The serial cultivation of human diploid cell strains.

The limited in vitro lifetime of human diploid cell strains

The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

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Nitric Oxide and Peroxynitrite in Health and Disease

▪ Abstract At the interface between the innate and adaptive immune systems lies the high-output isoform of nitric oxide synthase (NOS2 or iNOS). This remarkable molecular machine requires at least 17 binding reactions to assemble a functional dimer. Sustained catalysis results from the ability of NOS2 to attach calmodulin without dependence on elevated Ca2+. Expression of NOS2 in macrophages is controlled by cytokines and microbial products, primarily by transcriptional induction. NOS2 has been documented in macrophages from human, horse, cow, goat, sheep, rat, mouse, and chicken. Human NOS2 is most readily observed in monocytes or macrophages from patients with infectious or inflammatory diseases. Sustained production of NO endows macrophages with cytostatic or cytotoxic activity against viruses, bacteria, fungi, protozoa, helminths, and tumor cells. The antimicrobial and cytotoxic actions of NO are enhanced by other macrophage products such as acid, glutathione, cysteine, hydrogen peroxide, or superoxid...

Nitric oxide and macrophage function

http://theory.bio.uu.nl/immbio/Marks91.pdf

By-passing immunization: Human antibodies from V-gene libraries displayed on phage

Viruses are ubiquitous in plants, particularly those associated with agriculture Plant viruses have a range of features that extend from detrimental to potentially beneficial Economic loses to agriculture caused by viral infections led to the development of genetic systems that allow manipulation of the virus to manage plant diseases; however, these genetic systems have also led to the development of viruses as beneficial tools, exploiting the ability of the small plus-sense single-stranded RNA viruses that commonly infect higher plants to rapidly amplify virus-related RNAs and produce large amounts of proteins As early as 1983 Siegel described a strategy for the development of virus-based vectors to express foreign genes in plants, although at that time the technology for manipulation of viruses through cDNA cloning was not yet available, and the issue was controversial (Siegel 1983; van Vloten-Doting 1985) Since that time, in vitro genetic systems have been developed for viruses in different taxonomic groups and several of these have been manipulated to transiently express foreign genes (for review, see Kearney et al 1995)

Plant viral vectors based on tobamoviruses.

Human peripheral blood mononuclear cells and T-cell lines and clones from individuals immunized with rabies PM vaccine were tested for the ability to recognize antigenic determinants in rabies and rabies-related viruses in an antigen-induced proliferation assay. Some, but not all, of the T cells from these individuals cross-reacted with various laboratory strains of rabies virus with rabies-related viruses such as Duvenhage and Mokola. In addition, these T cells were shown to react with epitopes of either the ribonucleoprotein or the viral glycoprotein. Rabies-specific cytotoxic T-cell responses by a CD4+ T-cell line were evident against antigenic determinants of the ribonucleoprotein and glycoprotein.

Recognition of rabies and rabies-related viruses by T cells derived from human vaccine recipients.

Cultures of normal human fibroblasts are characterized by a finite lifetime in vitro.' Actively proliferating cells can usually be maintained in culture for only 35-40 transfers. Following this period the growth rate of the cultures gradually declines, and proliferation ceases completely after 5-10 further passages. 2 In previously reported studies3-6 on transformation of human cell cultures by simian virus 40 (SV40), either primary explants or actively proliferating established cell strains were exposed to SV40. In this study the susceptibility to SV40 of cultures approaching the end of their expected in vitro lifetime was assessed. It was found that these aging cultures transformed considerably earlier after infection than the primary explants and \"young\" actively proliferating cell strains examined before. Materials and Methods.-The WI-26 and WI-38 human fibroblast strains were originally obtained by cultivation of embryonic lung tissue.' Cells of two WI-26 substrains used in the present study (Nos. XII and XXVI, Table 1) could not be transferred beyond the 41st and 56th subcultures, respectively. Each of three parallel cultures of substrain XXVI was exposed to SV40 at the 55th subculture. Cells of the parent WI-38 strain and of its No. IV substrain did not survive subculturing beyond the 50th and 48th passages, respectively. As shown in Table 1, these cultures were exposed to SV40 at the level of the 50th and 47th subcultures. The W-5 CM strain originated from an explant of a fragment of buccal mucosa obtained from a 72-year-old man who had been treated with X rays and cytoxan after surgical removal of a bronchial carcinoma.' Following formation of a monolayer, the cell strain was propagated in actively proliferating cultures up to the 23rd transfer, when the cultures were frozen and stored, but could not be recovered in a viable state after thawing. Two substrains (D 90 and D 285) were established from the 12th passage of the parental W-5 CM strain after the culture flasks were left in an incubator for 90 and 285 days, respectively, without a change of nutrient medium. Although this prolonged incubation period led to a cessation of mitosis and some cell loss, the remaining cells of the two cultures started to proliferate again when fresh medium was supplied. However, cells of substrain D 90 could be subcultured only at weekly intervals as compared to biweekly transfers for the parent strain. The cell growth of D 90 slowed down even more at the level of the 19th passage, and the cultures could not survive beyond the 25th-30th passage (Table 1). Each of the three parallel cultures of this substrain was exposed to SV40 at the 21st, 21st, and 22nd passages, respectively (Table 1). Three parallel cultures of substrain W-5 D 285 (Table 1) were infected with SV40 at the 13th passage, i.e., the first passage following restoration of cultures after prolonged incubation. Although parallel noninfected cultures of this strain are still maintained, growth is so slow that they can be subcultured only at intervals of 2-3 weeks.

Rapid transformation of human fibroblast cultures by simian virus 40

This paper reports on laboratory studies made in guinea-pigs on puncture wounds infected with fixed rabies virus and treated one hour later with various substances, the purpose being to review the experience of previous workers and to explore new approaches to the problem of local treatment of wounds inflicted by rabid animals.AMONG THE MEASURES AFFORDING GREATER OR LESSER PROTECTION WERE: nitric acid cauterization; direct application of benzalkonium chloride to the wound or its infiltration, as well as that of methylbenzethonium chloride, proximal to the wound; repeated swabbing and flushing with 20% soap solution or benzalkonium chloride; local inoculation of procaine anaesthetics; infiltration of the leg wound, or inoculation of the opposite leg, with antirabies gamma-globulin; and infiltration of the wound with interferon prepared in guinea-pig tissue cultures.Protection was not afforded by flushing with 20% soap solution alone; topical application of aqueous or tincture preparations of iodine or thiomersal; inoculation of phenoxybenzamine, physostigmine or diphenhydramine hydrochloride; or infiltration of the wound with interferon prepared on monkey kidney tissue cultures.The authors conclude that, at the present time, very vigorous cleansing of the wound with 20% soap solution or 2% benzalkonium chloride, local infiltration of the wound with antirabies serum and-to minimize pain-the use of procaine in saline are indicated in the local treatment of wounds for the prevention of rabies.

Studies on the local treatment of wounds for the prevention of rabies.

THERE are no reports of the direct isolation of myco-plasmas from human leukaemic blood or blood-forming organs, although the direct isolation of mycoplasmas from the blood of a few septic patients has been claimed1–4. In the present investigation, mycoplasmas were isolated from broth cultures seeded with three bone marrow samples obtained on the same day from one of three children suffering from acute lymphoblastic leukaemia (Table 1).

Hilary Koprowski

Papers

Plant viral vectors based on tobamoviruses.

Recognition of rabies and rabies-related viruses by T cells derived from human vaccine recipients.

Rapid transformation of human fibroblast cultures by simian virus 40

Studies on the local treatment of wounds for the prevention of rabies.

Direct agar isolation of mycoplasmas from human leukaemic bone marrow.