Establishment of a swine monocyte cell line

A swine monocyte cell line was established from peripheral blood sample collected from a healthy adult male pig. The cloned cells grow actively in forming monolayers in both glass and plastic cell culture flasks with the growth medium reported previously (Kadoi, 2000) at 36.5 degrees C incubation. The plating efficiency is more than 95%. Densely grown cells in flasks show an epithelioid morphology. The fundamental properties of the cells were examined for cytological definition as monocytes. A positive property detected was guinea pig complement receptor, porcine IgG receptor, non-specific esterase, and acid phosphatase. A significant phagocytic activity proved by the inoculation of Saccharomyces cerevisiae is also one of the characteristics observed in the LPS-activated cells.     

Establishment of a canine monocyte cell line

A canine monocyte cell line was established from the peripheral blood sample collected from a healthy young male Beagle dog. The cloned cells grew easily and were serially passaged in vitro in the medium, a slight modification further made on Dulbecco's modified Eagle medium, supplemented with 10% fetal bovine serum. The morphology of single cell was shown in triangular or round form, however, it became epithelioid in a densely grown monolayer. Non-specific esterase was detected in all cells by a cytochemical examination. The cells reacted rapidly to the addition of a small amount of LPS and differentiated to the cells of morphologically typical macrophages. Both complement receptor (CD35) and Fc gamma receptor (CD64) were demonstrated on the cell membrane.     

Poly-L-histidine inhibits prion propagation in a prion-infected cell line

ABSTRACT

Transmissible spongiform encephalopathies (TSEs) are a group of lethal neurodegenerative diseases involving the structural conversion of cellular prion protein (PrP^C) into the pathogenic isoform (PrP^Sc) for which no effective treatment is currently available. Previous studies have implicated that a polymeric molecule with a repeating unit, such as pentosane polysulfate and polyamidoamide dendrimers, exhibits a potent anti-prion activity, suggesting that poly-(amino acid)s could be a candidate molecule for inhibiting prion propagation. Here, by screening a series of poly-(amino acid)s in a prion-infected neuroblastoma cell line (GT^FK), we identified poly-L-His as a novel anti-prion compound with an IC₅₀ value of 1.8 µg/mL (0.18 µM). This potent anti-prion activity was specific to a high-molecular-weight poly-L-His and absent in monomeric histidine or low-molecular-weight poly-L-His. Solution NMR data indicated that poly-L-His directly binds to the loop region connecting Helix 2 and Helix 3 of PrP^C and sterically blocks the structural conversion toward PrP^Sc. Poly-L-His, however, did not inhibit prion propagation in a prion-infected mouse when administered intraperitoneally, suggesting that the penetration of blood-brain barrier and/or the chemical stability of this polypeptide must be addressed before its application in vivo. Taken together, this study revealed the potential use of poly-L-His as a novel treatment against TSEs. (203 words)     

The propagation of a porcine epidemic diarrhea virus in swine cell lines

A strain of porcine epidemic diarrhea virus (PEDV), P-5V, utilized as a live virus vaccine in Japan was infected to a swine cell lines, KSEK6 and IB-RS-2 cells. Clear CPE, characterized by cellular destruction, started to appear in the infected cells on 2-3 days post infection (DPI) and affected cells was completely degenerated on 4 DPI. The virus was serially passaged in the cells even without addition of trypsin. Small but clear plaques were formed under an agar overlay medium on the cells. The infective titer in the order of 10(7.00-7.50) TCID50 per ml was obtained at usual incubation temperature.     

Cytostatic effect of interferon-gamma and monocyte against a human carcinoma cell line

Human interferon gamma (IFN-gamma) and normal peripheral blood monocytes had appreciable anticellular activities against KB cells such that in both cases, the rate of DNA synthesis, the number and percent viability of the cells were greatly reduced by 70-85% in about 3 days post-treatment. However, neither IFN-gamma nor monocyte alone was able to totally eliminate the tumor cells. Our results suggest that IFN-gamma and monocyte could effectively prevent the growth of residual tumor cells that would have escaped destruction when only one effector element was present.     

Activation of human monocyte cell line U937 via cell surface calreticulin

U937 cells were found to be activated by an antibacterial peptide, KLKLLLLLKLK-NH2 (L5), to generate superoxide anion (O2-)-like peripheral neutrophils. However, the state of cell surface calreticulin, a possible receptor for L5, was suggested to differ between neutrophils and U937 cells. Unlike the former, the latter ones were activated by anti-C-domain peptide antibody of calreticulin even in the absence of L5 and generated O2- in a GTP-binding protein (G-protein)-dependent manner.     

Scrapie strains maintain biological phenotypes on propagation in a cell line in culture.

Bovine spongiform encephalopathy (BSE) and its human equivalent, variant Creutzfeldt-Jakob disease (vCJD), are caused by the same strain of infectious agent, which is similar to, but distinct from, >20 strains of their sheep scrapie homologue. A better understanding of the molecular strain determinants could be obtained from cells in monoculture than from whole animal studies where different cell targeting is commonly a strain-related feature. Although a few cell types can be infected with different strains, the phenotypes of the emergent strains have not been studied. We have cured the scrapie-infected, clonal SMB cell line with pentosan sulfate, stably re-infected it with a different strain of scrapie and shown that biological properties and prion protein profiles characteristic of each original strain are propagated faithfully in this single non-neuronal cell type. These findings attest to the fact that scrapie strain determinants are stable and host-independent in isolated cells.     

Serial propagation of Pneumocystis carinii in cell line cultures.

Pneumocystis carinii was propagated on three cell lines routinely cultured in many laboratories; the method is practical and convenient. Organisms produced were found to be reactive to Pneumocystis antisera. Studies of antigenic relationships, life cycles, and diagnostic methods will be made easier by these cultures.     

Serial propagation of Pneumocystis carinii in cell line cultures.

Pneumocystis carinii was propagated on three cell lines routinely cultured in many laboratories; the method is practical and convenient. Organisms produced were found to be reactive to Pneumocystis antisera. Studies of antigenic relationships, life cycles, and diagnostic methods will be made easier by these cultures.     

Establishment of a Vero cell line persistently infected with African swine fever virus.

A Vero cell line persistently infected with African swine fever virus was established by infecting the cells in the presence of 10 mM NH4Cl (Vero-P cell line). The virus derived from the Vero-P cultures infected Vero cells, and virus titers were comparable to those obtained in Vero cells acutely infected with African swine fever virus. The structural proteins of the virus from Vero-P cells were similar to those of the virus produced in lytic infections. Virus production was low when the Vero-P cells were growing logarithmically and increased considerably in confluent cultures when lysis appeared in a fraction of the cell population.     

Cytological study of a continuous swine embryonic kidney cell line with a persistent Toxoplasma infection]

Invasion of the PEK cell culture by toxoplasma did not cause any sharp disruption of the vital activity of the cells and failed to lead to their rapid degeneration. The observed changes in the morphology of the host cells were apparently chiefly the result of a purely mechanical pressure of the vacuoles on the nucleus and the cytoplasm, although the extreme forms of disturbances of the cell morphology were possibly associated with their greater injury. No complete "selfpurification" of the cells apparently occurred, since despite the marked reduction in the number of parasites a possibility of reinfection with toxoplasma of the culture at the later periods after the infection could not be excluded.     

Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine

In vitro studies on the pathogenesis in swine have been hampered by the lack of relevant porcine cell lines. Since many bacterial infections are swine-specific, studies on pathogenic mechanisms require appropriate cell lines of porcine origin. We have characterized the permanent porcine intestinal epithelial cell line, IPEC-J2, using a variety of methods in order to assess the usefulness of this cell line as an in vitro infection model. Electron microscopic analyses and histochemical staining revealed the cells to be enterocyte-like with microvilli, tight junctions and glycocalyx-bound mucin. The functional integrity of monolayers was determined by transepithelial electrical resistance (TEER) measurements. Both commensal bacteria and important bacterial pathogens were chosen for study based on their principally different infection mechanisms: obligate extracellular Escherichia coli, facultative intracellular Salmonella and obligate intracellular Chlamydia. We determined the colonization and proliferation of the bacteria on and within the host cells and monitored the host cell response. We verified the expression of mRNAs encoding the cytokines IL-1α, −6, −7, −8, −18, TNF-α and GM-CSF, but not TGF-β or MCP-1. IL-8 protein expression was enhanced by Salmonella invasion. We conclude that the IPEC-J2 cell line provides a relevant in vitro model system for porcine intestinal pathogen–host cell interactions.     

Induction of acetyl-LDL receptor activity by phorbol ester in human monocyte cell line THP-1

Human monocytic cell line THP-1 incubated with as little as 10 ng/ml of phorbol myristate acetate bound and metabolized 1-2 micrograms of Ac-LDL over a 5-h period. In the absence of phorbol treatment, no specific metabolism of Ac-LDL occurred. Optimal levels of receptor were reached after 72 h of exposure. Induction of receptor was dependent on protein and RNA synthesis and was partially reversed upon removal of the phorbol. Induction of receptor required activation of the protein kinase C pathway. Metabolism of Ac-LDL by THP-1 cells at 37 degrees C was saturated at 25 micrograms/ml. Binding at 4 degrees C was saturable with an average Kd of 8.0 x 10(-9) M. Cell population studies by fluorescent activated cell sorting indicated that approximately 87% of the THP-1 population was expressing scavenger receptor activity 96 h after phorbol treatment as compared to 99% for murine macrophage cell line P388D1. Uptake of Ac-LDL by THP-1 resulted in an 11-fold increase in the rate of cholesterol esterification which was saturable at 50 micrograms/ml. Incubation of cells for 48 h with 50 micrograms/ml of Ac-LDL resulted in a 60% increase in free cholesterol and a 10-fold increase in the cholesteryl ester content of the cells. Lipid accumulation in THP-1 cells after Ac-LDL uptake was readily visible by Oil Red-O staining. Solubilization of THP-1 cells, before and after phorbol treatment, followed by ligand blotting with Ac-LDL detected the presence of a 250-kDa protein only in cells treated with phorbol. The protein comigrated with the scavenger receptor derived from mouse macrophage cell line P388D1.     

Long-distance propagation of forces in a cell

A fundamental question in the field of mechanotransduction is how forces propagate inside a cell. Recent experiments have shown that a force of a physiological magnitude, applied via a focal adhesion, can propagate a long distance into the cell. This observation disagrees with existing models that regard the cell as a homogeneous body. We show that this "action at a distance" results from the inhomogeneity in the cell: a prestressed and stiff actin bundle guides the propagation of forces over long distances. Our models highlight the enormous ratios of the prestress and the modulus of the actin bundle to the modulus of the cytoskeleton network. For a normal cell, the models predict that forces propagate over characteristic lengths comparable to the size of the cell. The characteristic lengths can be altered, however, by treatments of the cell. We provide experimental evidence and discuss biological implications.     

Decrease in the average size of replicons in a Werner syndrome cell line by Simian Virus 40 infection

We have measured the distance between replicon initiation sites as well as the rate of DNA chain elongation in Simian Virus 40 (SV40)-infected and uninfected Werner syndrome (WS) and normal cell lines by DNA fiber-autoradiography. There was no difference in the rate of chain elongation among these cell lines. On the other hand, the replicon center-to-center distance was clearly longer in WS fibroblasts than that in normal fibroblasts. SV40 infection changed the center-to-center distance in WS cells toward that in normal cells.