Formation Of Confluent Monolayer Research Articles

Effectiveness of cell therapy of acute radiation syndrome in mice with intravenous and intraperitoneal administration of a cellular product

The therapeutic effect of fibroblast-like cells obtained from the stromal vascular fraction of subcutaneous adipose tissue of mice and cultured for the treatment of bone marrow form of acute radiation syndrome was studied on a mouse experimental model. The cells were identified as multipotent mesenchymal stem (stromal) cells, owing to adhesion to plastic, confluent monolayer formation during cultivation, and the fact that osteogenic differentiation in vitro resulted in osteoblast maturation and calcium deposit formation, which indicated their multipotent nature. Irradiation of laboratory rodents was conducted using theX-ray therapy unit “RUM-17”. Stromal cells were obtained from subcutaneous adipose tissue of a mouse and grown in a culture of 3–4 passages and used as a cell product. Cell transplantation was performed 24 h after uniformX-ray irradiation of mice at a dose of 7.8 Gy. This is the first study to compare the therapeutic efficacy of allogeneic transplantation of multipotent mesenchymal stem cells with the different routes (intravenous and intraperitoneal) of cell suspension administration. A significant increase was found in the survival rate of mice during the 30-day follow-up period after lethal dose irradiation, which depended on the number of injected cells and delivery method of the biomedical cell product. Thus, with intravenous administration of 30 and 60 × 103multipotent mesenchymal stem cells, the 30-day survival rate of mice after irradiation at a dose of 7.8 Gy increased by 54.5% and 40%, respectively, compared with that of untreated animals (p= 0.03). An increase in the number of cells in the cell product to 120 × 103/mouse led to a decrease in therapy effectiveness. In intraperitoneal administration, the protection of animals from death was 57% after transplantation of 30 and 60 × 103cells (p= 0.039) and 50% after application of 120 × 103cells. On day 30 after irradiation, in the introduction of a cellular product in different schemes, 70%–80% of animals showed restoration of the values of the main indicators of the hematopoiesis system to initial levels. Thus, cell therapy using multipotent mesenchymal stem cells isolated from adipose tissue with intravenous and intraperitoneal delivery routes of the cellular product to the irradiated body protects mice from death after exposure toX-ray radiation in lethal doses, decreasing the severity of radiation damage to the hematopoietic system in mice, and provides prospects for further research as an effective and safe treatment for acute radiation sickness.

Rapid integration of screen-printed electrodes into thermoplastic organ-on-a-chip devices for real-time monitoring of trans-endothelial electrical resistance.

Trans-endothelial electrical resistance (TEER) is one of the most widely used indicators to quantify thebarrier integrity of endothelial layers. Over the last decade, the integration of TEER sensors into organ-on-a-chip (OOC) platforms has gained increasing interest for its efficient and effective measurement of TEERin OOCs. To date, microfabricated electrodes or direct insertion of wires has been used to integrate TEERsensors into OOCs, with each method having advantages and disadvantages. In this study, we developed aTEER-SPE chip consisting of carbon-based screen-printed electrodes (SPEs) embedded in a poly(methylmethacrylate) (PMMA)-based multi-layered microfluidic device with a porous poly(ethylene terephthalate)membrane in-between. As proof of concept, we demonstrated the successful cultures of hCMEC/D3 cellsand the formation of confluent monolayers in the TEER-SPE chip and obtained TEER measurements for 4days. Additionally, the TEER-SPE chip could detect changes in the barrier integrity due to shear stress oran inflammatory cytokine (i.e., tumor necrosis factor-α). The novel approach enables a low-cost and facilefabrication of carbon-based SPEs on PMMA substrates and the subsequent assembly of PMMA layers forrapid prototyping. Being cost-effective and cleanroom-free, our method lowers the existing logistical andtechnical barriers presenting itself as another step forward to the broader adoption of OOCs with TEERmeasurement capability.

Topographical influence of electrospun basement membrane mimics on formation of cellular monolayer

Functional unit of many organs like lung, kidney, intestine, and eye have their endothelial and epithelial monolayers physically separated by a specialized extracellular matrix called the basement membrane. The intricate and complex topography of this matrix influences cell function, behavior and overall homeostasis. In vitro barrier function replication of such organs requires mimicking of these native features on an artificial scaffold system. Apart from chemical and mechanical features, the choice of nano-scale topography of the artificial scaffold is integral, however its influence on monolayer barrier formation is unclear. Though studies have reported improved single cell adhesion and proliferation in presence of pores or pitted topology, corresponding influence on confluent monolayer formation is not well reported. In this work, basement membrane mimic with secondary topographical cues is developed and its influence on single cells and their monolayers is investigated. We show that single cells cultured on fibers with secondary cues form stronger focal adhesions and undergo increased proliferation. Counterintuitively, absence of secondary cues promoted stronger cell–cell interaction in endothelial monolayers and promoted formation of integral tight barriers in alveolar epithelial monolayers. Overall, this work highlights the importance of choice of scaffold topology to develop basement barrier function in in vitro models.

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier.

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of non-essential ions and toxic substances. Tight junctions (TJ) effectively seal the paracellular space in the monolayers resulting in an intact barrier. This study describes a LY-based fluorescence assay that can be used to determine its apparent permeability coefficient (Papp) and in turn can be used to determine the kinetics of the formation of confluent monolayers and the resulting tight junction barrier integrity in hCMEC/D3 monolayers. We further demonstrate an additional utility of this assay to determine TJ functional integrity in transfected cells. Our data from the LY Papp assay shows that the hCMEC/D3 cells seeded in a transwell setup effectively limit LY paracellular transport 7 days-post culture. As an additional utility of the presented assay, we also demonstrate that the DNA nanoparticle transfection does not alter LY paracellular transport in hCMEC/D3 monolayers.

Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells.

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great promise for these RPE cell therapies. The aim of the present study was to assess whether a flexible, elastic and biodegradable poly(trimethylene carbonate) (PTMC) film promotes the formation of functional hESC-RPE and performs better than often used biodegradable poly(d,l-lactide) (PDLLA) film. Human ESC-RPE maturation and functionality on PTMC films was assessed by cell proliferation assays, RPE-specific gene and protein expression, phagocytic activity and growth factor secretion. It is demonstrated that the mechanical properties of PTMC films have close resemblance to those of the native Bruch's membrane and support the formation hESC-RPE monolayer in serum-free culture conditions with high degree of functionality. In contrast, use of PDLLA films did not lead to the formation of confluent monolayers of hESC-RPE cells and had unsuitable mechanical properties for retinal application. In conclusion, the present study indicates that flexible and elastic biodegradable PTMC films show potential for retinal tissue engineering applications. Copyright © 2017 John Wiley & Sons, Ltd.

Reorganization of actin and microtubule systems in human vein endothelial cells during intercellular contact formation

Endothelial cells tightly joined to each other line the interior surface of all vessels in living organisms and ensure the regulation of vascular permeability and exchange between the blood circulating in vessels and tissue fluids of those organs in which these vessels are located. In vitro, the endothelial cell monolayer retains the basic barrier function of the native endothelium. We use endothelial cells growing in vitro as a model system to study the interaction between cytoskeleton and adhesion structures in endotheliocytes. In the present paper, cultured human vein endothelial cells were used to quantify cytoskeleton alterations from cell spreading and formation of the first cell–cell contacts to confluent monolayer formation. The actin filament system in vein endothelial cells consists of a cortical network and stress fibers arranged parallel to the substrate. This cells express two isoforms of nonmuscle actin: β- and γ-cytoplasmic actins. Antibodies to β-actin stain actin filament bundles, whereas antibodies to γ-actin visualize cortical and lamellar networks. To evaluate the state of actin structures we analyzed their fluorescence intensity near the free edge and zone of cell–cell contacts. Fluorescence intensity of β-actin structures was higher in the areas of cell–cell contacts. Fluorescence of γ-actin was the highest at the leading edges of lamellae and the weakest in the stable edges of the cells with formed cell–cell contacts. The endothelial monolayer formation was accompanied by microtubule system alteration: the number of microtubules increased at the cell edge, and what was more, the quantity of microtubules in the area of the already-formed cell–cell contact always being higher than in the leading edge region.

Standardization of serum neutralization assay of Japanese encephalitis virus (Nakayama NIH strain) on BHK-21 (Cl-13) cell line.

Potency testing of Japanese encephalitis (JE) vaccine has been acomplex process since its inception. To overcome difficulties encountered therein, an alternative assay, serum neutralization test (SNT), using Baby Hamster Kidney 21 cell line, has been standardized. The antibody response generated against JE vaccine was quantified and the assay was found to be sensitive and specific enough with significant accuracy and precision. On analysis of cell count, acell concentration of 1.5 x 104 was selected as the optimum, since concentrations above and below this resulted in problems of confluent monolayer formation and incomplete monolayer formation. Incubation time has also been standardized for measuring cytopathic effect (CPE). Out of the four different time points selected, 90 min was found to be adequate for 50% reduction in the amount of CPE. The accuracy of SNT assay is explained in terms of fiducial limits at 95% level. Inter- and intra-assay reproducibility testing was also performed. Acomparison of potency of JE vaccine by plaque reduction neutralization test (PRNT) and SNT method was conducted and it was found that SNT can be areliable approach for estimating the potency of JE vaccine. The results of this study throw alight on the utility of SNT assay for the potency estimation of JE vaccine in routine practice.

A novel high-speed production process to create modular components for the bottom-up assembly of large-scale tissue-engineered constructs.

To replace damaged or diseased tissues, large tissue-engineered constructs can be prepared by assembling modular components in a bottom-up approach. However, a high-speed method is needed to produce sufficient numbers of these modules for full-sized tissue substitutes. To this end, a novel production technique is devised, combining air shearing and a plug flow reactor-style design to rapidly produce large quantities of hydrogel-based (here type I collagen) cylindrical modular components with tunable diameters and length. Using this technique, modules containing NIH 3T3 cells show greater than 95% viability while endothelial cell surface attachment and confluent monolayer formation are demonstrated. Additionally, the rapidly produced modules are used to assemble large tissue constructs (>1 cm(3) ) in vitro. Module building blocks containing luciferase-expressing L929 cells are packed in full size adult rat-liver-shaped bioreactors and perfused with cell medium, to demonstrate the capacity to build organ-shaped constructs; bioluminescence demonstrates sustained viability over 3 d. Cardiomyocyte-embedded modules are also used to assemble electrically stimulatable contractile tissue.

Pro-angiogenic CD14++ CD16+ CD163+ monocytes accelerate the in vitro endothelialization of soft hydrophobic poly(n-butyl acrylate) networks

As the majority of the polymers used as cardiovascular grafts so far do not match the elasticity of human arteries (100–1000kPa) and the required endothelialization, a multifunctional material approach is needed to allow the adjustment of the mechanical properties while at the same time exhibiting a haemocompatible surface. Recently soft poly(n-butyl acrylate) networks (cPnBA) with adjustable mechanical properties were introduced as candidate materials with a surface that can be endothelialized. In this study, angiogenically stimulated intermediate CD163+ monocytes/macrophages (aMO2) were utilized as a cellular cytokine release system to realize the functional endothelialization of the hydrophobic cPnBA surface. We investigated the influence of co-cultured aMO2 on the morphology, density and cytokine secretion of human umbilical venous endothelial cells (HUVEC) seeded on cPnBA with an elastic modulus of around 250kPa (cPnBA0250). A functional confluent HUVEC monolayer could be developed in the co-culture within 3days. In contrast, the HUVEC in the monoculture exhibited stress fibres, broadened marginal filament bands and significantly more and larger cell-free areas in the monolayer, indicating incomplete cell–substrate binding. Remarkably, a functional confluent monolayer formation could only be achieved in co-cultures; it did not develop with the sole supplementation of recombinant VEGF-A165 to the HUVEC monocultures (unpublished data). The study demonstrated the multifunctional potential of cPnBA in combination with aMO2 as a cellular cytokine release system, adapting their secretion to the demand of HUVEC. In this way, a functional confluent monolayer could be generated within 3days.

Toxoplasma gondii antigens: Recovery analysis of tachyzoites cultivated in Vero cell maintained in serum free medium

Vero cells have been used successfully in Toxoplasma gondii maintenance. Medium supplementation for culture cells with fetal bovine serum is necessary for cellular growth. However, serum in these cultures presents disadvantages, such as the potential to induce hypersensitivity, variability of serum batches, possible presence of contaminants, and the high cost of good quality serum. Culture media formulated without any animal derived components, designed for serum-free growth of cell lines have been used successfully for different virus replication. The advantages of protozoan parasite growth in cell line cultures using serum-free medium remain poorly studied. Thus, this study was designed to determine whether T. gondii tachyzoites grown in Vero cell cultures in serum-free medium, after many passages, are able to maintain the same antigenic proprieties as those maintained in experimental mice. The standardization of Vero cell culture in serum-free medium for in vitro T. gondii tachyzoite production was performed establishing the optimal initial cell concentration for the confluent monolayer formation, which was 1×106 Vero cell culture as initial inoculum. The total confluent monolayer formatted after 96h and the best amount of harvested tachyzoites was 2.1×107 using parasite inoculum of 1.5×106 after 7days post-infection. The infectivity of tachyzoites released from Vero cells maintained in serum-free medium was evaluated using groups of Swiss mice infected with cell-culture tachyzoites. The parasite concentrations were similar to those for mice infected with tachyzoites collected from other infected mice. The data from both in vivo and in vitro experiments showed that in at least 30 culture cell passages, the parasites maintained the same infectivity as maintained in vivo. Another question was to know whether in the several continued passages, immunogenic progressive loss could occur. The nucleotide sequences studied were the same between the different passages, which could mean no change in their viability in the lysate antigen. Thus, the antigen production by cell culture has clear ethical and cost-saving advantages. Moreover, the use of culture media formulated without any human or animal derived components, designed for serum-free growth of cell lines, successfully produced tachyzoites especially for antigen production.

Optimized allylamine deposition for improved pluripotential cell culture

Deposition of allylamine (ALL) by plasma enhanced chemical vapor deposition has been optimized on silicon based models. Simultaneous energy recoil detection analysis and Rutherford backscattering spectra show that 100 W deposition is ideal in terms of polymerized film formation and H content while, lower or higher power induce reduced film retention or excessive cross linking, respectively. Surface composition of the ALL film was further probed by X-ray photoelectron spectroscopy revealing a monocomponent N 1s spectrum compatible with the presence of primary amines. Optimized ALL films were applied to polycaprolactone (PCL) surfaces after Ar plasma activation with implications in the chemistry and wettability of this biocompatible polymer. Human mesenchymal stem cells (hMSCs) were cultured on ALL coated PCL surface and controls. ALL functionalized PCL was found to be especially attractive for the formation of confluent monolayers of hMSCs after 72 h of culture.

The role of endothelial cell attachment to elastic fibre molecules in the enhancement of monolayer formation and retention, and the inhibition of smooth muscle cell recruitment

The endothelium is an essential modulator of vascular tone and thrombogenicity and a critical barrier between the vessel wall and blood components. In tissue-engineered small-diameter vascular constructs, endothelial cell detachment in flow can lead to thrombosis and graft failure. The subendothelial extracellular matrix provides stable endothelial cell anchorage through interactions with cell surface receptors, and influences the proliferation, migration, and survival of both endothelial cells and smooth muscle cells. We have tested the hypothesis that these desired physiological characteristics can be conferred by surface coatings of natural vascular matrix components, focusing on the elastic fiber molecules, fibrillin-1, fibulin-5 and tropoelastin. On fibrillin-1 or fibulin-5-coated surfaces, endothelial cells exhibited strong integrin-mediated attachment in static conditions (82% and 76% attachment, respectively) and flow conditions (67% and 78% cell retention on fibrillin-1 or fibulin-5, respectively, at 25 dynes/cm 2), confluent monolayer formation, and stable functional characteristics. Adhesion to these two molecules also strongly inhibited smooth muscle cell migration to the endothelial monolayer. In contrast, on elastin, endothelial cells attached poorly, did not spread, and had markedly impaired functional properties. Thus, fibrillin-1 and fibulin-5, but not elastin, can be exploited to enhance endothelial stability, and to inhibit SMC migration within vascular graft scaffolds. These findings have important implications for the design of vascular graft scaffolds, the clinical performance of which may be enhanced by exploiting natural cell–matrix biology to regulate cell attachment and function.

Evaluation of Proliferation and Functional Differentiation of LLC-PK1 Cells on Porous Polymer Membranes for the Development of a Bioartificial Renal Tubule Device

To develop a bioartificial renal tubule system using renal tubular cells and porous polymer membrane hollow fibers, long-term maintenance of a confluent monolayer and the functionally differentiated condition of cells is essential. We examined the proliferation and functional differentiation of LLC-PK1 (Lewis-lung cancer porcine kidney 1) cells on two types of membranes: polysulfone and cellulose acetate. Cell proliferation was significantly higher on the polysulfone membrane than on the cellulose acetate membrane, and was enhanced by coating the membranes with various extracellular matrices. Confluent monolayer formation of cells was observed on matrix-coated polysulfone membrane but not on matrix-coated cellulose acetate membrane within 1 week. Cell proliferation continued for 3 weeks after confluent monolayer formation. Messenger RNA (mRNA) expression of glucose transporters, indicators of the functional differentiation of the LLC-PK1 cells, was observed in the polysulfone and cellulose acetate membrane groups, but was not observed in the nonporous polystyrene plate group under subconfluent conditions. Expression of glucose transporters mRNA was maintained for 3 weeks after confluent monolayer formation. Polysulfone membrane is more suitable than cellulose acetate membrane for a bioartificial renal tubule system with regard to LLC-PK1 cell proliferation. Extracellular matrix coating of the membrane further improves cell proliferation.

The role of cytoskeleton in cell changes under condition of simulated microgravity

Single cells and cell culture are very good model for estimation of primary effects of gravitational changes. It is suggested that cell cytoskeleton plays a key role in mechanisms of adaptation to mechanical influences including gravitational ones. Our results demonstrated that cultured cells of human vascular endothelium are highly sensitive to hypogravity (clinorotation) and respond by significant decrease of cell proliferative activity. Simultaneously it was noted that the formation of confluent monolayer appeared early in cultures exposed to simulated microgravity due to accelerated cells spreading. Long-term hypogravity (several hours or days) leads to significant changes of cell cytoskeleton revealed as microfilament thinning and their redistribution within cell. Such changes were observed only in monolayer cells and not in cell suspensions. Gravitational forces as known to be modificators of cell adhesive ability and determine their mobility. Hypogravity environment stimulated endothelial cell migration in culture: 24–48 hrs pre-exposition to hypogravity significantly increased endothelial cell migration resulting in 2–3-fold acceleration of mechanically injured monolayer repair. Obtained results suggest that the effects of hypogravity on cultured human endothelial cells are, possibly, associated with protein kinase C and/or adenylate cyclase activity and are accompanied by noticeable functional cell changes.

Decreased Cholesterol Impairs Cell Junctions

Endothelial cells form tight junctions and adherins junctions both in vivo and in culture. In vivo this phenomenon is essential to the proper function of the endothelial cells as a barrier between the blood vessel and the extracellular space. The formation of confluent monolayers of endothelial cells in culture is associated with increases in membrane cholesterol, increased association of annexin II with membranes, and dephosphorylation of adherins junction proteins (cadherin, β-catenin, γ-catenin, and pp120). Corvera et al . found that removal of cholesterol from confluent endothelial cells by cyclodextrin increased the tyrosine phosphorylation of pp120 and γ-catenin and disrupted adherins junctions between endothelial cells in culture. Additionally, membrane-associated annexin II was solubilized by digitonin, which selectively solubilizes cholesterol from membranes, suggesting that the association of annexin II with the membrane was dependent on the level of cholesterol. The authors suggest that cholesterol itself may be part of the regulatory pathway leading to the formation of adherins junctions. Corvera, S., DiBonaventura, C., and Shpetner, H.S. (2000) Cell confluence-dependent remodeling of endothelial membranes mediated by cholesterol. J. Biol. Chem. 275 : 31414-31421. [Abstract] [Full Text]

Decreased Cholesterol Impairs Cell Junctions

Endothelial cells form tight junctions and adherins junctions both in vivo and in culture. In vivo this phenomenon is essential to the proper function of the endothelial cells as a barrier between the blood vessel and the extracellular space. The formation of confluent monolayers of endothelial cells in culture is associated with increases in membrane cholesterol, increased association of annexin II with membranes, and dephosphorylation of adherins junction proteins (cadherin, β-catenin, γ-catenin, and pp120). Corvera et al. found that removal of cholesterol from confluent endothelial cells by cyclodextrin increased the tyrosine phosphorylation of pp120 and γ-catenin and disrupted adherins junctions between endothelial cells in culture. Additionally, membrane-associated annexin II was solubilized by digitonin, which selectively solubilizes cholesterol from membranes, suggesting that the association of annexin II with the membrane was dependent on the level of cholesterol. The authors suggest that cholesterol itself may be part of the regulatory pathway leading to the formation of adherins junctions.Corvera, S., DiBonaventura, C., and Shpetner, H.S. (2000) Cell confluence-dependent remodeling of endothelial membranes mediated by cholesterol. J. Biol. Chem. 275: 31414-31421. [Abstract] [Full Text]

Partially Purified Soy Hydrolysates Retard Proliferation and Inhibit Bacterial Translocation in Cultured C2BBe Cells ,

Hydrolyzed soybean isolates SP-A and SP-B (Abbott Laboratories, OH), developed for use in enteral nutritional products, were tested in cultures of C2BBe cells, a colonic adenocarcinoma cell line with enterocytic differentiation, to evaluate effects on cell growth, maturation and ability to resist infection by enteric bacteria. SP-A delayed formation of confluent monolayers by 10 d compared with cells cultured without SP-A. SP-A also caused a retardation in the development of intercellular tight junctions as measured by transmonolayer electrical resistance (TER). SP-B had no effect on cell proliferation or TER of intestinal cell cultures. SP-A and SP-B enhanced the development of the brush border enzymes alkaline phosphatase and isomaltase over a 28 d period. By these criteria, SP-A and SP-B appear to affect intestinal epithelial cell development in culture. When C2BBe monolayers were exposed to the enteric bacteria, Salmonella typhimurium or Salmonella typhi, an inhibition of the passage of S. typhi was seen in cultures with SP-A and SP-B. No effect on the passage of S. typhimuriumwas seen with either soy isolate. Partially purified soy isolates therefore impart resistance to selected enteroinvasive bacteria. Addition of soy hydrolysates to the media of cultured intestinal cells may serve as a rapid and economical screening mechanism for preclinical trials that would test the therapeutic benefits of soybean isolates.

Crystallins and their synthesis in human lens epithelial cells in tissue culture

Explants of epithelial cells from young human lenses of 5–12 months of age, obtained from patients who underwent surgery for retinopathy of prematurity, were cultured in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum. Without exception, every piece of the anterior capsule explant showed cell outgrowth within 48–72 h and resulted in confluent monolayer culture within 2 weeks. From these monolayer cultures, two to three passages of subcultures were obtained by routinely seeding cells in a ratio of 1: 4. The doubling times for these human lens epithelium (HLE) cultures during the first 4 weeks of two passages were found to be 24–36 h. In a majority of cultures through the first three passages, more than 12 population doublings were attained. However, no lentoid bodies were formed during this period. These cells were studied for the presence of crystallins and their synthesis. Using SDS-polyacrylamide gel electrophoresis, the presence of alpha- and beta-crystallins was demonstrated in HLE cells through three passages. The amount of alpha-crystallin in the first two passages amounted to nearly 13% of the total protein, but decreased significantly in the third passage. The presence of crystallins was corroborated by antibody reaction to the specific crystallins. Indirect immunofluorescence revealed the presence of actin and vimentin in these cell cultures. The synthesis of crystallins in HLE cultures was shown by the incorporation of [ 35S]methionine which was time dependent. The crystallin synthesis was found to decrease in third passage when the cell growth slowed down without consistent formation of confluent monolayer. These studies have demonstrated that primary cultures of HLE cells can be successfully grown from young lenses through several passages which continue to express the characteristic crystallins of the epithelial cells.

Effect of Treatment with 5-Ethyl-2′-Deoxyuridine on Herpes Simplex Virus Encephalitis in Normal and Immunosuppressed Mice

5-Ethyl-2'-deoxyuridine (5-ethyl-dUrd), an analog of thymidine, was evaluated for its capacity to inhibit herpes simplex virus (HSV) replication in vitro and in vivo. The 50% effective dose concentration of 5-ethyl-dUrd for HSV types 1 and 2 (HSV-1 and -2) cultured in Vero cells was 6 and 9 mug/ml, respectively. Levels of 5-ethyl-dUrd 14-fold in excess of the 50% effective dose for HSV-1 did not inhibit the formation of confluent monolayers by Vero cells, suggesting that the compound was not cytotoxic or inhibitory for mammalian cells. In vivo studies showed that 5-ethyl-dUrd was effective in significantly reducing mortality when administered to young adult mice after subcutaneous infection with HSV-2. Intraperitoneal and intravenous inoculation of drug (250 mg/kg per day) resulted in a 50% survivor rate at 15 days. Comparative studies with adenine arabinoside at 250 mg/kg per day gave a 40% survivor rate. Intramuscular injection of 5-ethyl-dUrd at a concentration as high as 2,000 mg/kg per day for 10 days was well tolerated by uninfected animals, and HSV-2-infected mice treated at this dosage had a 100% survival rate. Treatment with 5-ethyl-dUrd at a concentration of 500 mg/kg per day significantly increased the mean survival times of HSV-1- and HSV-2-infected mice immunosuppressed by irradiation; however, the fatal course of the infection was not altered. Assay for virus in tissues showed that 5-ethyl-dUrd treatment delayed progression of the infection into the central nervous system, indicating suppression of virus replication in the tissues.

Nature of lymphocyte-tumor interaction. A general method for cellular immunoabsorption.

The binding of sensitized lymphocytes to tumor cells that leads to tumor cell lysis in vitro has been investigated using poly-L-lysine-fixed tumor cell monolayers and lymphocytes obtained from the anatomical site of tumor allograft rejection. The results show that magnesium is an important prerequisite for this interaction and that the extent of lymphocyte-tumor cell binding depends upon temperature as well as pH. Binding can occur in the absence of serum, although serum factors are necessary for the completion of the cytolytic process. The poly-L-lysine technique is applicable to the formation of confluent monolayers with virtually any normal or neoplastic cell type, including those that are otherwise nonadherent to surfaces. Cells immobilized by this technique can be used for the specific immunoabsorption and subsequent recovery of effector lymphocytes sensitized against transplantation or tumor cell antigens.