Human Amnion Cell Cultures Research Articles

Tissue culture of the sera in human ulcerative colitis

1. 1. Control human sera had no effect on human amnion cell culture. 2. 2. The sera from nineteen of twenty-one patients with ulcerative colitis had a marked cytopathic effect on cell cultures, suggesting an autoimmune effect. The sera of three of these showed an additional cytopathic effect on human amnion cell culture, suggesting a viral-like quality. 3. 3. The sera from patients after total colectomy and proctectomy for ulcerative colitis had no effect on cell cultures. 4. 4. Rabbit antisera did not produce demonstrable antibodies or protect against the cytopathic effect in cell culture.

Action of heparin on some mitotic indices of transplantable cultures of human amnion cells (line A1)

The effect of heparin was studied on the mitotic indices of transplantable monolayer cultures of human amnion cells (strain A1). Inhibition of mitotic activity of the cultures was found to be directly proportional to the heparin concentration in the medium. High doses of heparin blocked mitosis at the stage of transition from late telophase to interphase of the next cell cycle. Meanwhile numerous amitoses appeared. The cells blocked in late telophase or which took part in amitosis underwent pycnosis and destruction.

Synthese und Umsatz von Ribonukleinsäuren in suspendierten Säugetierzellen / Synthesis and Turnover of Ribonucleic Acids in Suspended Mammalian Cells

The influence of ionizing radiation on the turnover of high molecular weight RNA in mammalian cells was studied. Growing cultures of suspended human amnion cells were labeled with 32P and irradiated with different doses of X-rays. RNA was isolated by hot phenol and the high molecular weight RNA fractionated by chromatography on methylated serum albumin celite columns. The salt gradient eluted RNA with sedimentation coefficients of 16 S or greater, which were characterized by analysis of nucleotide composition. Irradiation with doses from 900—6000 R results in degradation of pre-labeled RNA. A quantitative determination reveals that more 32P-radioactivity leaves the high molecular weight RNA compared to labeled total material that is degraded to acid soluble products. This indicates that RNA is partly broken down to low molecular weight fragments. Nucleotide analysis of the high molecular weight RNA fractions indicate a preferred degradation of ribosomal precursor RNA. The experimental results give evidence that the analysis of RNA synthesis for a certain period after irradiation is complicated by interfering processes like the simultaneous degradation of RNA.

Morphological changes and resistance to vaccinia virus induced in human amnion cells by yeast extract.

Summary Secondary cultures of human amnion cells developed characteristic morpho-logical changes when re-fed with media containing yeast extract. When cultures so treated were inoculated with vaccinia virus, the usual cytopathic effect failed to develop although isolated microplaques were occasionally observed after prolonged incubation. Some studies were made of the nature of the active substance and its mode of action. We wish to thank the Research Grants Committee of the United Manchester Hospitals for supporting this project.

Irradiation of Primary Human Amnion Cell Cultures: Effects on DNA Synthesis and Progression through the Cell Cycle

The effects of radiation on progression through the life cycle were studied in very slowly proliferating, morphologically pure, cells derived directly from normal human tissue. Owing to a low mitotic activity balanced by some cell loss, these cells could be maintained for 2 months in the same culture tubes without wide changes in net cell count. During the first 3 weeks of growth the population consisted primarily of slowly proliferating cells in which S and G2 were relatively short fixed phases of the life cycle, each lasting less than 24 hours. Because the estimated generation times averaged 14-20 days, most cells were in the presynthetic G1 phase. The most prominent finding which differentiated the response of these cells from that of rapidly proliferating mammalian cells was the marked radiosensitivity of the initiation of DNA synthesis. There was a dose-dependent decrease in the rate of flow of G1 cells into S with 10-100 R, and progression was almost completely stopped for 3-10 days by 300-1000 R. O...

Replication of varicella-zoster virus in cell culture: An ultrastructural study

In cultures of primary human amnion cells, varicella-zoster (V-Z) viruses first appear in nuclei as capsids which are freely dispersed, clustered, or in crystalline arrays. These capsids may be “empty” or contain several morphologically identifiable components including a dense core composed of deoxyribonucleic acid (DNA). Virions become morphologically mature when capsids containing DNA (nucleocapsids) acquire an envelope from the inner nuclear membrane during passage of the nucleocapsid to the cytoplasm. Once outside the nucleus, V-Z viruses appear pleomorphic, are often surrounded by a dense material, and exhibit a paucity of central dense cores. In thin sections, intact virions appear morphologically identical to other herpesviruses.

SV40 transformation of human amnion cells under different culture conditions.

SummarySV40 transformation of primary cultures of human epithelial amnion cells depended on multiplicity of virus exposure, culture age, and medium. Under optimal conditions transformed foci were observed 12 days after infection. Morphological transformation frequency was 10-4. The percentage of cells containing T antigen early after infection was higher than reported for SV40 infected human fibroblasts. Early SV40 production was pronounced and similar virus titers were observed after high and low virus multiplicity within 3 weeks. The amount of growth and the period of time before “crisis” depended on the transfer schedule, but also showed a direct relationship to the original multiplicity of virus exposure.

Differential response of rapidly and slowly proliferating human cells to X irradiation.

MOST STUDIES of the effects of ionizing radiations on mitosis and on the progression of mammalian cells through the replicative cycle in vitro have been carried out with rapidly proliferating cell systems, as have in vivo investigations in normal and tumor tissue. Very little is known specifically about these effects in slowly proliferating cells, although many tissue complexes are comprised of populations of slowly turning-over cells. For this reason, we have been studying the effects of x irradiation on progression through the life cycle in primary human amnion cell cultures in vitro (1, 2). These cultures are derived from amnionic membranes of normal human placentas delivered by cesarean section. They consist of morphologically pure diploid cells which, owing to their slow rate of proliferation, may be maintained in the same culture tube for several months without wide changes in cell count (3). In various experiments, the average generation time of the amnion cells during the first month, as calculated from the mitotic index and time, ranged from sixteen to thirty-one days; at any given time, more than 90 per cent of the cells in a culture were in the G1 or postmitotic resting phase. When primary human amnion cell cultures were exposed to 10–1,000 R of x rays, the effects on progress through the cell cycle and on the first postirradiation mitosis appeared similar to those observed in rapidly proliferating human cells, with one important exception: the initiation of DNA synthesis, usually considered a relatively radioresistant process. The rate of passage of G1 cells into the DNA-synthetic (S) phase was significantly depressed by exposures as low as lOR and completely blocked for five to ten days by 300–1,000 R (4). If such an effect occurred in slowly turning-over cell populations in vivo, it could be of considerable importance in determining their net response to fractionated or continuous irradiation. The object of the present investigation was to determine whether this effect on the initiation of DNA synthesis occurs in other slowly proliferating human cell systems or is peculiar to the primary amnion cells grown under the particular cultural conditions we have employed. Recently, Hahn and his co-workers (5) have shown that when monolayer cultures of Chinese hamster cells are allowed to become confluent, they enter a plateau phase of growth in which the cell concentration shows no further increase, despite regular replenishment of nutrient media. During this time the average cell generation time more than doubles, while the rate of DNA synthesis per cell falls to about 10 per cent of its former value. The latter finding has also been observed in other mammalian cells allowed to reach this growth stage (6, 7).

Rubella Virus

THE observations of Dr. N. McAlister Gregg in 1941, 1 linking the occurrence of rubella in early pregnancy and the congenital defects resulting in the newborn, stimulated renewed interest in the disease and in measures which might lead to its control. It was not until 1962, however, that recovery of the virus was accomplished by Weller and Neva 2 at the Harvard School of Public Health and by Parkman et al 3 at the Walter Reed Army Institute of Research. Each group worked independently and used different techniques. At Harvard, the presence of rubella virus was recognized in human amnion cell cultures by the appearance of a subtle cytopathic effect (CPE). At Walter Reed, it was found that rubella virus infected African green monkey kidney (GMK) cells were resistant to the CPE induced by echovirus type 11. These findings were rapidly confirmed by other investigators. 4-7 In this report these

Laboratory diagnosis of epidemic keratoconjunctivitis.

SUMMARY During an outbreak of epidemic keratoconjunctivitis in 1967, adenovirus type 8 was recovered from 57 of 186 patients investigated. Primary human embryo kidney and human amnion cell cultures were more efficient than either HeLa or rhesus monkey kidney cultures for isolation of this virus. Serological tests detected 21 additional infections. Haemagglutination inhibition, using human group-O cells, provided a convenient technique for detecting adenovirus-8 infections.

Thymidine kinase activity in human amnion cell cultures infected with Shope fibroma virus.

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Labile coat: reason for noninfectious cell-free varicella-zoster virus in culture.

Experiments designed to determine why cell-free varicella-zoster virus replicated in cell culture is noninfectious were performed. Electron micrographs in which varicella-zoster virus (a herpesvirus) was compared to herpes simplex virus in primary human amnion cell cultures showed that the viruses were morphologically indistinguishable inside the nucleus. However, extranuclear varicella-zoster viruses were distinguished from herpes simplex virus by the presence of pleomorphism, incomplete coats, and a resultant loss of central dense cores. This result indicates that varicella-zoster virus possesses a labile coat which is degraded outside the nucleus. It is suggested that the labile coat is a principal reason for the lack of cell-free infectious virus in this system.

Observations on the fine structure and replication of varicella virus in cultivated human amnion cells.

Serial propagation of varicella virus in tissue culture was accomplished first by Weller (1953) using human embryonic tissue in roller tubes, and later by Taylor-Robinson (1959) in stationary cultures of human fibroblasts and human amnion cells. However, the virus showed a strange anomaly in these systems in that serial transfer of the agent could not be achieved with culture fluids but only by means of intact infected cells. The virus was thus avidly cell-associated and its infectivity and stability appeared to be entirely dependent on cell integrity and viability (Gold, 1965); cell-disruption by any of a variety of procedures resulted in inactivation of the virus. In marked contrast, the virus in human skin vesicles was found to be mostly extracellular since cell-free filtrates of vesicular fluid sometimes had an infective titre almost as high as the unfiltered fluid (Taylor-Robinson, 1959).

The cytotoxic effect of fowlpox virus on primary human amniotic cell cultures.

This report describes a cytotoxic effect of fowlpox virus on human amnion cells. Changes began after 48 hours and were complete within a week following inoculation. The infective property and the cytotoxic property were compared. Suspensions which were cytotoxic nearly always contained at least 103 PFU/ml. Titers of both properties fell to undetectable levels during 30 days in human amnion cell culture. Titers of both properties were reduced by centrifugation, filtration, heat, ultraviolet light, acid, chloroform, and ether. Exposure to ultraviolet light reduced infectivity more rapidly than cytotoxicity. Treatment with ultrasound regularly increased cytotoxicity titer; infectivity increased to a much smaller and less predictable degree. Cytotoxicity-neutralizing serum antibodies developed in guinea pigs inoculated with fowipox virus. Cytotoxicity was identical in normal and IDUR-treated human amnion cells, but no plaques appeared in IDUR-treated chick embryo cells. The data suggest a mechanism of cytotoxicity that requires contact between virus and cell that does not require assembly of infectious viral particles.

Observations on the Transformation of Human Amnion Cell Cultures by Simian Virus 40<xref ref-type="fn" rid="FN2">2</xref>

Experiments were performed to evaluate the potential of human amnion cell cultures as a tool in the investigation of in vitro transformation of normal human cells. Simian virus 40 (SV40) was the transforming agent used. The amnion cultures were readily transformed by SV40, and the transformed foci were easily recognized. Study of environmental factors that may affect the process of transformation reveals the following positive findings: 1) X irradiation blocks cell transformation but not virus synthesis; 2) hydrocortisone delays the appearance of the transformed foci; 3) some older cultures are more easily transformed than some younger cultures, although a linear relationship between the in vitro age of a culture and its susceptibility to transformation cannot be demonstrated, and 4) overcrowding in a culture also may delay the appearance of transformed foci.

Transformation of epithelial cells in vitro.

DURING attempts to demonstrate replication of herpes simplex virus in cultures of human leucocytes1, we observed that supernatants from uninoculated leucocyte cultures induced a morphological transformation when added to primary cultures of human amnion cells. When the leucocyte culture fluid was added, the human amnion cells became transformed from epithelial to fibroblast-like cells. This leucocyte factor was produced in cultures of human blood cells which contained the mitogenic agent phytohaemagglutinin (PHA; phytohaemagglutinin-M, Difco), and was found only in the extracellular medium. Neither extracts of leucocytes nor supernatants of leucocyte cultures prepared without PHA possessed this activity. We have called this factor which induces metamorphosis, “MF”. Preliminary observations indicate that it is produced by lymphocytes.

Effect of interferon on deoxyribonuclease induction in chick fibroblast cultures infected with cowpox virus.

An exonuclease which degrades native deoxyribonucleic acid at pH 9.2 was induced in chick embryo fibroblast cultures and in human amnion cells by infection with cowpox virus. Highly purified chick embryo interferon suppressed the induction of the enzyme in the homologous cell system but not in the human amnion cell cultures. "Mock" interferon prepared from uninfected chicken eggs and purified in the same manner as biologically active interferon preparations had no effect on the induction of the enzyme.

Toxic effects of various penicillins and cephalosporin derivatives on human amnion cell cultures.

SummaryThe cytotoxicity of a number of penicillin and cephalosporin congeners for cultures of human amnion was determined. The least injurious compounds were found to be 7-aminocephalosporanic and 6-aminopeni-cillanic acids. Addition of a side chain to position 6 of the basic nucleus of penicillin or to position 3 or 7 of cephalosporanic acid appeared to be associated with an increase in capacity to injure cultured cells. Destruction of the antimicrobial activity of penicillin by treatment with penicillinase resulted in an increase in toxicity, while inactivation of cephalothin by exposure to cephalosporinase decreased the tissue damaging effect. These observations suggest a lack of relation between antibacterial effects and the potential for producing cellular injury. Addition of tetracycline to penicillin or cephalothin reduced the cytotoxicity of these agents.

On the mechanism of the cytopathic changes produced in human amnion cell cultures by the molluscum contagiosum virus

On the mechanism of the cytopathic changes produced in human amnion cell cultures by the molluscum contagiosum virus

In vitro cultivation of Eimeria acervulina (Coccidia)

Eimeria acervulina oocysts were sterilized with 50 ppm chlorine and viable sporozoites were released mechanically with the aid of trypsin and bile. These sporozoites, when suspended in the appropriate cell culture nutrient, were used as inoculum for chick embryo kidney, chick embryo fibroblast, mouse fibroblast, human amnion, and HeLa cell cultures. Infection of the host cell by sporozoites occurred in cultures of each cell type. Apparent cell infection occurred as early as one hour post inoculation, and forms recognized as trophozoites were observed at 18 to 24 hours. Further development failed to occur.