Preparation and neutralization characteristics of an anti-CSF antibody

Nine of ten rabbits immunized with a partially purified L-cell CSF had demonstrable titers of anti-CSF activity. In vitro the antibody was markedly inhibitory to both post-endotoxin mouse sera and several mouse tissue extracts. CSF containing conditioned media prepared from a number of sources showed variable inhibition suggesting that murine CSF's may be characterized by marked antigenic differences. Human sources of CSF were also inhibited thus indicating a degree of cross-reactivity between murine and human factors. These studies may provide the initial steps toward definition of the role of CSF in vivo.     

Tissue sources of bone marrow colony stimulating factor

Possible tissue sources in C57BL mice of the serum factor stimulating colony formation in vitro by mouse bone marrow cells have been investigated. A reproducible technique employing batch chromatography on calcium phosphate gel was developed for the extraction and assay of material with colony stimulating activity from mouse tissues. Sixteen hematopoietic and non-hematopoietic tissues from C57BL mice were found to vary widely in their content of extractable activity. Characterisation of the colony stimulating factors (CSF's) from these tissues by assay of stepped concentrations of eluate showed that CSF's from most tissues were similar in chromatographic behavior, but all differed significantly from those of serum in being both more disperse and more firmly bound to calcium phosphate gel. Male submaxillary salivary gland gave the richest yield of CSF. CSF from this source displayed a greater dispersity on and affinity to calcium phosphate, a lower electrophoretic mobility and a smaller average sedimentation coefficient than that from any other source investigated. Colony morphology appeared to be identical for all tissue sources investigated.     

A low molecular weight factor in lung-conditioned medium stimulating granulocyte and monocyte colony formation in vitro

Medium conditioned by excised whole lungs from endotoxin-injected C₅₇BL mice was highly active in stimulating hemopoietic colony formation, particularly of granulocytic type, in agar cultures of mouse bone marrow cells. The colony stimulating factor (CSF) in this material had an α₁-α₂ electrophoretic mobility, was eluted from calcium phosphate gel by 0.04 M phosphate buffer and had an unusually low apparent S₂₀W of 1.9. Sequestered polymor-phonuclear neutrophils were excluded as a major source of this CSF. The high specific activity and ease of preparation of lung conditioned medium make it valuable both for the large scale production of CSF and as a source of an unusual type of CSF.     

Purification and properties of colony-stimulating factor from mouse lung-conditioned medium.

Colony-stimulating factor, which specifically stimulates mouse bone marrow cells to proliferate in vitro and generate colonies of granulocytes, or macrophages, or both, was purified 3500-fold from mouse lung-conditioned medium. Analysis by discontinuous polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate indicated that there was a single protein component. All of the colony-stimulating activity was coincident with the protein band. The molecular weight of colony-stimulating factor estimated by gel filtration was approximately 29,000 and by electrophoresis approximately 23,000. The specific activity of purified colony-stimulating factor from mouse lung-conditioned medium bound to concanavalin A-Sapharose, indicating that it is a glycoprotein. The small percentage of colony-stimulating factor in mouse lung-conditioned medium which did not bind to concanavalin A-Sepharose appeared to represent molecules which lacked the carbohydrate moieties required for binding to this lectin. It was necessary to include low concentrations (less than 0.01%, v/v) of polymers such as gelatin and polyethylene glycol, or nonionic detergents such as Triton X-100, in all of the buffers used throughout the purification scheme, otherwise colony-stimulating factor was lost from solution. At high concentrations (greater than 20 mug/ml) the factor stimulated the formation of granulocytic, macrophage, and mixed colonies from C57BL mouse bone marrow cells. As the concentration of purified colony-stimulating factor was decreased, the frequency of colonies containing granulocytes also decreased. At low concentrations of colony-stimulating factor (less than 70 pg/ml) only macrophage colonies were stimulated.     

Capacitation and the acrosome reaction in squirrel monkey (Saimiri sciureus) spermatozoa evaluated by the chlortetracycline fluorescence assay

Capacitation and the acrosome reaction in squirrel monkey seminal spermatozoa diluted in Tyrode's medium (TALP) and TC-199 were monitored by a chlortetracycline (CTC) fluorescence assay. Four CTC patterns, similar to those found in human sperm, were readily characterized by fluorescent staining on the heads of the spermatozoa. The appearance of the capacitated (CP) pattern was dependent on the concentration of the bovine serum albumin. Acrosomal loss was observed in a maximum of 15% of the sperm in the populations studied here. Calcium ionophore A23187 (5 μM to 20μM) induced acrosomal loss in 60–70% of capacitated spermatozoa. However in freshly ejaculated sperm incubated under capacitating conditions or in spermatozoa incubated in Ca^{+ +}-free medium, A23187 failed to induce acrosomal loss. Furthermore, spermatozoa incubated in the presence of seminal plasma or spermatozoa obtained following a 1-hour “swim-up” procedure showed an identical timecourse of appearance of the CP pattern, indicating the lack of effect of seminal plasma on capacitation in the squirrel monkey.     

Engineering superactive granulocyte macrophage colony‐stimulating factor transferrin fusion proteins as orally‐delivered candidate agents for treating neurodegenerative disease

Intravenously injected granulocyte macrophage colony‐stimulating factor (GM‐CSF) has shown efficacy in Alzheimer's Disease (AD) and Parkinson's Disease (PD) animal studies and is undergoing clinical evaluation. The likely need for dosing of GM‐CSF to patients over months or years motivates pursuit of avenues for delivering GM‐CSF to circulation via oral administration. Flow cytometric screening of 37 yeast‐displayed GM‐CSF saturation mutant libraries revealed residues P12, H15, R23, R24, and K72 as key determinants of GM‐CSF's CD116 and CD131 GM‐CSF receptor (GM‐CSFR) subunit binding affinity. Screening combinatorial GM‐CSF libraries mutated at positions P12, H15, and R23 yielded variants with increased affinities toward both CD116 and CD131. Genetic fusion of GM‐CSF to human transferrin (Trf), a strategy that enables oral delivery of other biopharmaceuticals in animals, yielded bioactive wild type and variant cytokines upon secretion from cultured Human Embryonic Kidney cells. Surface plasmon resonance (SPR) measurements showed that all evaluated variants possess decreases in CD116 and CD131 binding K_D values of up to 2.5‐fold relative to wild type. Improved affinity led to increased in vitro bioactivity; the most bioactive variant, P12D/H15L/R23L, had a leukocyte proliferation assay EC₅₀ value 3.5‐fold lower than the wild type GM‐CSF/Trf fusion. These outcomes are important first steps toward our goal of developing GM‐CSF/Trf fusions as orally available AD and PD therapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:668–677, 2015     

Comparison of strains and culture media used for mouse in vitro fertilization

Success rates of superovulation in response to gonadotropic hormone treatment and in vitro fertilization (ie, mitotic cleavage following insemination) of mouse eggs from outbred CD-1, hybrid CB6F_l, or hybrid B6CBAF₁, mice were compared using either a mouse inseminationmedium, modified Krebs-Ringer-bicarbonate (m-KRB), or a human insemination medium, Ham's F10 nutrient mixture. Inseminations were performed in either organ culture dishes or screw-top, flat-side tissue culture tubes. Mean superovulation rates (± SD) were 24.2 (5.1) for CD-1, 33.0 (5.8) for CB6F₁, and 16.3 (6.6) for B6CBAF₁ mice. For in vitro cleavage the best combination of mouse strain, insemination medium, and culture container was achieved using CB6F₁, mice, m-KRB medium, and culture tubes. However, Ham's medium used with either hybrid mouse strain was shown to be employable for fertilization of mouse eggs in vitro as a quality control assay and/or experimental model system for testing the human in vitro fertilization procedure.     

Isolation and characterization of wheat ribulose-1,5-diphosphate carboxylase

Wheat ribulose-1,5-diphosphate carboxylase purified to homogeneity had a MW of 540 000, sedimentation coefficient (S_{20, W}) of 18.5 S, apparent diffusion constant (D_app) of 3.07 × 10^?7 cm²/sec, Stoke's radius 5.44 nm, and fractional ratio of 1.17. Electron microscopy revealed particles of 10–12 nm diameter. The enzyme was dissociated by sodium dodecyl sulphate into two subunits of MW 53 000 (S_{20, W} = 3.0 S) and 13 500 (S_{20, W} = 1.7 S). The total amino acid residues in the large and small subunits were 481 and 117, respectively. Tryptic peptide maps of the two subunits confirmed the estimated numbers of Arg and Lys residues. Although the amino acid pattern of the large subunit closely resembled that from barley, rather than that for spinach, beet or tobacco, the pattern of the small subunit was markedly different from those of all the other species.     

Studies on the bacteriophage MS2. XXIV. Hydrodynamic properties of the native and acid MS2 RNA structures

The molecular weight of native 26.6S MS2 RNA, of acidic 53.1S MS2 RNA and of formaldehyde-treated 36.1S MS2 RNA was determined by light-scattering studies. The three forms examined correspond to monomers. Also νv values were determined, and in conjunction with our previous estimates for S_20,W and [η], led again to the conclusion that formaldehyde-cross-linked 37S MS2 RNA is a monomer. On the basis of the experimentally observed R_G, [η], S_20,W, and νv values, and taking a prolate ellipsoid as a model, the hydrodynamic volume, the solvation water and the axial ratio could be deduced for the three conformations. In the “native” 26S form the RNA occupies only 8.2% of the hydrodynamic volume. The formation of the 36S and the 53S structures correspond largely to a loss of solvation water.     

Lost in translation: The search for an in vitro screen for spermatogenic toxicity

The last two decades have seen an increasing search for in vitro models that can replace the use of animals for safety testing. We adapted the methods from a recent nonquantitative report of spermatogenesis occurring in ex vivo mouse testis explants and tried to develop them into a screening assay. The model consisted of small pieces of neonatal mouse testis (testis “chunks”), explanted and placed on pillars of agarose or chamber inserts, and cultured at the air–liquid interface. A peripheral torus‐shaped zone in these explants would often contain tubules showing spermatogenesis, while the middle of each chunk was often necrotic, depending on the thickness of the tissue. The endpoint was histology: what proportion of tubules in the “permissive torus” actually contained healthy pachytene spermatocytes or spermatids? Extensive statistical modeling revealed that a useful predictive model required more than 60% of these tubules to show spermatogenesis. Separately, the logistics of running this as a predictive assay require that the controls consistently produce ≥ 60% tubules with pachytenes and round spermatids, and achieving this level of spermatogenesis reliably and consistently every week proved ultimately not possible. Extensive trials with various media additions and amendments proved incapable of maintaining the frequency of spermatogenic tubules at consistently ≥ 60%. Congruent with Schooler's “decline effect”; generally, the more often we ran these cultures, the worse the performance became. We hope that future efforts in this area may use our experience as a starting point on the way to a fully productive in vitro model of spermatogenesis.     

In vitro differentiation of tooth buds from embryos and adult lizards (L. gravenhorsti): An ultrastructural comparison

It is well established that the capacity for teeth to differentiate “in vitro” depends upon: (a) the age of the embryonic rudiments at the time of excision and (b) the number of cells within each tissue type which are capable of differentiating into organ culture. This paper studies ultrastructural aspects of tooth buds grown in vitro from lizard embryos and compares these characteristics with those observed in dental germs grown in situ in older lizard embryos. Moreover, we report the self-differentiation in vitro dental tissues from adult lizard and compare this phenomenon with the main features of a morphogenetic field. Our results suggest that approximately in the first third of gestation in L. gravenhorsti the dental buds has already acquired the capacity for self-differentiation in vitro. The ultrastuctural observations show that there are no significant differences between odontoblasts and ameloblasts in situ and in vitro. The tooth from “adult lizards,” isolated by combined microsurgical and enzymatic procedure and cultured in semisolid-liquid medium were also able to differentiate teeth. This phenomenon implies that self-differentiation is not rigidly determined, and that in these animals the tooth tissues represents a continuous morphogenetic field throughout the animal's life. This property is intrinsic, resides in the isolated tooth tissues, and is relatively independent of external factors. In addition, these studies indicate that the chick chorio–allantoic membrane and the semisolid-liquid culture medium supply the majority of the factors required for development of these tissues.     

Differentiation of human umbilical cord Wharton's jelly‐derived mesenchymal stem cells into germ‐like cells in vitro

Recent studies have demonstrated that mesenchymal stem cells could differentiate into germ cells under appropriate conditions. We sought to determine whether human umbilical cord Wharton's jelly‐derived mesenchymal stem cells (HUMSCs) could form germ cells in vitro. HUMSCs were induced to differentiate into germ cells in all‐trans retinoic acid, testosterone and testicular‐cell‐conditioned medium prepared from newborn male mouse testes. HUMSCs formed “tadpole‐like” cells after induction with different reagents and showed both mRNA and protein expression of germ‐cell‐specific markers Oct4 (POUF5), Ckit, CD49_f (α6), Stella (DDPA3), and Vasa (DDX4). Our results may provide a new route for reproductive therapy involving HUMSCs and a novel in vitro model to investigate the molecular mechanisms that regulate the development of the mammalian germ lineage. J. Cell. Biochem. 109: 747–754, 2010. © 2010 Wiley‐Liss, Inc.     

Calcium transport and cellular distribution in quiescent and serum-stimulated primary cultures of bone cells and skin fibroblasts

Primary cultures of bone cells and skin fibroblasts were examined for their Ca⁺⁺ content, intracellular distribution and Ca⁺⁺ fluxes. Kinetic analysis of ⁴⁵Ca⁺⁺ efflux curves indicated the presence of three exchangeable Ca⁺⁺ compartments which turned over at different rates: a “very fast turnover” (S₁), a “fast turnover” (S₂), and a “slow turnover” Ca⁺⁺ pool (S₃). S₁ was taken to represent extracellular membrane-bound Ca⁺⁺, S₂ represented cytosolic Ca⁺⁺, and S₃ was taken to represent Ca⁺⁺ sequestered in some intracellular organelles, probably the mitochondria. Bone cells contained about twice the amount of Ca⁺⁺ as compared with cultured fibroblasts. Most of this extra Ca⁺⁺ was localized in the “slow turnover” intracellular Ca⁺⁺ pool (S₃). Serum activation caused the following changes in the amount, distribution, and fluxes of Ca⁺⁺: (1) In both types of cells serum caused an increase in the amount of Ca⁺⁺ in the “very fast turnover” Ca⁺⁺ pool, and an increase in the rate constant of ⁴⁵Ca⁺⁺ efflux from this pool, indicating a decrease in the strength of Ca⁺⁺ binding to ligands on cell membranes. (2) In fibroblasts, serum activation also caused a marked decrease in the content of Ca⁺⁺ in the “slow turnover” Ca⁺⁺ pool (S₃), an increase in the rates of Ca⁺⁺ efflux from the cells to the medium, and from S₃ to S₂, as well as a decrease in the rate of influx into S₃. (3) In bone cells the amount of Ca⁺⁺ in S₃ remained high in “serum activated” cells, the rate of efflux from S₃ to S₂ increased, and the rate of influx into S₃ also increased. The rate of efflux from the cells to the medium did not change. The results suggest specific properties of bone cells with regard to cell Ca⁺⁺ presumably connected with their differentiation. Following serum activation we investigated the time course of changes in the amount of exchangeable Ca⁺⁺ in bone cells and fibroblasts, in parallel with measurements of ³H-thymidine incorporation and cell numbers. Serum activation caused a rapid decrease in the content of cell Ca⁺⁺ which was followed by a biphasic increase lasting until cell division.     

Triamcinolone acetonide induces transient changes in accumulation and fluxes of calcium in cultured bone cells

The effects of triamcinolone acetonide on the pattern of ⁴⁵calcium efflux were investigated in cultured bone cells. Efflux was measured after equilibrating the cells with ⁴⁵calcium for 24 hours and a short (3 hours) or a long (24 hours) preincubation with the hormone. The results were analyzed by fitting them to a model of three exponential terms, using a computer program based on the non-linear least square method. As reported previously the results indicated the presence of three exchangeable calcium pools which differ in their rates of calcium exchange. A short preincubation with the hormone (3 hours) caused the following changes: (1) A marked increase in the amount of exchangeable calcium in the “slow turnover” calcium pool (S₃) which is probably in the mitochondria. (2) A lesser but significant increase in the amount of calcium in the “fast turnover” calcium pool (S₂) which is probably the calcium in the cytosol. (3) An increase in the rates of calcium exchange between S₂ and the medium (?₂₀) and between S₃ and S₂ (?₃₂). All these changes were transient. After 24 hours of preincubation with triamcinolone acetonide the amounts of calcium in S₂ and S₃ decreased below the control levels and the fluxes were not significantly different from those of the controls.     

A granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by carrageenin-induced inflammatory cells of mice

Seven days after subcutaneous injection of 2% carrageenin solution in mice, the induced inflammatory tissue was isolated and treated with 0.1% trypsin. When the dispersed cells were incubated in a nutrient medium containing 5--20% calf serum, the cells grew adhering to the culture-dish surface and colony-stimulating factor (CSF) was accumulated in the medium gradually. Addition of lipopolysaccharide (Escherichia coli endotoxin) in the cell culture did not enhance the CSF production. It was shown by isoelectrofocusing that the majority of the produced CSF was an acidic molecule (pI = 3.8), while the treatment of this CSF with neuraminidase yielded a less acidic CSF species (pI = 5.1). Upon gel-filtration chromatography in the presence of 6 M guanidine HCl, the CSF exhibited an apparent molecular weight of 42,000. On the other hand, polyacrylamide gel-electrophoresis in the presence of 0.1% sodium dodecyl sulfate gave a molecular weight estimate of 65,000. Microscopic examination of the bone marrow cell culture showed that about one-third of the colonies were granulocytic. Addition of prostaglandin E₁(PGE₁) in the bone marrow cell culture significantly inhibited the action of the CSF, while prostaglandin D₂ was less inhibitory than PGE₁. The result suggests that the cells isolated from the inflammatory tissue are capable of producing CSF, which may have a role for proliferation and maturation of the mononuclear phagocytes at the site of inflammation.     

Total Mercury Concentrations in Lake and Streams Sediments Related to Wet-Area Coverage and Geogenic Sources within Upslope Basins

Total mercury concentrations (THg) in lake and stream sediments generally decrease with wet-area coverage (A_W) per upslope basin area (A_B). This was determined by delineating the wet-area component of 12,653 basins above as many sediment-sampling locations of the Geological Survey of Canada. These locations represent four climate regions (maritime, boreal, arctic, alpine) comprising six stream and six lake study areas. The dependence of sediment THg on A_W/A_B was examined by dividing the 0 < A_W/A_B < 1 range into 40 equal segments, and obtaining the mean sediment THg value for each segment. The results were evaluated by way of regression analysis using the following equation: mean sediment THg = a (1 ? A_W/A_B)^b + c A_W/A_B, with a, b and c as area-specific coefficients. The “a” and “c” coefficients could – in part – be inferred from bedrock type, annual atmospheric Hg deposition, and mean monthly air temperatures, and mean annual precipitation. Both “a” and “c” increased with increasing atmospheric Hg deposition for lake sediments. For stream sediments, only “a” did so. The geogenic influence on the THg variations per study area was addressed through multiple regression analyses, using sediment concentrations of other heavy elements and organic matter as independent variables.     

Purification and characterization of a Mr approximately 66,000 lung-derived (paracrine) growth factor that preferentially stimulates the in vitro proliferation of lung-metastasizing tumor cells

In medium containing low concentrations of serum, rat 13762NF mammary adenocarcinoma cell lines and clones (MTPa and MTC; isolated from the locally growing tumor) of low metastatic potential to lung did not exhibit a growth response to lung-conditioned medium, whereas a highly metastatic cell clone isolated from a spontaneous lung metastasis (MTLn3) did. The major growth-promoting factor for MTLn3 cells from porcine and rat lung-conditioned media was isolated by using a five-step procedure (anion exchange chromatography, Affi-gel blue affinity chromatography, chromatofocusing, size exclusion chromatography, and preparative native gel electrophoresis). The lung-derived factor that stimulated the growth of highly metastatic MTLn3 cells was a glycoprotein of Mr approximately 66,000 (non-reduced) or Mr approximately 72,000 (reduced) and possessed a pI of 6.9-7.0. It preferentially promoted the growth of lung-metastasizing tumor lines over their poorly lung-metastasizing counterparts in three tumor systems: rat 13762NF mammary adenocarcinoma, murine B16 melanoma, and murine RAW117 large-cell lymphoma. The factor's growth-stimulatory affect was inactivated by reduction or exposure to high temperature (95 degrees C). Although the growth factor appears to be glycosylated, its molecular weight was not altered by treatment with the protein-deglycosylating agent, trifluoromethane sulfonic acid. Cleavage of the protein by cyanogen bromide resulted in the formation of five fragments. Malignant cell response to this lung-derived paracrine growth factor may be important in the successful formation of lung metastases.     

Calcium fluxes and intracellular distribution in clonal osteosarcoma cell lines

Calcium efflux patterns were investigated in two clonal osteosarcoma cell lines, ROS $\text{17}\text{2}$ and ROS $\text{2}\text{3}$. Efflux was measured after equilibrating the cells with ⁴⁵calcium in medium containing 1% or 10% serum. The results of ⁴⁵calcium efflux were analyzed by fitting them to a model of three exponential terms, using a computer program based on the non-linear least square method. The results indicated the presence of three exchangeable calcium pools which differ in their rate constants of calcium efflux: a “very fast turnover” (S₁) a “fast turnover” (S₂) and a “slow turnover” (S₃). Washing the labelled cells with sucrose solutions, at pH 7.8 and pH 4, removed most of the calcium localized in S₁, indicating that this calcium is membrane bound. The parameters of calcium efflux in ROS cells were found to be different from those measured previously in cultured bone cells: (1) There was no difference between efflux patterns in cells incubated in medium containing 1% and 10% serum; (2) Rates of calcium fluxes were much lower in ROS cells than those in bone cells; and (3) The amount of calcium in S₃ was very small.     

The effect of mouse lung granulocyte-macrophage colony-stimulating factor and other colony-stimulating activities on the proliferation and differentiation of murine bone marrow cells in long-term cultures

The roles of colony-stimulating factors in long-term bone marrow cultures were studied and compared. After single additions of high concentrations of unpurified colony-stimulating activities to the cultures, rapid deterioration of the cultures was observed. This appears to result from contaminants in the stimulatory preparations. Cultures to which one purified colony-stimulating factor (CSF) from endotoxin mouse lung-conditioned medium was added did not run down ten weeks after addition and were found to be the same as the controls. The deterioration of the cultures to which unpurified stimulators were added could not be accounted for by accelerated granulopoiesis leading to subsequent exhaustion of the cultures. The inability of purified CSF to affect the cellularity of the suspension cells did not result from instability or masking of the activity in the cultures, nor did CSF preferentially stimulate the cells within the adherent layer. The suspension cells responded to purified CSF after separation from the adherent cells. The data suggest that if CSFs are marrow stimulators, their effects in turn may be stringently regulated within the marrow.     

Induction of colony-stimulating factors by Plasmodium cynomolgi components

Plasmodium cynomolgi total parasite antigens soluble in culture medium (P.c.SA), when injected in monkeys (Macaca mulatta) intravenously, induced the synthesis and secretion of serum colony-stimulating factors (CSFs). In vitro cultured monkey splenic macrophages and blood monocytes, following incubation with P.c.SA, also elaborated CSFs: the splenic macrophages responded more. Peak CSFs levels, both in vivo and in vitro, were attained after 8 hours of P.c.SA stimulation, and thereafter declined to baseline values within 48 hours. CSFs, both in serum and in conditioned medium, induced the formation of macrophage, granulocyte and granulocyte-macrophage colonies in vitro, in the same proportion, indicating that committed progenitor cells responded to CSF from both sources in a similar way. Polymyxin B treatment had no effect on P.c.SA stimulated CSF elaboration by macrophages, suggesting an LPS-independent mechanism of CSF induction. CSF synthesis appeared to be de novo, as cycloheximide treatment of macrophages completely inhibited CSF production. These observations indicate that P. cynomolgi components can induce CSF synthesis.