Showing papers on "In vivo published in 1989"

In Vivo Stimulation of Bone Formation by Transforming Growth Factor-β

Abstract: The effect of transforming growth factor-β (TGFβ) on bone in vivo was examined. Twelve daily injections of 1 μg TGFβ directly onto the periostea of parietal bones of neonatal rats stimulated the formation of periosteal woven bone. The thickness of the treated parietal bones increased at least 2- fold in a dose-dependent manner. This TGFβ effect was localized at the sites of injection, and no change was observed in contralateral parietal bones and tibiae. The body weight in these growing rats was not affected by TGFβ1. TGFβ2 had effects similar to those of TGFβ1 on the parietal bones in vivo. These results reveal for the first time that TGFβ stimulates bone formation in vivo and indicate its anabolic role in local bone metabolism. (Endocrinology 124: 2991-2994, 1989)

Recombinant gene expression in vivo within endothelial cells of the arterial wall

Abstract: A technique for the transfer of endothelial cells and expression of recombinant genes in vivo could allow the introduction of proteins of therapeutic value in the management of cardiovascular diseases. Porcine endothelial cells expressing recombinant beta-galactosidase from a murine amphotropic retroviral vector were introduced with a catheter into denuded iliofemoral arteries of syngeneic animals. Arterial segments explanted 2 to 4 weeks later contained endothelial cells expressing beta-galactosidase, an indication that they were successfully implanted on the vessel wall.

In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection.

Abstract: Azithromycin, a novel azalide antibiotic, concentrated in human and mouse polymorphonuclear leukocytes (PMNs), murine peritoneal macrophages, and mouse and rat alveolar macrophages, attaining intracellular concentrations up to 226 times the external concentration in vitro. In murine peritoneal macrophages, azithromycin achieved concentration gradients (internal to external) up to 26 times higher than erythromycin. The cellular uptake of azithromycin was dependent on temperature, viability, and pH and was decreased by 2,4-dinitrophenol. Azithromycin did not decrease phagocyte-mediated bactericidal activity or affect PMN or macrophage oxidative burst activity (H2O2 release or Nitro Blue Tetrazolium reduction, respectively). Azithromycin remained in cells for several hours, even after extracellular drug was removed. However, its release was significantly enhanced by phagocytosis of Staphylococcus aureus (82 versus 23% by 1.5 h). In vivo, 0.05 micrograms of azithromycin was found in peritoneal fluids of mice 20 h after oral treatment with a dose of 50 mg/kg. Following caseinate-induced PMN infiltration, there was a sixfold increase in peritoneal cavity azithromycin to 0.32 micrograms, most of which was intracellular. Therefore, the uptake, transport, and later release of azithromycin by these cells demonstrate that phagocytes may deliver active drug to sites of infection.

A derivative of staurosporine (CGP 41 251) shows selectivity for protein kinase C inhibition and In vitro anti‐proliferative as well as In vivo anti‐tumor activity

Abstract: Analogues of staurosporine were synthesized and their ability to inhibit protein kinases was examined. Staurosporine is a potent but non-selective inhibitor of in vitro protein kinase C(PKC) activity (IC50 6.0 nM). The derivative CGP 41 251 had reduced PKC activity with an IC50 of 50 nM but showed a high degree of selectivity when assayed for inhibition of cyclic AMP-dependent protein kinase (IC50 2.4 microM), S6 kinase (IC50 5.0 microM) and tyrosine-kinase-specific activity of epidermal growth factor receptor (IC50 3.0 microM). Staurosporine and CGP 41 251 exerted growth inhibition in the human bladder carcinoma line T-24, human promyelocytic leukemia line HL-60 and bovine corneal endothelial cells at concentrations which correlated well with in vitro PKC inhibition. In addition, both compounds inhibited the release of H2O2 from human monocytes pre-treated with 12-O-tetradecanoyl-phorbol-13-acetate at non-toxic concentrations. In vivo anti-tumor activity was examined in T-24 human bladder carcinoma xenografts in athymic nude mice. Tumor growth inhibition tests revealed significant anti-tumor activity (2p less than 0.001) at 1/10 of the maximum tolerated doses for both compounds. By contrast, a closely related derivative of staurosporine (CGP 42 700) was inactive at concentrations of over 100 microM in all in vitro enzyme and anti-proliferative assays as well as in animal tumor models. Our data suggest an association between PKC inhibition and anti-proliferative and anti-tumor activity.

Production of interleukin-8 by human dermal fibroblasts and keratinocytes in response to interleukin-1 or tumour necrosis factor.

Abstract: Cultured normal human fibroblasts were stimulated to produce neutrophil-activating protein/interleukin-8 (IL-8) in response to IL-1 alpha (0.1-1000 U/ml) or tumour necrosis factor (TNF) alpha (0.1-1000 U/ml). Induction of mRNA for IL-8 in fibroblasts was rapid (within 30 min) and maximal responses were obtained with either 100 U/ml IL-1 alpha or 100 U/ml TNF alpha. Expression of mRNA for IL-8 was accompanied by the production of high levels of neutrophil chemotactic activity. IL-1 alpha (1000 U/ml), but not TNF alpha, induced mRNA for IL-8 in cultured normal human keratinocytes. The relevance of production of IL-8 by these cell types was evaluated further by comparing the local inflammatory effects of IL-1 alpha, TNF alpha and IL-8. Intradermal injection of either recombinant IL-8, IL-1 alpha or TNF alpha lead to a similar in vivo effect, i.e. dose-dependent accumulation of lymphocytes and polymorphonuclear leucocytes at sites of injection. The in vivo attraction of neutrophils and lymphocytes to the site of injection by TNF or IL-1 (which is not chemotactic for neutrophils or lymphocytes in vitro), may be partly mediated by locally produced IL-8. Thus, IL-8 may be a vital participant in the cascade of interacting cytokines that is induced by tissue injury and immunologically induced inflammation.

Function and regulation of the neutrophil MEL-14 antigen in vivo: comparison with LFA-1 and MAC-1.

Abstract: The CD11/18 (LFA-1, Mac-1) molecules participate in neutrophil adhesion to cultured endothelium in vitro and are critical for effective neutrophil localization into inflamed tissues in vivo. More recently, the MEL-14 Ag, which was first defined as a lymphocyte homing receptor, has also been implicated in inflammatory neutrophil extravasation. Here we compare the regulation and function of these adhesion molecules on neutrophils during the in vivo inflammatory response. The MEL-14 Ag is expressed at high levels on bone marrow and peripheral blood neutrophils, but is lost on neutrophils isolated from the thioglycollate-inflamed peritoneal cavity. In contrast, Mac-1 is up-regulated on inflammatory neutrophils and little change is seen in the level of LFA-1 expression. In vitro activation of bone marrow neutrophils with PMA or leukotriene B4 results in a dose dependent increase in Mac-1 and decrease in MEL-14 Ag expression within 1 h after treatment, thus reflecting what is found during inflammation in vivo. Neutrophils activated in vitro or in vivo (MEL-14Low, Mac-1Hi) do not home to inflammatory sites in vivo, correlating with the loss of the MEL-14 Ag and the increased Mac-1 expression. Anti-LFA-1, anti-Mac-1, or MEL-14 antibody given i.v. suppress neutrophil accumulation within the inflamed peritoneum (38%, 30%, and 37% of medium control, respectively) without affecting the levels of circulating neutrophils. However, when FITC-labeled cells are precoated with the mAb and injected i.v., only MEL-14 inhibits extravasation into the inflamed peritoneum (25% of medium control). Finally, in ex vivo adhesion assays of neutrophil binding to high endothelial venules in inflamed-lymph node frozen sections MEL-14 inhibits greater than 90%. anti-LFA-1 20 to 30% and anti-Mac-1 less than 10% of the binding of bone marrow neutrophils to inflamed-lymph node high endothelial venules. These results confirm that both the MEL-14 antigen and Mac-1/LFA-1 are important in neutrophil localization to inflamed sites in vivo, but suggest that their roles in endothelial cell interactions are distinct.

DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues

Abstract: 20(S)-Camptothecin, the 20(S)-camptothecin sodium salt, and 12 analogues with substituents on the A ring differ widely in their effectiveness in the treatment of murine L1210 lymphoblastic leukemia in vivo. The drugs were screened in the following systems: System 1, the cleavage of DNA in the presence of purified topoisomerase I; System 2, drug-induced trapping of topoisomerase I in a covalent complex with DNA; and System 3, the induction of protein-associated DNA breaks in drug-treated L1210 leukemia cells. 9-Amino-20(S), 10-amino-20(RS), and 10,11-methylenedioxy-20(RS), drugs effective against murine L1210 leukemia in vivo, stabilize topoisomerase I-DNA cleavable complexes in a purified system and in cultured L1210 cells. Other analogues, inactive against L1210 leukemia in vivo, were totally ineffective in topoisomerase I-directed screens. The rest of the analogues were intermediate in terms of their antitumor and topoisomerase I-directed activities. The study shows that the drug-induced accumulation of enzyme-DNA cleavable complexes is directly proportional to drug cytotoxicity and antitumor activity.

L-663,536 (MK-886) (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid), a novel, orally active leukotriene biosynthesis inhibitor

Abstract: L-663,536 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2, 2-dimethylpropanoic acid) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human polymorphonuclear leukocytes (PMN) (IC50, 2.5 nM). Similarly, L-663,536 inhibited A23187-induced LTB4 formation by rat peripheral blood and elicited PMN. At concentrations where inhibition of leukotriene biosynthesis occurred in human whole blood (1.1 microM), no effect was seen on cyclooxygenase or 12-lipoxygenase, an effect also observed in washed human platelets. The compound had no effect on rat or porcine 5-lipoxygenase indicating that L-663,536 is not a direct 5-lipoxygenase inhibitor. When administered in vivo L-663,536 was a potent inhibitor of antigen-induced dyspnea in inbred rats pretreated with methysergide (ED50, 0.036 mg/kg p.o.) and of Ascaris-induced bronchoconstriction in squirrel monkeys (1 mg/kg p.o.). The compound inhibited leukotriene biosynthesis in vivo in a rat pleurisy model (ED50, 0.2 mg/kg p.o.), an inflamed rat paw model (ED50, 0.8 mg/kg), a model of leukotriene excretion in rat bile following antigen provocation, and a model in the guinea-pig ear where leukotriene synthesis was induced by topical challenge with ionophore A23187 (ED50, 2.5 mg/kg p.o. and 0.6 micrograms topically). The results indicate that L-663,536 is a potent inhibitor of leukotriene biosynthesis both in vitro and in vivo indicating that the compound is suitable for studying the role of leukotrienes in a variety of pathological situations.

An L-arginine-dependent mechanism mediates Kupffer cell inhibition of hepatocyte protein synthesis in vitro.

Abstract: The hepatic failure associated with severe sepsis is characterized by specific, progressive, and often irreversible defects in hepatocellular metabolism (1). Although the etiologic microbe can often be identified, the direct causes and mechanisms of the hepatocellular dysfunction are poorly understood. We have hypothesized that Kupffer cells (KC), which interact with ambient septic stimuli, respond by providing signals to adjacent hepatocytes (HC) in sepsis . Furthermore, we have provided evidence (2, 3) that KC activated by LPS from Gram-negative bacteria can induce profound changes in the function of neighboring HC in coculture. In our model, coculture of either KC (2) or peritoneal macrophages (Mφ)(3) with HC normally promotes HC protein synthesis ([(3)H]leucine incorporation). The addition of LPS or killed Escherichia colt' to such cocultures induces a profound decrease in HC protein synthesis, as well as qualitative changes ([(35)S]methionine, SDS-gel electrophoresis) in protein synthesis without inducing HC death (2, 3) . In this report we show that the inhibition in protein synthesis is mediated via an L-arginine-dependent mechanism. The metabolism of L-arginine by activated Mφ to substances with cytostatic and even lethal effects on target cells is a relatively recent discovery. After the description by Stuehr and Marletta (4, 5) that LPS- triggered Mφ produced nitrite/nitrate (NO(2)(-)/NO(3)(-)), Hibbs et al. (6, 7) and Iyengar et al. (8) demonstrated that L-arginine was the substrate for the formation of both these nitrogen end products and citrulline. A role for the arginine-dependent mechanism in Mφ tumor cytotoxicity (6, 7) and microbiostatic activity (9) has been suggested. However, the in vivo functions of this novel Mφ mechanism have not yet been defined, but it is possible that there are both physiologic as well as pathologic roles. Our in vitro results raise the possibility that some metabolic responses to microbial invasion maybe partially mediated by the L-arginine-dependent mechanism. What other metabolic responses are affected and the possible pathologic consequences remain to be studied.

Mechanism of calcification: Role of collagen fibrils and collagen‐phosphoprotein complexes in vitro and in vivo

Abstract: Samples of decalcified chicken bone together with varying concentrations of phosphoproteins from bone or egg yolk (phosvitin) were used in vitro as heterogenous nucleators for the induction of Ca-P apatite crystals. The lag time between exposure of the collagen-phosphoprotein complexes and the time nucleation of crystals occurred decreased as the concentration of Ser(P) and Thr(P) increased. Enzymatic cleavage of the phosphate groups by wheat germ and phosphatase reversed this effort, indicating that the phosphate group per se principally facilitated the nucleation of Ca-P crystals by the phosphoprotein complex and collagen.

IL-6 modulates the synthesis of a specific set of acute phase plasma proteins in vivo.

Abstract: Human rIL-6, produced either in COS cells or Escherichia coli, similarly stimulates the production of acute phase plasma proteins in cultured human and rat hepatoma cells. This anabolic effect in hepatoma cells suggested a potential in vivo role of the cytokine in mediating the hepatic response to inflammation. Injection of IL-6 into adult male rats elicited a cytokine-specific change in the liver expression of acute phase proteins. As predicted from in vitro studies, glucocorticoids were needed to achieve a maximal IL-6 response in vivo. Optimal conditions were found to be two i.p. injections of 35 to 120 micrograms IL-6 and 65 micrograms dexamethasone per kg body weight administered at 12-h intervals. Within 24 h, the plasma concentrations for alpha 2-macroglobulin, fibrinogen, thiostatin, and hemopexin were increased to levels approximating those observed in acute phase animals. These results support the notion that direct interaction of IL-6 with the liver is an essential part in initiating the hepatic acute phase reaction.

Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese hamster ovary cells.

Abstract: Two forms of erythropoietin, EPO-bi and EPO-tetra, with different biological activities were isolated from the culture medium of a recombinant Chinese hamster ovary cell line, B8-300, into which the human erythropoietin gene had been introduced. EPO-bi, an unusual form, showed only one-seventh the in vivo activity and 3 times higher in vitro activity of the previously described recombinant human EPO (standard EPO). In contrast, EPO-tetra showed both in vivo and in vitro activities comparable to those of the standard EPO. EPO-bi, EPO-tetra, and the standard EPO had the same amino acid composition and immunoreactivity. However, structural analyses of their N-linked sugar chains revealed that EPO-bi contains the biantennary complex type as the major sugar chain, while EPO-tetra and the standard EPO contain the tetraantennary complex type as the major sugar chain. From examination of various preparations of recombinant human EPO, we found a positive correlation between the in vivo activity of EPO and the ratio of tetraantennary to biantennary oligosaccharides. These results suggest that higher branching of the N-linked sugar chains is essential for effective expression of in vivo biological activity of EPO.

A new in vitro assay for quantitating tumor cell invasion.

Abstract: The attachment to and penetration of basement membranes by tumor cells is required to complete the metastatic cascade which culminates in the establishment of secondary tumor foci. Therefore, basement membranes are critical barriers to the passage of disseminating tumor cells. We have developed a simple, in vitro model using matrigel-coated transwell chambers (Costar) for use in a tumor cell invasion assay. Two variants of the K1735 UV-induced murine melanoma cell line were assayed for their invasive capabilities and compared with their ability to colonize the lung in an experimental metastasis assay. The K1735-M2 cells, which are highly metastatic in vivo, invaded through basement membrane matrigel at a significantly higher rate than the low metastatic cells, K1735-16, in a 72-hour assay. As a negative control, normal murine fibroblasts were incapable of penetrating the barrier. Tumor cell invasion in vitro correlated with lung colonization in vivo. Therefore, this model may provide a valuable tool to study the mechanisms involved in the pathogenesis of tumor cell invasion during hematogenous dissemination.

Induction of ovalbumin-specific cytotoxic T cells by in vivo peptide immunization.

Abstract: CTL recognize peptide forms of processed, foreign antigens in association with class I molecules encoded by the MHC and are usually directed against endogenously synthesized "cellular antigens," such as those expressed by virus-infected cells. In vitro studies have shown that small exogenous peptides can directly associate with class I molecules on the cell surface and mimic the target complex derived by intracellular processing and presentation. We have recently generated OVA-specific, H-2Kb-restricted CTL by immunizing C57BL/6 mice with a syngeneic tumor line transfected with the OVA cDNA. The CTL recognize the OVA transfectant E.G7-OVA and the synthetic peptide OVA258-276, but fail to recognize the native protein. We reasoned that given the potential for direct peptide/class I association observed in vitro, OVA258-276 may induce CTL after in vivo priming. However, we found that this is not the case. OVA258-276 and peptides of increasing lengths up to OVA242-276 and OVA242-285, which are all able to form the target complex in vitro, are inefficient at priming E.G7-OVA-specific CTL responses after intravenous injection. This is also true for both native and denatured OVA. In contrast to these results the synthetic peptide OVA229-276 corresponding to a peptide in a partial tryptic digestion of OVA can efficiently prime C57BL/6 mice in vivo after intravenous injection. This peptide elicits CTL that appear identical to those derived from animals immunized with syngeneic cells producing OVA endogenously. These results are discussed in terms of separate class I and class II antigen presentation pathways and the ability of only certain, exogenous antigens to enter the cytoplasmic, class I pathway.

Relation between phosphate metabolites and oxygen consumption of heart in vivo.

Abstract: The relation between induced increases in cardiac work and phosphate metabolites was investigated in the canine heart in vivo to evaluate the role of ATP hydrolysis products, ADP and inorganic phos...

Recombinant retroviruses with amphotropic and ecotropic host ranges

Abstract: Packaging cell lines useful for the generation of helper-free recombinant retroviruses with amphotropic or ecotropic host ranges, methods of constructing such packaging cell lines and methods of using the recombinant retroviruses to introduce DNA of interest into eukaryotic cells, both in vitro and in vivo.

Bovine Brain Endothelial Cells Express Tight Junctions and Monoamine Oxidase Activity in Long‐Term Culture

Abstract: The passage of substances across the blood-brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies a number of characteristics of the in vivo system are lost. To provide an in vitro system for studies of brain capillary functions, we developed a method of isolating and producing a large number of bovine brain capillary endothelial cells. These cells, absolutely free of pericyte contamination, are subcultured, at the split ratio of 1:20 (20-fold increase of the cultured surface), with no apparent changes in cell morphology up to the fiftieth generation (10 passages). Retention of endothelial-specific characteristics (factor VIII-related antigen, angiotensin-converting enzyme, and nonthrombogenic surface) is shown for brain capillary-derived endothelial cells up to passage 10, even after frozen storage at passage 3. Furthermore, we showed that bovine brain capillary endothelial cells retain, up to the fiftieth generation, some of the characteristics of the blood-brain barrier: occurrence of tight junctions, paucity of pinocytotic vesicles, and monoamine oxidase activity.

In vivo inflammatory activity of neutrophil-activating factor, a novel chemotactic peptide derived from human monocytes.

Abstract: Neutrophil-activating factor (NAF), a 72-amino acid peptide produced by human monocytes, induced plasma leakage and neutrophil accumulation after intradermal injection in rabbits (10(-11) to 10(-9) mol/site). NAF was about three times more potent than fMet-Leu-Phe, but considerably less potent than endotoxin. The response to NAF was not inhibited by the endotoxin inhibitor polymyxin B or the protein synthesis inhibitor actinomycin D. Histology of NAF-induced lesions showed large numbers of neutrophils, but no monocytes, eosinophils, basophils, or lymphocytes were observed. Intravascular neutrophil accumulation, aggregate formation, and venular wall damage were also apparent. In vitro, NAF stimulated rabbit neutrophils as shown by the release of N-acetyl-beta-glucosaminidase. This study demonstrates that NAF elicits a rapid inflammatory response in vivo with massive neutrophil emigration, which is qualitatively similar to that observed with other chemotactic agonists.

Effects of in vivo administration of anti-CD3 monoclonal antibody on T cell function in mice. II. In vivo activation of T cells.

Abstract: Anti-CD3 mAb are known to be both immunosuppressive and mitogenic to T cells in vitro. However, only immunosuppression has been observed after in vivo administration of these mAb. The present study demonstrates that T cell activation does occur after in vivo administration of anti-CD3 mAb to mice, evidenced by increased IL-2R expression on T cells, CSF secretion, and extra-medullary hematopoiesis in the spleen. These effects required multivalent cross-linking of the mAb, since F(ab')2 fragments failed to induce them. However, the F(ab')2 fragments did induce modulation of CD3/TCR from the surface of T cells, demonstrating that TCR modulation is not sufficient to induce activation. In addition, interaction of the TCR with either intact or F(ab')2 fragments of the mAb led to increased expression of CD8 in vivo, suggesting that the F(ab')2 fragments of anti-CD3 mAb might be capable of inducing a T cell to undergo some, but not all, of the changes involved in reaching a fully activated state. Further study of the activating effects of anti-CD3 mAb might increase the understanding of the mechanisms of in vivo T cell activation and might also be exploited clinically to stimulate T cell function in immunocompromised states and to enhance hematopoiesis in myelodysplastic disorders.

Bone marrow-derived stromal cell line expressing osteoblastic phenotype in vitro and osteogenic capacity in vivo.

Abstract: Marrow stroma has been shown to have osteogenic potential. Here we report the characterization of a unique stromal cell line derived from mouse bone marrow (MBA-15), which expresses osteoblastic phenotype in vitro and forms bone in vivo. More than 70% of cells in culture were histochemically positive for alkaline phosphatase. The enzyme levels were enhanced threefold when cultures were treated with dexamethasone. Gel electrophoresis of [3H]-proline-labeled cultures showed that MBA-15 cells produced only type I collagen. These cells were responsive to PTH, as indicated by a 50-fold increase in intracellular cAMP. Prostaglandin E2, but not calcitonin, stimulated cAMP up to 70-fold. When cultures were grown to confluence and fed daily with ascorbic acid and beta-glycerophosphate, the cells formed a Von Kossa positive, thick extracellular matrix, shown to contain hydroxyapatite crystals. MBA-15 cells produced mineralized bone when implanted in diffusion chambers. These results indicate that the MBA-15 cell line possesses osteoblastic features in vitro and osteogenic capacity in vivo.