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• A 61-year-old woman with progressive blindness, initially responsive to steroid therapy, was found to have an undifferentiated neoplasm in the cervix uteri. Visual fields demonstrated ring scotomas and the electroretinographic pattern eventually became flat. The interval from onset of visual symptoms to blindness was 19 months. Eye pathologic findings included loss of retinal photoreceptors, a macular hole, and normal optic nerves. Antibodies were demonstrated in the patient's serum to normal fresh human retinal photoreceptors. An autoimmune mechanism may have been responsible for the retinal degeneration in this patient, and such a hypothesis raises questions about (1) autoimmune mechanisms in patients with retinitis pigmentosa and other retinal degenerative diseases and (2) autoimmune mechanisms in other cases of remote effects of carcinoma.

Experiments designed to characterize an unidentified transmissible agent brought forth the following findings: The cytopathology consisted of the formation of intranuclear globules, collapse of the involved nuclei, and the extrusion of nuclear materials. The relatively dormant primary human amnion cells were less susceptible than the rapidly growing cell lines. Similarly, the slowly multiplying ribose variants were less susceptible than their corresponding parent cell lines. Interferon-like activity was released from infected cells. Infectivity was readily demonstrated following storage at 0–4°C for at least 8 months or at 37°C for at least 2 weeks. Freeze-thawing, however, markedly reduced or completely destroyed its infectivity. Infectivity was destroyed completely by ether and chloroform; partially by desoxycholate, and not affected by trypsin, papain, RNAse, DNAse, hyaluronidase, lysozyme, lecithinase, or pancreatic lipase. The rate of inactivation by 0.025 per cent formalin was much slower than that of vaccinia and herpes viruses. Its synthesis was suppressed by 5-fluorodeoxyuridine. This suppression was not reversed by thymidine and/or uracil. Heat-stable neutralizing antibody could not be demonstrated in 379 human and animal serums, in human gamma globulins, or in serums from animals "immunized" with this agent. Heat-labile inhibitors (lipoprotein-like) capable of inhibiting the infectivity of this agent were demonstrated in 154 of the 157 serums tested. Experimental evidence indicated the non-identity of this ubiquitous inhibitor and the properdin system. The non-infectious complex between this agent and the ubiquitous serum inhibitor may be dissociated (hence, become infectious) by simple dilution. Repeated attempts to reisolate a similar agent have not been successful. We have hypothesized that this agent is a virus consisting of DNA wrapped in a surface coat rich in lipid, and suggest that this virus be referred to tentatively as a lipovirus.

The release of a toxic product from cultured human cells infected by a new cytopathic agent (the lipovirus) was described. This toxin was dissociable from the infectious particles. It induced sudanophilia of human and mouse cells, an increase in the total fatty acid content, and a change in the major constituent fatty acids. Similar toxin was not demonstrated in cultures infected by the vaccinia herpes simplex, adeno 3, polyoma, polio 1 and 2, Coxsackie B1, parainfluenza A, and Rous sarcoma viruses. Preliminary characterization indicated that this toxin was resistant to tryptic digestion and could not be dialyzed or neutralized by human gamma globulin. It was inactivated at 58°C for 30 minutes, but stable at 37°, 4°, and –60°C. The significance of these findings is discussed.

DNA and thymine degradation on cultured human, mouse, and chick cells were studied. Significant increase in DNA-degrading activity was demonstrated in human embryonic cells killed by freeze-thawing, liver cells killed with mitomycin C, mouse embryonic cells infected with encephalomyocarditis virus, and in all cells killed by the lipovirus. Twelve other viral agents, actinomycin D, and 5-fluorodeoxyuridine failed to produce a similar increase. Thymidine-2-C14-labeled cultures, either live, killed, or infected by 19 different physical-chemical and biological agents, did not release detectable quantity of C14C2. Following infection with the lipovirus 20 to 60 per cent of the total radioactivity of thymidine-2-C14-labeled cultures was liberated as C14O2. It was postulated that the lipovirus introduced into the host cells the missing genetic information necessary for the synthesis of one or more enzymes responsible for the reductive catabolism of thymine.

Under the culture conditions described in this report, 20 lines of cells morphologically similar to the lymphoid cells were derived from the blood of eight healthy adults. Four of the 20 lines have undergone 85 to 107 serial divisions and are still growing vigorously. Fifteen lines showed an unexplained loss in growth vitality after the 3rd to 48th serial division. One line was contaminated by fungi in the 8th serial division.

The relative capacity of several types of human cells and tissue to produce interferon was studied. Types of cells and tissue included were fibroblasts from embryos, foreskins, and biopsied skins; amnion cells; peripheral leukocytes; established lymphoid cell lines; established heteroploid cell lines; and chorioamniotic membrane. When Newcastle disease virus was used as the inducer, fibroblasts and amnion cells produced more interferon per 106cells than leukocytes, lymphoid cells, and heteroploid cells. Only minor variations in interferon-producing capacity were observed among fibroblasts from 36 persons. Culture passage level, cell concentration, and inducer were factors that significantly affected interferon production.

The isolation of carbohydrate variants from cultures of HeLa and conjunctival cells was described. Factors inherent in the cell culture system, such as parent populations and dialyzed serums, have been shown to influence the outcome of variant isolations. Established stable variants incorporated significantly more pentoses or lactate into various cell fractions than the parent cultures. Besides their abilities to propagate continuously in the selecting environments, the variants multiplied slower, were more susceptible to sub-zero preservation and the cytotoxic effect of D-2-deoxyglucose, showed lower cloning efficiencies and were less susceptible to the deleterious effect of glucose oxidase. The ribose variants also differed from the parent cultures in morphological appearance such as formation of multinucleated cells and ring-shaped colonies. They converted more ribose into other component sugars of mucopolysaccharides than the parent cultures. Preliminary analyses of the mucopolysaccharides extracted from the ribose variants and parent cultures showed large difference in their carbohydrate (Molisch-positive materials) and DNA ratios. Evidence suggests that a sequence of interrelated events from genetic selection to primitive morphogenesis has been established.

The propagation of a strain of Coxsackie virus, group B type 1, in human cell cultures depleted of bicarbonate has been studied. Under the described experimental conditions, bicarbonate depletion suppresses the propagation of this virus. This suppressive effect may be reversed by the addition of the following compounds to the bicarbonate-depleted cultures: (a) bicarbonate; (b) adenosine, guanosine, cytidine, and uridine; (c) adenylic, guanylic, cytidylic, and uridylic acids; (d) enzymatically degraded RNA prepared from yeasts or human embryo, or (e) RNA. The following compounds are unable to reverse the suppressive effect of bicarbonate depletion: (a) adenine, guanine, cytosine, and uracil, with or without ribose; (b) adenine, guanine, cytosine, and thymine; (c) deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine; (d) deoxyadenylic, deoxyguanylic, deoxycytidylic, and thymidylic acids; (e) enzymatically degraded DNA, or (f) DNA. The same general results as with the Coxsackie virus have been obtained with a strain of poliovirus and vaccinia virus. The failure of bicarbonate depletion to suppress completely the propagation of the poliovirus under the described condition constitutes a major difference. The significance of these findings is discussed.

This report describes experiments designed to study changes in metabolic patterns associated with the senescence of human amnion cells in vitro. Senescence has been defined as the increase in the chronological age of a population of cultured cells which are predominantly nondividing. This definition is based on the concept formulated by Comfort and Shock.1 The general scheme of these experiments consists of the determination of radioactivity in various cell components obtained from cultures previously labeled with C14 substrates. MATERIALS AND METHODS AMNION CULTURE.— Cell suspensions were prepared from amniotic membranes carefully dissected from placentae obtained at normal full-term deliveries.2 Approximately 5 million cells suspended in 10 ml. of nutrient medium, consisting of 10% human and 5% calf or 20% horse serum in Eagle's basal medium,3 were implanted in milk dilution bottles of 200 ml. capacity. The media were renewed on the 4th and 7th days. On the

The growth characteristics, nutritional requirements, and metabolic activities of primary explants (chiefly amnion) and transformed human cells (derived from amnion, conjunctiva, and cervical cancer) were compared. Major differences observed are as follows: 1. Five types of human tissue when freshly explanted in vitro were found to have varying but limited growth potential in contrast to the autonomous and apparently unlimited growth of the transformed cell. 2. A requirement for an external source of inositol or glutamine could not be demonstrated for the slowly multiplying primary amnions in contrast to the rapidly growing transformed cells which degenerated in the absence of either metabolite. 3. Unlike the transformed cells, the primary amnion cells fixed an insignificant amount of CO2; when infected by the Coxsackie virus, however, a requirement for CO2 in the formation of more virus became readily demonstrable. 4. The biggest difference in the incorporation of various C14 substrates occurred in the nucleic acid fractions. 5. The difference in the incorporation of glycine into the protein fraction was very small. 6. The utilization of glucose, ribose, and xylose as well as the oxidation of lactic acid into CO2 by the primary amnion cells occurred at a rate much lower than that of the transformed cells. 7. The cytotoxic dose of 5-fluorodeoxyuridine for the primary amnion was at least 100 times that of the transformed cells although growth inhibition was readily demonstrated at 0.1 µg. per ml. for both types of cells. Based on these observations, several hypotheses are put forth for consideration.

Nineteen lines of human fibroblasts were inoculated with a filtrate prepared from the ST feline sarcoma. Seven lines showed morphological alteration and released focus-forming activity for feline cells, 2 lines showed morphological alteration but did not release focus-forming activity, and 11 lines showed no morphological alteration and released no focus-forming agent. Morphologically altered cells appeared enlarged, hyper-refractile, and intensely stained by hematoxylin. They neither assumed a crisscross pattern nor piled up to form a visible focus. Time-lapse cinegraph showed that the morphologically altered cells did not divide and were motile. The fluid from two human fibroblast cultures, inoculated 4 and 14 weeks previously with the ST sarcoma filtrate, induced fibrosarcoma in newborn kittens.