Development and Characterization of a Model Latent Herpes Simplex Virus Infection in Cultured Cells

Herpes simplex virus is the classic example of a virus which persists indefinitely in the host following the initial infection. Recurrent episodes of active virus growth characterize the infection in some individuals . In addition to the periodic flair-ups of active virus growth, the persistence of virus may be expressed in a more subtle way. It is possible that one or more types of human cancer result from the persistence of this virus in the host. The exact mechanism by which the virus is maintained in the host between outbreaks of active growth is not known. It is usually not possible to isolate the virus between the outbreaks and the virus is said to be latent during this period. Most of the available evidence indicates that the virus exists in a nonreplicating form when it is latent. This dissertation describes the establishment and characterization of a model latent herpes simplex virus infection in cell cultures . Cultures of rabbit kidney cells were infected with approximately one infectious unit of virus per 100 cells and incubated at 41C for 1-7 days. No evidence of virus growth was seen at 4 1C and infectious virus disappeared from the cells very rapidly at this temperature. Two-hundred infected cultures were transferred to 37C after incubation at 41C. In 43% of these cultures virus was recovered within 3 days after incubation at 3 7C. In 16% of these cultures no virus was recovered after incubation at 3 7C for up to 150 days. In the remaining 41% of the cultures, however, virus growth occurred but only after lag periods of variable length. The longest lag period was 45 days and the average lag period was 15.3 days. During the period when no evidence of virus growth was seen, treatment of the cells by freeze-thawing or by sonication failed to yield infectious virus. Once active virus growth occurred, virus was able to be isolated from the cultures and this virus was neutralized by commercial herpes simplex virus antiserum. Quantitative studies indicated that 0.25-1% of the inoculum virus was able to survive incubation at 41C for up to 6 days and replicate after transfer to 3 7C. Immunofluorescence studies indicated that virus-specific antigens were produced at 4 1C. Fluorescence microscopy of cells stained with acridine orange indicated that the block in virus replication occurred early in the infection cycle. Characteristic features of cells infected with virus at 3 7C were absent from the cells infected at 41C. Although virus failed to replicate in rabbit kidney cells at 41C, control cells preincubated at 41C were fully competent to replicate exogenous virus immediately after transfer to 3 7C . An attempt was made to establish the latent infection in cells directly at 3 7C by using a very small inoculum of virus . The latent infection could not be established in this way. When virus was added directly onto cells at 3 7C, active virus growth was always seen within 3 days or no growth occurred at all in cultures maintained for up to 30 days . A latent infection similar to that established in rabbit kidney cells was established in Wistar-38 cells. However, attempts to establish a similar infection in human kidney cells were unsuccessful. The basis for this cell-dependent difference was not investigated. Cultures were treated with certain hormones and chemical agents in attempts to modify the latent infection. Hormones that were used include hydrocortisone, 17 B-estradiol, progesterone, L-thyroxin and L- epinephrine. Treatment of cultures with progesterone at the time that the cultures were infected with virus and incubated at 41C resulted in a significant reduction in the number of cultures from which virus was recovered after transfer to 3 7C. The other hormones did not affect the latent infection in this way. Three chemical agents, 5-bromo-2-deoxyuridine, 5-iodo-2- deoxyuridine, and puromycin increased the reactivation rates of virus from cultures when the cultures were treated with the agent at the time of infection with virus and incubation at 41C. When cultures were treated with 5-bromo-2-deoxyuridine at the time of transfer from 41C to 3 7C (after the latent infection had already been established) the overall recovery rate was the same from treated cultures as from control cultures. However, the average lag period of the treated cultures was significantly increased. Finally, treatment of cultures with neutral red dye and exposure of these cultures to strong visible light had no effect on the overall recovery rate but did increase the average lag period of the treated cultures

Modulation of fibronectin synthesis and fibronectin binding during transformation and differentiation of mouse AKR fibroblasts

In previous studies it was shown that transformation of AKR fibroblasts with 3-methylcholanthrene was associated with a loss of surface fibronectin and that induction of differentiation of the transformed cells with N,N-dimethylformamide (DMF) was associated with reacquisition of surface fibronectin (Chakrabarty et al., J. Cell. Physiol. 133:415, 1987). It is shown in the present study that changes in surface fibronectin reflect altered fibronectin synthesis and altered fibronectin binding. Both the nontransformed cells (AKR-2B) and their transformed counterparts (AKR-MCA) bound 125 I-fibronectin in a receptor-like fashion, but the AKR-MCA cells had only 20% of the receptors found on the AKR-2B cells. Whole cell extracts prepared from the AKR-2B cells and separated by sodium dodecyl sufate-polyacrylamide gel electrophoresis under reducing conditions were examined for 125 I-fibronectin binding. Under these conditions, the majority of binding occurred to moieties with molecular weights of 180 kD, 150 kD, and 97 kD. Binding to similar moieties on the AKR-MCA cells was virtually absent but occurred rapidly after treatment with DMF. The appearance of these moieties paralleled the acquisition of 125 I-fibronectin binding activity by whole cells. Antibodies to the fibronectin receptor isolated from human placenta reacted with the DMF-sensitive moieties in immunoblot assays. Both the appearance of the fibronectin binding moieties and the acquisition of 125 I-fibronectin binding activity by whole cells occurred within 6 hr of DMF treatment and increased over the suosequent 4 day period. The time course of these events paralleled closely the time course for induction of fibronectin biosynthesis by DMF. These changes in fibronectin binding and fibronectin production were associated with alterations in cell-substrate adhesion. The AKR-2B cells rapidly attached and spread on bovine serum albumin-coated dishes and on fibronectin-coated dishes, whereas the AKR-MCA cells were less adhesive on both substrates. Capacity to attach and spread was regained concomitantly with the induction of fibronectin binding and fibronectin production. Adhesion on both substrates was partially inhibited by antibodies to the fibronectin receptor and by RGDS. These studies suggest that fibronectin production and fibronectin binding are coregulated in AKR fibroblasts and that they function together to bring about changes in cell-substrate adhesion.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49878/1/1041430307_ftp.pd

Mechanisms of Neutrophil‐Mediated Killing of Endothelial Cells

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141751/1/jlb0097.pd

Arachidonic Acid Metabolism In Murine Fibrosarcoma Cells With Differing In Vivo And In Vitro Characteristics

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144314/1/ijc1985363383.pd

Induction of calcium sensing receptor in human colon cancer cells by calcium, vitamin D and aquamin: Promotion of a more differentiated, less malignant and indolent phenotype

The calcium sensing receptor (CaSR) is a robust promoter of differentiation in colonic epithelial cells and functions as a tumor suppressor. Cancer cells that do not express CaSR (termed CaSR null) are highly malignant while acquisition of CaSR expression in these cells circumvents the malignant phenotype. We hypothesize that chemopreventive agents mediate their action through the induction of CaSR. Here, we compare the effectiveness of Ca2+, vitamin D, and Aquamin (a marine algae product containing Ca2+, magnesium and detectable levels of 72 additional minerals) on the induction of CaSR in the CBS and HCT116 human colon carcinoma cell lines and the corresponding CaSR null cells isolated from these lines. All three agonists induced CaSR mRNA and protein expression and inhibited cellular proliferation in the parental and CaSR null cells. Aquamin was found to be most potent in this regard. Induction of CaSR expression by these agonists resulted in demethylation of the CaSR gene promoter with a concurrent increase in CaSR promoter reporter activity. However, demethylation per se did not induce CaSR transcription. Induction of CaSR expression resulted in a down‐regulated expression of tumor inducers and up‐regulated expression of tumor suppressors. Again, Aquamin was found to be most potent in these biologic effects. This study provides a rationale for the use of a multi‐mineral approach in the chemoprevention of colon cancer and suggests that induction of CaSR may be a measure of the effectiveness of chemopreventive agents. © 2013 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111913/1/mc22123.pd

Interaction of viable group a streptococci and hydrogen peroxide in killing of vascular endothelial cells

Previous studies have shown that the streptococcal hemolysin, streptolysin S, is capable of interacting with hydrogen peroxide (H2O2) to injure vascular endothelial cells (Free Radic. Biol. Med. 7:369-376; 1989). To extend these observations, intact group A streptococci (strain 203S) were examined for ability to injure endothelial cells alone and for ability to injure the same cells in the presence of sublethal concentrations of H2O2 (generated from glucose oxidase). While neither control bacteria nor bacteria that had been pretreated with poly--histidine to render them cationic were cytotoxic to endothelial cells by themselves under the conditions of the experiment, endothelial cells were injured by combinations of streptococcal cells and sublytic amounts of H2O2. Taken together, these data suggest that the sequelae which often occur following primary infection with group A streptococci may be the result of a combined assault of host inflammatory cells and the invading bacteria on the vascular lining cells of the host.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30821/1/0000481.pd

Matrix Metalloproteinase-1 is the Major Collagenolytic Enzyme Responsible for Collagen Damage in UV-irradiated Human Skin ¶

Punch biopsies of human skin were obtained 1 day after irradiation with two minimal-erythema doses (MED) from either a UVB light source or a Solar Simulator and incubated in organ culture for 72 h. Organ culture fluids obtained at 24, 48 and 72 h were analyzed for collagenolytic activity and for reactivity with antibodies to matrix metalloproteinase-1 (MMP-1; interstitial collagenase) and MMP-13 (collagenase-3). High levels of collagenolytic activity were seen in organ culture fluid from skin exposed to either light source. MMP-1 was strongly induced in parallel, increasing from less than 100 ng/ml in organ culture fluid from control skin to approximately 1.1 mg/ml in culture fluid from UV-treated skin. Whereas most of the detectable MMP-1 in control culture fluid was represented by the latent form of the enzyme, approximately 50% of the enzyme was present as the active form in organ culture fluid of UV-exposed skin. In contrast, there was no detectable MMP-13 in control organ culture fluid and very little change after UV exposure (less than 100 ng/ml in both cases). Finally, neutralization studies with a blocking antibody to MMP-1 removed 95 ± 4% of the collagenolytic activity in the organ culture fluid from UV-treated skin. These findings strongly implicate MMP-1 rather than MMP-13 as the major collagenolytic enzyme responsible for collagen damage in photoaging.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73069/1/0031-8655_2003_0780043MMITMC2.0.CO2.pd

Cell growth on microcarriers: comparison of proliferation on and recovery from various substrates

Three commercially-important types of cell were grown on four different microcarrier substrates. The cells, which included normal human diploid fibroblasts (MRC-5), primary chick embryo cells and Madin-Darby bovine kidney cells (MDBK), were compared with regard to proliferation on the substrates and with regard to recovery of viable cells from the same substrates. The substrates used included glass-coated microcarriers (Biosil), collagen microcarriers (Ventregel), DEAE-dextran microcarriers (Cytodex I) and collagen-linked DEAE-dextran microcarriers (Cytodex III). The established cell line (MDBK) grew well on all of the substrates and a high percentage of viable cells could be harvested from each substrate. The MRC-5 cells also grew well on all four substrates but high recovery rates were achieved only with cells grown on the glass-coated microcarriers or collagen microcarriers. In contrast, the primary chick embryo cells grew well only on the glass microcarriers and the recovery rate of cells harvested from this substrate was high. In some industrial operations, the re-utilization of cells after removal from the substrate is necessary. In these situations the appropriate choice of microcarriers for the cultivation of the cells may be critical.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26400/1/0000487.pd

Out-of-equilibrium collective oscillation as phonon condensation in a model protein

In the first part of the present paper (theoretical), the activation of out-of-equilibrium collective oscillations of a macromolecule is described as a classical phonon condensation phenomenon. If a macromolecule is modeled as an open system, that is, it is subjected to an external energy supply and is in contact with a thermal bath to dissipate the excess energy, the internal nonlinear couplings among the normal modes make the system undergo a non-equilibrium phase transition when the energy input rate exceeds a threshold value. This transition takes place between a state where the energy is incoherently distributed among the normal modes, to a state where the input energy is channeled into the lowest frequency mode entailing a coherent oscillation of the entire molecule. The model put forward in the present work is derived as the classical counterpart of a quantum model proposed long time ago by H. Fr\"ohlich in the attempt to explain the huge speed of enzymatic reactions. In the second part of the present paper (experimental), we show that such a phenomenon is actually possible. Two different and complementary THz near-field spectroscopic techniques, a plasmonic rectenna, and a micro-wire near-field probe, have been used in two different labs to get rid of artefacts. By considering a aqueous solution of a model protein, the BSA (Bovine Serum Albumin), we found that this protein displays a remarkable absorption feature around 0.314 THz, when driven in a stationary out-of-thermal equilibrium state by means of optical pumping. The experimental outcomes are in very good qualitative agreement with the theory developed in the first part, and in excellent quantitative agreement with a theoretical result allowing to identify the observed spectral feature with a collective oscillation of the entire molecule.Comment: 49 pages, 10 figures; Physical Review X, (2018) in pres

Inhibitory Effect of Gamma Interferon on Cultured Human Keratinocyte Thrombospondin Production, Distribution, and Biologic Activities

Rapidly proliferating keratinocytes (KCs) maintained in low calcium, serum-free medium produce and utilize thrombospondin (TSP) as an attachment and spreading factor. To begin to understand the modulation of KC TSP metabolism, gamma interferon (IFN-γ), a product of activated T lymphocytes, was added to KC cultures. IFN-γ; was chosen because activated T cells appear at sites of cutaneous injury. Two additional cytokines including tumor necrosis factor (TNF) and IFN-β were also examined. IFN-γ (600 U/ml), but not TNF (500 U/ml) or IFN-β (103 U/ml), as single agents decreased KC TSP biosynthesis, secretion, and utilization as an attachment factor. IFN-γ alone did not detectably decrease TSP mRNA levels suggesting a post-transcriptional effect in KCs. However, the combination of IFN-γ (600 U/ml) and TNF (500 U/ml) inhibited TSP mRNA production. These results demonstrate the modulation of KC TSP metabolism and biologic activity