Characteristics of human arterial smooth muscle cell cultures infected with cytomegalovirus

Summary Distinct, sequential events occurring during the destruction and simultaneous regrowth of human arterial smooth muscle cell (SMC) cultures infected with cytomegalovirus (CMV, AD169 strain) were characterized. The events were influenced by the typical phenotypic diversity reflecting relative states of differentiation of the SMC cultures. Progenitors of regeneration were a surviving population of small, undifferentiated or relatively undifferentiated SMCs. As these cells reached confluence focally, the number of cells reactive with antismooth muscle serum, i.e. differentiating, increased, and in some postconfluent foci the organization of SMCs resembled the topography of uninfected cultures. Thus, infected SMC cultures had a limited capacity to repopulate, to organize typically, and to differentiate. However, continuing cytopathic effects gradually destroyed much of the regrowth, and a relatively large, nondividing SMC with prominent cytoplasmic filaments, similar to SMCs in terminal, uninfected cultures, predominated. Infected cultures consisting overwhelmingly of the large terminal phenotype were far less productive of infectious CMV than cultures populated by SMCs with continuing capacity to divide. Gradually, cultures consisting of the terminal phenotype deteriorated as a result of sporadic cytopathic effects of CMV and an effect resembling “senescent” degeneration in uninfected, nondividing cultures in late passage. The infected, terminal phenotype could be a latent or persistent source of CMV antigen or nucleic acid-positive cells detected by different investigators in normal and in atheromatous, human tissue, assuming that it exists and survives for an extended period in vivo after infection of vascular SMC. The derivation of smooth muscle cell lines used in this investigation was supported through National Research and Demonstration Center grant HL-17269-07 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

Previously unrecognized virus from submaxillary glands of gnotobiotic and conventional rats

Ashe, Warren K. (National Institute of Dental Research, Bethesda, Md.), Henry W. Scherp, and Robert J. Fitzgerald. Previously unrecognized virus from submaxillary glands of gnotobiotic and conventional rats. J. Bacteriol. 90:1719-1729. 1965.-A serially transmissible cytopathic agent was isolated from histologically normal submaxillary glands, but not from various other tissues or specimens, from 74 of 97 gnotobiotic and conventional rats. Triturates of the glands or subsequent culture supernatant fluids induced specific cytopathic effects (CPE) in monolayer cultures of primary rabbit kidney cells (14 passages), a line of human skin cells (8 passages), and HeLa cells (17 passages). Transfer of supernatant fluids containing infected cells enhanced transmissibility. Neutralization of the CPE was demonstrated with sera of gnotobiotic and conventional rats and with homologous rabbit antiserum. A cold hemagglutinin specific for rabbit erythrocytes is associated with, but separable from, the infectious particle. Cultures of the agent induced no discernible effect on inoculation by various routes into suckling, weanling, or adult conventional mice, rats, and hamsters. Weanling and adult rabbits were also unaffected. Cultures for bacteria in gland extracts and infected cell supernatant fluids were uniformly negative. Negative cultures on PPLO media and negative arginine deiminase tests indicated that this agent is not a Mycoplasma. The data indicate that it is a virus whose biological and physical properties distinguish it from the known cytomegaloviruses. Because it has been found so far only in rat submaxillary glands, this agent is designated provisionally as RSMG virus.     

Epitheliocystis,a new infectious disease of the bluegill (Lepomis macrochirus)

A new infectious, benign and chronic disease, histopathologically distinct from the viral disease lymphocystis, has been found in the bluegill. The symptoms are much enlarged cells in skin and gill epithelium, filled with basophilic granules. By electron microscopy these granules were shown to be organisms with the morphological characteristics ofBedsonia (Miyagawanella). During an attempt to isolate the agent in the bluegill fibroblast cell line BF-2, a cytopathic effect somewhat resembling the lesionsin vivo was found in the first passage. In later passages the agent was lost, but a true virus eliciting a distinctive fusiform cytopathic effect was recovered from these passages. The virus was found to be cytopathogenic also for the rainbow trout cell line RTG-2, but no recognizable disease could be evoked in young bluegills by inoculation of infected cell culture material. While the larger sized, non-viral organism is almost certainly the etiological agent of epitheliocystis, the role of the virus is, therefore, conjectural. Electron microscopy of thin sections has shown the virus to resemble influenza virus in morphology.     

Chicken kidney cell culture in medium without serum

When chicken kidney cell (CKC) culture in a petri dish was prepared in medium with or without serum and incubated in a humidified incubator at 38 degreesC with no addition of CO2, monolayers of CKCs were formed completely on the 5th day of cultivation. Growth medium used for CKC culture was Eagle's minimum essential medium containing 0.3% of dehydrated tryptose phosphate broth. The number of cells in both cultures prepared in medium with or without serum was the same when measured on the 5th day of cultivation. Monolayers of CKC culture prepared in medium with or without serum were maintained up to 21 days of cultivation, while maintenance medium was changed every 4th day. The time of appearance and degree of cytopathic effect, plaque-forming ability, and propagation of some avian viruses were similar in both cultures prepared in medium with or without serum.     

Virus Pneumonia of Pigs; Attempts at Propagation of the Causative Agent in Cell Cultures and Chicken Embryos

Two strains of the agent of virus pneumonia, were tested for the ability to propagate in 12 types of cell cultures and in chicken embryos. The 5 primary cell cultures used were: swine kidney, lung, bone marrow, testicle, and chicken embryo kidney; and the 7 serial passage cell cultures were: swine kidney, kidney-tumor, testicle, bone-marrow, bovine kidney, and human cervical carcinoma (HeLa). The agent of virus pneumonia was propagated in primary swine kidney and in HeLa cell cultures as shown by the production of typical gross and microscopic lesions in pigs inoculated with cell future fluids. Third passage cell culture fluids, produced typical gross lesions in pigs, but fourth passage cell culture fluids produced only microscopic lesions, and no lesions were produced by sixth and eleventh passage fluids. Control pigs receiving fluids from uninoculated cell cultures remained free of gross or microscopic lesions, as did uninoculated controls. Cytopathic effects were not detected in any of the inoculated cell cultures and no cellular changes were detected by staining with Giemsa stain or acridine orange. Neither lesions nor deaths occurred in chicken embryos inoculated with both strains of virus pneumonia virus. Pneumonia was not produced in pigs inoculated with suspensions from second chicken embryo passage of the 2 strains inoculated by the chorioallantioic sac, the amniotic sac, and the yolk sac routes. Identical gross and microscopic lesions were produced in pigs inoculated with either pneumonic lung suspensions or with virulent cell culture fluids. Gross lesions consisted of areas of light to reddish-purple consolidation usually limited to the anterior, cardiac, and intermediate lobes of the lungs. Pleuritis and pericarditis were never present in experimentally produced virus pneumonia. The microscopic lesions were characterized by: 1. perivascular and peribronchiolar lymphoid infiltration and hyperplasia, 2. alveolar interstitial thickening and infiltration, and 3. alveolar exudates consisting of alveolar cells, lymphocytes, plasma cells, and neutrophiles.     

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.     

Herpesvirus simiae infection in Macaca radiata

Forty of 79 bonnet macaques (Macaca radiata) housed in an outdoor structure became infected with a respiratory disease, and 16 died. The most conspicuous lesions were those of hemorrhagic interstitial lobar pneumonia and focal hepatic necrosis with monocytic infiltration and eosinophilic intranuclear inclusions. A virus, in high titer, was obtained from the lung and liver of two fatal cases (10⁷ TCID₅₀ × gram of tissue) by inoculating tissue homogenates in primary vervet monkey kidney, BSC-1, and MA104 cell cultures. The cytopathic effect was identical with that induced by Herpesvirus simiae in the same cell cultures. Similar cellular changes were seen in LLC-MK2 cell cultures. Infected cells contained eosinophilic intranuclear inclusions, and intranuclear herpes-like virus particles were seen by electron microscopy. The virus could not be passed serially in mice by the intracerebral route of inoculation. Bonnet monkeys (herpes antibody-free), inoculated intravenously with the virus, developed vesicular lesions on the arms, face, hands, and soles of the feet; and the virus was recovered from the vesicular fluid. All lesions disappeared within three weeks after inoculation, and the animals later recovered. On the basis of host range, cytopathic effect, electron microscopy, mouse susceptibility, and the results of neutralization tests in tissue cultures, the virus was identified as Herpesvirus simiae.     

INHIBITION OF DNA SYNTHESIS IN CHICK EMBRYO CULTURES BY DEPRIVATION OF EITHER SERUM OR ZINC

The rate of DNA synthesis in cultures of chick embryo cells is proportional to the concentration of serum added. The concentration of serum required to stimulate DNA synthesis increases with cell population density and with the duration of culture after trypsinization. The increase of the serum requirement with population density is not caused by the depletion of serum constituents. The requirement of cells for external zinc in DNA synthesis also increases with population density and duration of culture. The kinetics of inhibition of DNA synthesis by deprivation of serum or zinc are similar. Serum deprivation, however, inhibits 2-deoxyglucose uptake and cell movement, but zinc deprivation does not. The deprivation of either serum or zinc inhibits RNA synthesis about twofold. Very low concentrations of actinomycin D prevent the resumption of RNA and DNA synthesis upon restoration of serum or zinc to deprived cultures.     

Mycoplasmas in diseases of humans.

The roles of Mycoplasma pneumoniae, M. hominis and Ureaplasma urealyticum in diseases of humans are currently under investigation. M. pneumoniae, which causes primary atypical pneumonia, is a well established pathogen of the respiratory tract. Complications of infection by this organism are also being recognized; they include disorders of the hematopoietic, cardiovascular, central nervous, musculoskeletal, cutaneous and gastrointestinal systems. The roles of the genital mycoplasmas M. hominis and U. urealyticum are controversial but may include infections of the genitourinary tract and in pregnancy as well as diseases of the newborn, such as neonatal pneumonia and meningitis. In this review atypical pneumonia due to M. pneumoniae is described and the role of mycoplasmas in other diseases is discussed.     

细胞壁缺陷结核分支杆菌致细胞病变作用的观察

目的：探讨细胞壁缺陷结核分支杆菌的致细胞病变作用。方法：用利福平诱导结核分支杆菌形成稳定L型后感染Vero细胞,直接在显微镜下和抗酸染色观察细胞病变情况以及结核分支杆菌同宿主细胞的关系。结果：Vero细胞受结核分支杆菌L型感染72h后形成空泡、变圆、脱落和裂解,结核分支杆菌稳定L型细胞粘附或侵入细胞内。结论：细胞壁缺陷结核分支杆菌仍然能够引起Vero细胞发生病变但致细胞病变的作用较其亲代细菌型明显减弱,L型能够粘附于宿主细胞表面或进入宿主细胞内生长繁殖,引起缓慢的细胞病变。     

Elimination of mycoplasma contaminants from cell cultures with animal serum

Repeated treatment with guinea pig or rabbit serum, but not with human serum, was found to eliminate mycoplasma contaminants from mammalian cell cultures as judged by staining with the fluorescent dye Hoechst 33258. Following treatment with rabbit serum and several passages, M. hyorhinis could not be detected by staining, isolation on agar, or specific immunofluorescence in a human prostate carcinoma cell line heavily contaminated with this organism. There was no evidence for the involvement of antimycoplasma antibodies in the bactericidal activity of rabbit serum. Mycoplasmacidal activity of rabbit serum was associated with a heat-labile component(s) which could be inactivated by incubation of the serum with goat antirabbit complement component C3.     

Direct effects of IL-4 on the in vitro differentiation and proliferation of hematopoietic progenitor cells

The purpose of this study was to analyze the effects of recombinant human interleukin 4 (IL-4) on the differentiation and proliferation in vitro of human granulocyte/macrophage (GM) and erythroid progenitors. IL-4 was added to either fetal bovine serum (FBS)-supplemented or to FBS-deprived cultures of unfractionated human marrow cells or marrow cells depleted of adherent and/or T cells. Paradoxical effects similar to those reported in the murine system were detected in these experiments. In FBS-supplemented cultures, IL-4, which had no effect on the growth or erythroid bursts (from burst-forming cells; BFU-E) detected in the presence of Epo alone, decreased by 46% the number of erythroid bursts detected in the presence of Epo and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). In contrast, in FBS-deprived cultures, IL-4 increased by 30-700% the number of erythroid bursts in cultures containing Epo alone or containing Epo, IL-3, and GM-CSF. The stimulatory effect of IL-4 on erythroid burst growth under FBS-deprived conditions was particularly evident when adherent cells were removed. Under the conditions investigated, IL-4 had little effect on the growth of GM colonies. In FBS-deprived suspension cultures of nonadherent, T-cell-depleted marrow cells, IL-4 maintained both the number of BFU-E and CFU-GM for at least 8 days. In these cultures, IL-4 antagonized the capacity of IL-3 to increase the number of BFU-E but IL-4 and IL-3 acted together to maintain the number of CFU-GM. To determine if IL-4 acted directly or indirectly, its effects on the growth of factor-dependent subclones of the murine progenitor cell line 32D were analyzed. Three subclones were studied: the original IL-3-dependent clone 32D cl.3, the Epo-dependent erythroid clone 32D Epo-1, and the G-CSF-dependent myeloid clone 32D G-1. IL-4 alone failed to induce colony growth from these cell lines. However, IL-4 inhibited by 25% the number of colonies formed by 32D cl.3 in the presence of IL-3 while increasing by 25% and 25-50% the number of colonies formed by 32D Epo-1 and 32D G-1 in the presence of Epo or G-CSF, respectively. These results indicate that human IL-4, as its murine counterpart, is a multilineage growth factor with paradoxical effects which are mediated by the direct action of IL-4 on progenitor cells.     

REVERSAL OF d-CYCLOSERINE INHIBITION OF BACTERIAL GROWTH BY ALANINE.

Zygmunt, Walter A. (Mead Johnson & Co., Evansville, Ind.). Reversal of d-cycloserine inhibition of bacterial growth by alanine. J. Bacteriol. 84:154-156. 1962.-Reversal of the antibacterial activity of d-4-amino-3-isoxazolidone by alanine in bacterial cultures actively growing on chemically defined media was compared in cultures requiring exogenous alanine and those capable of its synthesis. dl-Alanine was the most effective reversal agent in Pediococcus cerevisiae, an alanine-requiring organism, and d-alanine was effective in Escherichia coli and Staphylococcus aureus, organisms synthesizing alanine. With all three cultures, l-alanine was the least effective reversal agent.     

Pleuropneumonialike organisms in tissue cultures

Of 55 continuous cell lines 32 gave growth of P.P.L.O. whereas 26 primary cell cultures were free from this contamination.Biochemical and serological typing proved that 31 of these 32 P.P.L.O. wereMycoplasma hominis I. One strain was identical with a recently described oralMycoplasma.It was demonstrated that insufficiently rigorous techniques tend to cause spreading of P.P.L.O. in tissue culture laboratories.There was no indication that either the sera or other ingredients of the media used might have introduced this contamination. HeLa cells, however, probably are the source.The minor differences between genital strains propagated in the laboratory, and tissue culture strains, are probably due to differences between the two media.By treatment of a contaminated cell line with serum againstM. hominis I a double infection with P.P.L.O. could be demonstrated. The cells were freed from the remaining P.P.L.O. by treatment with the serum against this strain.Contamination of cell lines withM. hominis I did not affect the growth rate of the wild poliovirus I strains tested, nor that of a Sabin type I strain. M. fermentans grows well in tissue cultures but has no cytopathic effect.M. salivarium cannot be propagated in ordinary tissue cultures unless Fildes extract, which contains catalase, is added. In cultures with this extractM. salivarium has a cytopathic effect.     

Viral transgenesis of embryonic cell cultures from the freshwater microcrustacean Daphnia

The aim of this study was to determine whether a recombinant vesicular stomatitis virus (VSV) vector encoding a transgene could be used to infect and express a foreign gene in embryonic primary cell cultures derived from the freshwater microcrustacean Daphnia, the most widely used ecotoxicological model organism. To facilitate the evaluation of gene transfer, a reproducible method for establishing primary cultures from Daphnia embryonic tissues was developed. Within 24 hr after infection, transgene expression could be detected in cell culture. VSV was found to replicate in the cells with no apparent cytopathic effect. Here we report the first evidence of gene transfer and foreign gene expression in cultures of Daphnia embryonic cells using a recombinant viral vector.     

Failure to detect interfering agents in febrile respiratory illnesses

Throat swab specimens from 57 military recruits with febrile respiratory illness in whom no virologic or serologic evidence of infection with respiratory viruses orMycoplasma pneumoniae was found were inoculated and passaged into fresh cultures of monkey kidney cells and continuous lines of human, simian and rabbit cells. Cultures of second passage were challenged with poliovirus type 2 or vaccinia virus and subsequently tested for interference by reading of cytopathic effect and titration of challenge virus. No interference was shown.     

Causes of atypical pneumonia: results of a 1-year prospective study.

In a protocol study of cases of atypical pneumonia over a 1-year period an etiologic agent was established in 16 cases: Legionella pneumophila in 8, Coxiella burnetii in 3, Chlamydia trachomatis in 2, Mycoplasma pneumoniae in 1, para-influenza 3 virus in 1 and cytomegalovirus in 1. In the remaining 11 cases no agent was identified; the illnesses in these cases tended to be less severe. The pneumonia took much longer to resolve in the patients with Legionnaires'' disease than in all the other patients (mean interval from onset of symptoms to clearing of the chest roentgenogram: 69 days v. an average of 16 days). However, the length of stay in hospital was similar for the three groups: those with Legionnaires'' disease, those with atypical pneumonia of unknown cause and those with atypical pneumonia of various other established causes. L. pneumophila infection may explain a proportion of atypical pneumonias that previously could not be diagnosed, although in this series the cause of 41% of the pneumonias remained unexplained.     

Pitfalls in the diagnosis of herpes simplex infection in respiratory cytology

OBJECTIVE: To evaluate the prevalence and potential pitfalls in making an accurate diagnosis of respiratory herpetic infection. STUDY DESIGN: Eighteen cases with the diagnosis of herpes simplex virus (HSV) infection were identified from a total of 7,501 (0.24%) respiratory specimens. All cases were evaluated for classic cytomorphologic features of HSV infection and associated cytologic findings. The parameters studied included number of cells with HSV cytopathic effect, intranuclear inclusions, multinucleation, presence of atypical squamous cells, reparative changes, presence and degree of inflammation and associated obscuring factors. RESULTS: Only a minority of cases (28%) had numerous cells with classic viral cytopathic change. Four (22%) of 18 cases showed atypical squamous cells, and 5 (28%) revealed reparative changes. The majority of the cases were associated with inflammation, which was severe in 4 cases (22%). Blood and degenerative changes obscured the cytologic findings in 3 cases (17%). One case showed a necrotic background. CONCLUSION: Due to the low prevalence of HSV infection in respiratory cytology, a high index of suspicion is necessary for an HSV diagnosis. Pitfalls for a false negative diagnosis include limited number of cells with viral cytopathic change, only mononuclear cells with viral changes and obscuring inflammation or blood. Pitfalls for a false positive diagnosis of malignancy include atypical keratinized squamous cells, atypical repair, cellular degeneration and necrotic background.