulation in suspensions of PMN pretreated with cytochalasin B, in which case aggregation becomes irreversible during the 20-min burst . If fMLP is slowly infused into a suspension of cells at 37 degrees C or if PMN are challenged at 0 degrees C, and then warmed to 37 degrees C, O-2 release greatly decreases or becomes undetectable . Suspended PMN do not respond to a second challenge by the same stimulus regardless of the rate or temperature at which the first stimulus was added, a phenomenon formerly described as desensitization . However, if PMN challenged with fMLP in suspension undergo the short respiratory response and then are later placed in petri dishes, they adhere and resume production of O-2 without further stimulation . Chemotactic factor-induced adherence and O-2 release of PMN on a surface is entirely independent of either the mode of activation or prior O-2 release during preincubation in suspension . Human C5a also promotes PMN adherence and prolonged O-2 release in petri dishes . Furthermore, lipid A increases O-2 release and adherence of settled PMN, but fails to elicit either response from suspended PMN . These results indicate that cell surface contact plays an essential role in triggering the respiratory burst of PMN activated by soluble stimuli . This long-lasting