Superantigen activation and kinetics of cytokines in the Long-Evans rat.

This study was conducted to identify and quantify, over time, selected cytokine responses in Long-Evans rats that were exposed to staphylococcus enterotoxin B (SEB). The kinetics of selected cytokines [interleukin-2 (IL-2), IL-6, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF)] and phenotype and cell cycle analysis of T lymphocytes were determined in Long-Evans rats administered a single intraperitoneal (i.p.) dose of either 50 microg or 500 microg of SEB. Rats injected with 50 microg SEB had significantly elevated levels of IL-2, IL-6 and IFN-gamma in their serum 2 hr post-injection. IL-2 serum levels were significantly elevated at 2 hr and returned to near control values by 12 hr while both IL-6 and IFN-gamma peaked at 6 hr but remained significantly increased at 24 hr post SEB exposure. A 500 microg dose of SEB did not further enhance these cytokine responses. When spleen cells were collected for culture 2 hr after rats were injected i.p. with 50 microg SEB and cocultured with SEB, TNF and IL-6 levels were significantly increased after 2 hr incubation, while IL-2 and IL-6 were significantly elevated at 6 hr. Production of all these cytokines in spleen cell cultures continued to increase over the 24 hr sampled. Peritoneal cells were collected for culture either at 1 hr or 2 hr after injection of either 50 microg or 500 microg of SEB. IL-6 was significantly increased after 1 hr in culture while TNF was significantly increased by 2 hr regardless of whether the cells were harvested 1 or 2 hr after SEB injection. The greatest response for both IL-6 and TNF occurred when cells from animals injected with 50 microg SEB were restimulated in vitro with SEB. The peak levels for IL-6 were at 12 hr post SEB exposure while TNF peaked at 6 hr. The percentage of CD4+ cells was significantly increased at 48 hr and 72 hr post SEB (50 microg) administration while the percentage of CD8+ cells remained similar to control values for the 168-hr test period. A similar pattern was observed in cell cycling where the CD4+ cells proliferated up to 2 days post SEB injection and then were significantly suppressed at day 3. The CD8+ cells were comparable to control values. These studies demonstrate that the cytokine responses in Long-Evans rats exposed to a superantigen are somewhat similar to those that occur in mice and humans, e.g. a rapid short increase in the production of IFN-gamma and TNF that was accompanied by an increase in the production of IL-2. Additional responses noted in this species, however, were a marked increase in IL-6 production, as well as an early increase in the number and cycling of CD4+ cells followed by a down-regulation of these events. These activities occurred in the absence of notable histopathological alteration of lymphoid organs. The results indicate that the Long-Evans rat is an acceptable animal model to investigate the pathogenesis of superantigen-induced disease and that IL-6 may be an active mediator of this process.     

Glycine-like immunoreactivity in the cerebellum of rat and Senegalese baboon,Papio papio: a comparison with the distribution of GABA-like immunoreactivity and with [3H]glycine and [3H]GABA uptake

Summary An antiserum against conjugated glycine was characterized and applied to cerebellar sections of rats and baboons that had been perfusion-fixed with glutaraldehyde. After immunosorbent purification the serum reacted with brain protein-glutaraldehyde-glycine conjugates, but did not stain similar test conjugates prepared from other amino acids, including GABA and -alanine. In the rat cerebellum the glycine antiserum selectively labelled a subpopulation of Golgi neurons. Adjacent Vibratome sections treated with an antiserum against conjugated GABA revealed an about equally large subpopulation of immunopositive Golgi cells. A proportion of the Golgi cells that were cleaved by the plane of section contained both immunoreactivities. Additional evidence for a colocalization of glycine and GABA was obtained by postembedding staining of alternate semithin sections with the GABA antiserum and glycine antiserum, respectively. The ability of the antisera to distinguish between fixed glycine and GABA was corroborated by preincubation of the antisera with glutaraldehyde-amino acid fixation complexes: glycine complexes abolished staining with the glycine antiserum but had no effect on the GABA antiserum. The opposite effects were obtained with the GABA complexes. Matching the distributions of the respective immunoreactivities, [³H]glycine uptake was restricted to glomerulus-like structures in the granule cell layer whereas [³H]GABA uptake also occurred in punctate and fibrous profiles in the molecular layer. The baboon showed a distribution of glycine-like immunoreactivity similar to that in the rat, except that a few immunopositive neurons occurred in the molecular layer. The latter neurons were interpreted as outlying Golgi neurons; however, the possibility that they represent a subpopulation of basket cells could not be excluded. The Purkinje cells were negative in both species. Glial cells were weakly stained with the glycine antiserum but were strongly immunopositive after incubation with an antiserum raised against conjugates of the structurally similar amino acid -alanine. The present data suggest that glycine and GABA occur in about equally large subpopulations of Golgi neurons. A subpopulation of the Golgi neurons appears to contain both glycine and GABA.     

The protective effects of hyperimmune anti-murine cytomegalovirus antiserum against lethal viral challenge: the case for passive-active immunization

The administration of 0.2 ml of hyperimmune anti-mouse cytomegalovirus (CMV) antiserum intraperitoneally (ip) or intravenously provided complete protection against lethal challenge (10(5.8) PFU ip) with murine CMV. Antiserum protection was complete when the antiserum was administered as long as 24 hr after viral challenge. The administration of antiserum had little effect on the titers of virus in the organs of these animals. Ammonium sulfate-treated antiserum provided similar complete protection. Animals rechallenged with 10(6)-10(6.5) PFU of murine CMV 1 month after initial challenge, at a time when the administrated antiserum was no longer detectable, all survived. We conclude that hyperimmune antiserum can provide significant protection against otherwise lethal murine CMV infection, that the protecting material lies within the immunoglobulin fraction, and that long-term immunity results from the combined exposure to virus and antiserum. Such passive-active protection could be useful in protecting against human CMV infection.     

GnRH in the spinal subarachnoid space potentiates lordosis behavior in the female rat

Gonadotropin releasing hormone (GnRH) (100 ng) infused directly into the spinal subarachnoid space via a chronically implanted catheter, induced a prompt facilitation of lordosis behavior in estrogen-primed ovariectomized female rats. Facilitation occurred within 5 min and lasted for 3 1/2 hr. This effect of GnRH could be blocked by a GnRH antagonist analog, [D-Phe2, Pro3, D-Phe6] GnRH (250 ng) which also completely abolished lordosis within 2 1/2 hr. The antagonist analog (250 ng) but not a high titre specific anti-GnRH antiserum (enough to neutralize 24 micrograms of GnRH) abolished lordosis when infused alone in the subarachnoid space in estrogen-primed rats. The antagonist but not the anti-GnRH antiserum also significantly suppressed lordosis in estrogen-progesterone primed female rats. Supporting experiments showed that the effects of GnRH and its antagonist may not be due to their effects on supraspinal structures by diffusion from the spinal subarachnoid space. These results may indicate a direct effect of GnRH and its antagonist at the level of the spinal cord and may suggest a possible role for GnRH in the processing of somatosensory information necessary for the trigger of lordosis.     

Specific elimination of the T lineage cells: Effect of in vitro treatment with anti-Thy 1 serum without complement on the adoptive cell transfer system

Thymocytes from C57BL/6(B6) mice treated with anti-Thy 1 antiserum without complement in vitro were transferred to lethally irradiated AKR mice. Five days following transfer, the proportion of Thy 1.2(+) cells recovered from the recipient spleen was significantly lower (7%) than that from the control mice which had received untreated cells (64%). The B6 spleen cells were treated in the same manner and transferred with SRBC (T-dependent antigen) or DNP-Ficoll (T-independent antigen) to irradiated syngeneic recipients. The recipients developed a response to SRBC which was significantly lower than that observed in control mice, but showed the same number of plaque-forming cell (PFC) against TNP-SRBC as the control group of mice which had received untreated B6 spleen cells.These results clearly show that in vitro pretreatment of lymphocytes with anti-Thy 1 serum without complement specifically resulted in elimination or inactivation of the T lineage cells in the host environment. The mechanisms of the elimination are discussed in this study.     

Distinguishing characteristics of interferon induction with poly(I)-poly(C) and Newcastle disease virus in human cells.

Exposure of a human diploid foreskin cell strain (FS-4) to polyinosinate-polycytidylate [poly(I)·poly(C)] resulted in an early rise of interferon production that peaked at about 4 hr after induction and decreased rapidly thereafter. Irradiation of cells with low to moderate doses of ultraviolet (uv) light immediately before induction with poly(I)·poly(C) increased the amount of interferon produced up to about tenfold. This enhancement was apparently due to interference with the shut-off process which in unirradiated cells leads to early termination of interferon production; in irradiated cells interferon production continued for much longer.Inoculation of FS-4 cells with Newcastle disease virus (NDV) resulted in interferon production that showed a slower rise and peaked only at about 10–15 hr after inoculation. Irradiation of cells at the time of induction with NDV resulted in a dose-dependent decrease of interferon production. However, a small fraction of the total amount of interferon produced in response to NDV, which appeared by about 5 hr after virus inoculation, was resistant to uv. This uv-resistant early peak of NDV-induced interferon was greatly enhanced in cells which 6 hr before virus inoculation had either been induced with poly(I)·poly(C) or incubated with interferon, while the appearance of the major, uv-sensitive peak of NDV-induced interferon was inhibited or delayed after the same treatments. In its characteristics the early peak of NDV-induced interferon resembled the poly(I)·poly(C)-induced interferon response. Poly(I)·poly(C)-induced, as well as the early and late NDV-induced interferons were all neutralized by an antiserum raised against poly(I)·poly(C)-induced interferon, suggesting that they represent products of the same structural gene(s). It is concluded that there may be more than one mechanism of interferon induction by a single virus.     

Production of interleukin-1 by draining lymph node cells during the induction phase of contact sensitization in mice.

Biologically active interleukin-1 (IL-1) has been detected in supernatants of draining lymph node cells isolated from contact-sensitized mice. Induction of IL-1 was dependent upon the concentration of sensitizer applied and occurred within 2 hr of exposure. The IL-1 activity could not be attributed to other interleukins and was neutralized by a specific antiserum. Reduced concentrations of IL-1 were produced by cells isolated from the draining nodes of mice that had been pre-exposed to the sensitizer. Since IL-1 has the potential to influence several aspects of lymphocyte activation, the production of significant concentrations of biologically active IL-1 by draining lymph node cells indicates that it is likely to play an important role in the afferent phase of contact sensitization.     

Hepatotoxicity of d-Galactosamine in the isolated perfused rat liver

The hepatotoxicty of d-galactosamine was evaluated using the isolated perfused rat liver. In controls, ATP- and UDPglucose-levels decreased by about 20% during 12 hr of perfusions; UTP + UDP as well as the energy charge remained constant. d-Galactosamine reduced the contents of UDPglucose and UTP + UDP to 10 to 20% of normal within 30 min, whereas the sum of all acid-soluble uracil nucleotides increased linearly for the first 8 hr. The leakage of intracellular enzymes into the perfusion medium was small and almost linear in controls; in livers treated with d-galactosamine, however, a sharp increase occurred after 6 hr. Addition of uridine 3 hr after d-galactosamine prevented cell damage as judged by enzyme release. E. coli endotoxin, either alone or after preincubation with rat serum, did not enhance enzyme leakage. Lightmicroscopically, liver cell plates were partially dissociated and necroses of groups of liver cells were found in galactosamine-treated livers. In electron microscopy, degenerative changes of hepatocytes were seen after galactosamine perfusion.     

Impact of cell cycle delay on micronucleus frequency in TK6 cells

Previous studies with TK6 cells have shown that extending the recovery period after pulse treatment allows for greater micronucleus expression for some compounds. This study explores the role of cell cycle delay in micronucleus expression after pulse treatment with three model genotoxins [mitomycin C, etoposide (ETOP), vinblastine]. Cells were treated for 4 hr and allowed to recover for 36 hr with samples removed at various time points during the recovery period and analyzed for cell cycle distribution, apoptosis and micronucleus frequency. Our results show that mitomycin C causes cell cycle delay for 20 hr after pulse treatment and cell cycle perturbation is no longer evident after 36 hr of recovery. The micronucleus frequency of cells sampled at 36 hr is doubled when compared with cells sampled at 20 hr after mitomycin C removal. When cells were treated with indirect acting genotoxins (ETOP, vinblastine), cell cycle perturbation was not observed at the 20 hr time point. Micronucleus frequency after treatment with either ETOP or vinblastine did not differ between the 20 hr and the 36 hr time point. All three compounds induced similar levels of apoptosis ranging from 4.5 to 5.6% with maximum induction occurring at the 36‐hr time point. We conclude that TK6 cells exhibit extended cell cycle arrest after exposure to MMC and can go on to express micronuclei, after overcoming cell cycle arrest. Environ. Mol. Mutagen. 55:64–69, 2014. © 2013 Wiley Periodicals, Inc.     

The effects of insulin replacement and withdrawal on hepatic ultrastructure and biochemistry

This study examines the early hepatic biochemical and ultra-structural responses to insulin replacement in streptozotocin-diabetic rats and insulin withdrawal from insulin-maintained diabetic rats. Insulin administration rapidly lowered plasma glucose and the elevated glucose-6-phosphatase (G-6-Pase) specific activity of the diabetic rats. However, hepatic glycogen did not increase until after 3 hr of insulin treatment. Hepatic ultrastructure responded to insulin replacement after the decline in glucose and G-6-Pase. This was seen in periportal hepatocytes as a reduction in the close association between smooth endoplasmic reticulum (SER) and glycogen particles in the diabetic animals. The treated rats showed hepatic SER restricted to the periphery of glycogen masses, as is characteristic of these cells from normal rats, in many cells by 6 hr and all cells by 18 hr. Insulin withdrawal from insulin-treated diabetic rats elicited nearly a total reversal of the above events. Plasma insulin declined to a value half that of the normal rats by 6 hr after withdrawal; concurrently, plasma glucose rose sharply to hyperglycemic values as hepatic glycogen content dropped. Following the rise in plasma glucose and fall in glycogen content, G-6-Pase specific activity increased and by 16 hr reached the high values characteristic of the diabetic animal. Hepatic ultra-structure was also changed as evidenced by an intrusion of elements of the SER into the dense glycogen masses; the result was dispersed glycogen closely associated with SER as seen in the diabetic animal. It is concluded that the hepatic response to insulin replacement in diabetic animals and diabetic onset in insulin-withdrawn animals is rapid and occurs through defined stages.     

Serological analysis of wheat striate mosaic virus and its soluble antigen

An antiserum prepared against purified wheat striate mosaic virus (WSMV) reacted specifically with WSMV-soluble antigen present in the supernatant obtained after removing the virus from the crude sap of infected wheat plants (Triticum durum). In agar gel diffusion tests, the supernatant and also the soluble antigen purified by an acid precipitation method produced one band midway between the antigen and antiserum wells in 24–36 hr. Recovery of WSMV-soluble antigen after purification was estimated to be only 10%.Purified WSMV at concentrations ranging from 0.75 to 6 mg/ml produced one band close to the antigen well in 3–5 days. Partially purified virus preparations, however, produced three bands at concentrations of either 3 or 6 mg/ml, and one at 0.75 and 1.5 mg/ml. Both purified and partially purified virus treated with lipase, sodium deoxycholate, sodium dodecyl sulfate, or Tween-ether produced one band midway between the antigen and antiserum wells in 24–48 hr when the final concentration of treated virus was 0.188 mg/ml. With a final virus concentration of 0.375 mg/ml, treatment with lipase produced two bands, with Tween-ether, three bands, and with other agents, only one band.     

大蒜乳剂对小鼠S180腹水癌细胞作用的电镜和电镜细胞化学观察

本文证实大蒜乳剂具有显著抑制肿瘤的作用,荷瘤小鼠的生命延长率和抑瘤率分别为81％和72％。在此基础上,我们利用电镜和电镜细胞化学技术,观察大蒜乳剂对S180腹水癌细胞超微结构及几种酶活性的影响。单次用药后2、6小时,部分肿瘤细胞表面微绒毛肿胀、脱落,核染色质浓缩,胞质内出现大量空泡;48小时后,肿瘤细胞膜表面微绒毛肿胀,脱落现象仍很严重,但细胞核和细胞质的受损程度已经减弱;72小时受损肿瘤细胞恢复正常。连续4次给药后,肿瘤细胞数量明显减少,细胞体积变小,部分肿瘤细胞的损伤程度与单次用药后6小时基本相似。连续4次给药后,肿瘤细胞的碱性磷酸酶(AlPase)、5核苷酸酶(5Nase),葡萄糖6磷酸酶(G6-Pase)活性下降。实验结果证明,大蒜乳剂对小鼠S180腹水癌细胞有明显的细胞杀伤作用。     

Labeling of hepatic glycogen after short- and long-term stimulation of glycogen synthesis in rats injected with 3H-galactose

The effects of short-and longterm stimulation of glycogen synthesis elicited by dexamethasone were studied by light (LM) and electron (EM) microscopic radioautography (RAG) and biochemical analysis. Adrenalectomized rats were fasted overnight and pretreated for short- (3 hr) or long-term (14 hr) periods with dexamethasone prior to intravenous injection of tracer doses of ³H-galactose. Analysis of LM-RAGs from short-term rats revealed that about equal percentages (44%) of hepatocytes became heavily or lightly labeled cells increased slightly 6 hr after labeling, and unlabeled glycogen became apparent in some hepatocytes. The percentage of heavily labeled cells had decreased somewhat 12 hr after labeling, and more unlabeled glycogen was evident. In the long-term rats 1 hr after labeling, a higher percentage of heavily labeled cells (76%) was observed compared to short-term rats, and most glycogen was labeled. In spite of the high amount of labeling seen initially, the percentage of heavily labeled hepatocytes had decreased considerably to 55% by 12 hr after injection; and sparsely labeled and unlabeled glycogen was prevalent. The EM-RAGs of both short- and long-term rats were similar. Silver grains were associated with glycogen patches 1 hr after labeling; 12 hr after labeling, the glycogen patches had enlarged; and label, where present, was dispersed over the enlarged glycogen clumps. Analysis of DPM/mg tissue corroborated the observed decrease in label 12 hr after adminstration in the long-term animals. The loss of label observed 12 hr after injection in the long-term pretreated rats suggests that turnover of glycogen occurred during this interval despite the net accumulation of glycogen that was visible morphologically and evident from biochemical measurement.     

Effects of heterologous anti-lymphocyte serum on the distribution of 51Cr-labelled lymph node cells in mice

The effects of heterologous anti-lymphocyte serum (ALS) were studied in a syngeneic cell transfer system, in which lymph node cells from donor CBA mice were labelled in vitro with ⁵¹Cr and transferred intravenously into syngeneic recipients. Labelled cells treated in vitro with ALS were unable to migrate to lymph nodes or spleens of recipients, as did normal cells, but instead distributed themselves very similarly to cells which had been killed by exposure to heat. It is thus likely that cells treated in vitro with ALS are killed after transfer by the cytotoxic action of ALS mediated by the complement of the recipient.

ALS administered directly to the recipients of labelled lymphocytes could also reduce their uptake into lymphoid tissue; however, the magnitude of this effect appeared to be critically dependent upon the timing of the antiserum dose with respect to the labelled cell dose. ALS given immediately prior to labelled cells showed the greatest effect, while treatment given either 24 hours before or after the labelled cells was much less effective. While with prior treatment the reduced effect could be due to a fall off in antibody titre during the interval between the dose of antiserum and cells, in the latter situation no drop in titre would have occurred. It thus seems that lymphocytes that have already established themselves in lymphoid tissue may be less susceptible to the action of ALS.

ALS given chronically to lymphoid cell donors resulted in a population of cells which upon transfer to normal recipients were distributed differently from either normal or NRS-treated donor cells.

These data support the hypothesis that the effects of ALS may be exerted preferentially on circulating lymphocytes, and that ALS may act to selectively reduce the representation of this cell type in lymphoid tissue.

     

Mechanism of interferon action. Kinetics of interferon action in mouse L929 cells: translation inhibition, protein phosphorylation, and messenger RNA methylation and degradation

The effect of the duration of interferon treatment of mouse L929 fibroblasts on the expression of ribosome-associated and cell-sap-localized translation inhibition, protein phosphorylation, and messenger RNA (mRNA) methylation and degradation was investigated. The following results were obtained: (1) Ribosomal salt-wash fractions prepared from L929 cells treated for 12 or 18 hr significantly inhibited the translation of methylated reovirus mRNA catalyzed by the cell-free system prepared from untreated ascites tumor cells. The translation of reovirus mRNA was slightly stimulated by the ribosomal salt-wash fractions prepared from untreated cells and cells treated for 2 hr and was slightly inhibited by the salt-wash fraction from cells treated for 6 hr. (2) Cell-sap fractions prepared from untreated cells and from cells treated for 2 or 6 hr did not appreciably affect the translation of reovirus mRNA in the absence of double-stranded RNA (dsRNA); cell-sap fractions prepared from cells treated for 12 or 18 hr were slightly inhibitory. (3) Neither reovirus genome dsRNA nor polyriboinosinic acid-polyribocytidylic acid inhibited the translation of reovirus mRNA by the untreated ascites system. The inhibitory activity of ribosomal salt-wash fractions from interferon-treated cells was dsRNA independent, whereas dsRNA enhanced the inhibitory activity of cell-sap fractions from interferon-treated cells. (4) Interferon treatment significantly enhanced a cell-sap-independent phosphorylation of at least three proteins present in the ribosomal salt-wash fractions. Phosphorylation of an approximately 50,000-dalton component was maximally enhanced after 12 hr of interferon treatment and was increased by dsRNA; phosphorylation of 30,000- and <20,000-dalton components was enhanced earlier and was dsRNA independent. (5) An increase in the nuclease-mediated degradation of [3H]uridine-labeled methylated reovirus mRNA catalyzed by ribosomal salt-wash fractions was detectable after 6 hr of interferon treatment and was maximally enhanced after 12 hr of interferon treatment to about threefold the level of the untreated ribosomal salt-wash fraction. The degradation was not enhanced by dsRNA. The levels of reovirus mRNA degradation catalyzed by cell-sap fractions from untreated cells and from cells treated with interferon for 2 to 18 hr were comparable. (6) No significant difference was observed in the ability of cell-sap fractions from untreated cells or from cells treated with interferon for 2 to 18 hr to catalyze the methylation of unmethylated reovirus mRNA. In addition, none of the ribosomal salt-wash fractions significantly inhibited reovirus mRNA methylation catalyzed by untreated ascites cell-free extracts. These results suggest that interferon treatment may mediate multiple biochemical changes in murine cells, including the specific phosphorylation of protein(s) associated with ribosomes that possess interferon-mediated inhibitor(s) of viral mRNA translation.     

Mechanically altered embryonic chicken endothelial cells change their phenotype to an epithelioid phenotype

Monolayers of retracted endothelial cells exhibiting wounds or zones denuded of cells were obtained from aortic explants from 10- to 12-day-old chicken embryos. Using time-lapse videomicroscopy, we investigated the sequence of events that occurred both during and after closure of the monolayer wounds. Such wound closure (re-endothelialization process) occurred 4-12 hr after removing the explants, depending on wound width and presence of serum. The cells from along the wound edges appeared to move toward one another. We suggest an important role for bFGF and TGFbeta-2 and -3 during this process. Twenty-five hours after removal there were still some areas of retracted cells, and many of the cells displayed a weak von Willebrand's Factor (vWf) immunoreactivity. Surprisingly, after 63-65 hr many of the endothelial cells had become epithelioid in shape and the vWf immunoreactivity appeared increased. This epithelioid phenotype is currently considered typical of cultured vascular non-muscle-like cells and intimal thickening cells. By 5-7 days, the vast majority of cells in the monolayer had acquired an epithelioid morphology, showing a cobblestone appearance. These cells were significantly smaller than polygonal cells. Most importantly, they showed strong vWf immunoreactivity. At the edge of the monolayers we found that the majority of the cells had become epithelioid. Some of them detached from their neighbors and became round in shape and acquired mesenchymal characteristics, some expressing smooth muscle alpha-actin (SM alpha-actin). These findings demonstrate not only that embryonic endothelial cells that are transiently mechanically altered may change their phenotype to an epithelioid phenotype, but also that these cells may eventually transdifferentiate into mesenchymal cells expressing SM alpha-actin. Since some aspects of endothelial cell behavior have been shown to be regulated by locally released growth factors such as TGFbeta and FGF, we also investigated TGFbeta-2 and -3 and bFGF expression. Presence of TGFbeta-2 and -3 and bFGF-immunoreactive epithelioid and mesenchymal cells indicates that these growth factors may be involved in the changes described.     

Electron microscope observations on the entry of avian infectious bronchitis virus into susceptible cells

Summary Infectious bronchitis virus was observed to enter cells of chicken chorioallantoic membrane by viropexis. There was no support for the suggestion that entry took place by fusion of viral and plasma membranes. The results of electron microscopy showed that virus attachment occurred both at 4° and at 37° C. Viropexis was not observed until the preparations were warmed. Similar results were obtained using chicken kidney cells. Quantitative data obtained from a plaque counting system employing chicken kidney cells indicated that attachment was the same at both temperatures and that some virus particles were taken up at 4° C.Virus uptake was triggered by attachment of the virus to the cell membrane and the subsequent process of virus entry visualised by E. M. appeared to proceed without the involvement of lysosomal enzymes. No intracellular virus was located by electron microscopy in warmed preparations when virus was treated with specific antiserum, either before or after adsorption to the cells.With 6 Figures