Human Amnion Cell Cultures Research Articles

Effect of Lactobacillus rhamnosus GR-1 Supernatant on Cytokine and Chemokine Output From Human Amnion Cells Treated With Lipoteichoic Acid and Lipopolysaccharide.

Preterm birth occurs in 9% to 13% of all human pregnancies and accounts for 80% of all neonatal morbidities and mortalities. Approximately 40% of all preterm births are idiopathic and about half are associated with infection and/or an activated inflammatory process. Further to studies showing anti-inflammatory effects of supernatant from the probiotic Lactobacillus rhamnosus GR-1 (GR-1), we tested its ability to modulate cytokine and chemokine production from amnion cells in response to stimulation by bacterial wall components, lipopolysaccharide (LPS), and lipoteichoic acid (LTA). Placentae were collected from women undergoing elective cesarean section at term. Amnion cells were cultured for 48 hours to confluence, serum starved for 12 hours, and then treated with GR-1 supernatant (1:20 dilution), followed after 12 hours by LPS (100 ng/mL) or LTA (10 ng/mL) for an additional 12 hours. Both LTA and LPS caused significant increases in the concentration of the pro-inflammatory cytokine, tumor necrosis factor α (TNF-α; 103.9 ± 67.5 pg/mL and 368.3 ± 65.7 pg/mL, respectively) in medium from cultured amnion cells compared to control (<4 pg/mL). There was no significant effect of GR-1 supernatant alone on TNF-α output, but there was significant reduction after LPS treatment. The basal output of the immunomodulatory cytokine, interleukin 6, was 613 ± 170 pg/mL and increased significantly after addition of GR-1 supernatant, LTA, LPS, and combinations of LTA/LPS with GR-1 supernatant. In conclusion, probiotic L rhamnosus GR-1 attenuates the effect of both LPS and LTA in stimulating the output of the pro-inflammatory cytokine TNF-α from mixed cultures of human amnion cells in keeping with previous findings in human trophoblast cells.

Cyclic Adenosine Monophosphate Regulation of Aquaporin Gene Expression in Human Amnion Epithelia

Cell membrane aquaporins (AQPs) may con t r i b u t e importantly to the regulation of intramembranous absorption of amniotic fluid. Recently, the authors demonstrated that human amnion AQP3 expression is upregulated by second-messenger cyclic adenosine monophosphate (cAMP). The present study was undertaken to determine the cAMP regulation of other AQP types, specifically AQP1, 8, and 9, in human amnion epithelia in vitro. Human amnion epithelial cell cultures were prepared from amnion of normal-term pregnancy. To investigate the effect of cAMP on AQP expression, primary human amnion cell cultures were incubated for 2, 10, and 20 hours with culture medium containing either 50 microM forskolin, an adenylate cyclase activator that stimulates cellular production of cAMP, or 100 microM SP-cAMP, a cAMP agonist that stimulates protein kinase A. Total RNA was isolated from the cultured cells, and semiquantitative real-time reverse transcription polymerase chain reaction was carried out to determine the relative level of AQPs mRNA expression. In primary amnion epithelial cell culture, AQP1 mRNA expression increased significantly at 10 hours (0.219 +/- 0.006 to 0.314 +/- 0.008, P < .05) and remained elevated for 20 hours (0.223 +/- 0.004 to 0.323 +/- 0.012, P < .05) following forskolin treatment. AQP8 mRNA expression increased significantly at 2 hours (0.069 +/- 0.003 to 0.086 +/- 0.012, P < .05) and remained upregulated for 20 hours following forskolin treatment. Forskolin stimulation of AQP9 mRNA expression was evidenced by 10 hours (0.098 +/- 0.005 to 0.115 +/- 0.006, P < .05) and maintained for 20 hours. In contrast to forskolin, SP-cAMP incubation resulted in no change in AQP1, 8, or 9 mRNA expression. Human amnion epithelial cell AQP1, 8, and 9 mRNA expression is upregulated by cAMP as their expression is simulated by forskolin. Lack of effect of SP-cAMP, the protein kinase A activator, on AQP1, 8, and 9 mRNA expression suggests that cAMP upregulates human amnion AQP1, 8, and 9 mRNA expression via the protein kinase A independent pathway.

Human amnion cell culture: current limitations and potential for regenerative medicine

Human amnion cell culture: current limitations and potential for regenerative medicine

Aquaporin 3 Expression in Human Fetal Membranes and its Up-regulation by Cyclic Adenosine Monophosphate in Amnion Epithelial Cell Culture

The cell membrane water channel protein aquaporins (AQPs) may be important in regulating the intramembranous (IM) pathway of amniotic fluid (AF) resorption. The objective of the present study was to determine whether aquaporin 3 (AQP3) is expressed in human fetal membranes and to further determine if AQP3 expression in primary human amnion cell culture is regulated by second-messenger cyclic adenosine monophosphate (cAMP). AQP3 expression in human fetal membranes of normal term pregnancy was studied by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). To determine the effect of cAMP on AQP3 expression, primary human amnion cell cultures were treated in either heat-inactivated medium alone (control), or heat-inactivated medium containing: (1) SP-cAMP, a membrane-permeable and phosphodiesterase resistant cAMP agonist, or (2) forskolin, an adenylate cyclase stimulator. Total RNA was isolated and multiplex real-time RT-PCR employed for relative quantitation of AQP3 expression. We detected AQP3 expression in placenta, chorion, and amnion using RT-PCR. Using IHC, we identified AQP3 protein expression in placenta syncytiotrophoblasts and cytotrophoblasts, chorion cytotrophoblasts, and amnion epithelia. In primary amnion epithelial cell culture, AQP3 mRNA significantly increased at 2 hours following forskolin or SP-cAMP, remained elevated at 10 hours following forskolin, and returned to baseline levels by 20 hours following treatment. This study provides evidence of AQP3 expression in human fetal membranes and demonstrates that AQP3 expression in primary human amnion cell culture is up-regulated by second-messenger cAMP. As AQP3 is permeable to water, urea, and glycerol, modulation of its expression in fetal membranes may contribute to AF homeostasis.

Nuclear factor kappa B activation and regulation of cyclooxygenase type-2 expression in human amnion mesenchymal cells by interleukin-1beta.

Interleukin-1beta (IL-1beta) has been shown in numerous studies to increase prostaglandin (PG) output by up-regulating the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in PG synthesis. In this study, we investigated the possible role of the nuclear factor kappa B (NFkappaB) in IL-1beta signaling, leading to the expression of COX-2 in human amnion cell culture. Fetal amnion was obtained following vaginal delivery and digested with collagenase, and the subepithelial (mesenchymal) cells were isolated. Cultures were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Confluent cells were stimulated with human recombinant IL-1beta, and activation of NFkappaB was assessed by measuring changes in the inhibitory protein IkappaB (total IkappaB and phosphorylated IkappaB) using Western blot analysis as well as by nuclear binding of NFkappaB using an electrophoretic mobility shift assay. COX-2 protein levels were determined by Western blot analysis. After 5 min of stimulation with IL-1beta, phosphorylated IkappaB began to appear, 90% of which was degraded within 15 min. This was temporally associated with decreased total IkappaB and increased nuclear NFkappaB DNA-binding activity. In the IL-1beta-treated group, COX-2 protein began to increase after 6 h; this response was time-dependent, with a significant increase until 24 h after IL-1beta stimulation. When NFkappaB translocation was blocked by using SN50 (a cell-permeable inhibitory peptide of NFkappaB translocation), the synthesis of COX-2 protein was inhibited. These results suggest that NFkappaB is involved in the IL-1beta-induced COX-2 expression in the mesenchymal cells of human amnion.

Cellular specificity of interleukin-1beta-stimulated expression of type-2 prostaglandin H synthase in human amnion cell cultures.

Interleukin-1beta (IL-1beta) has been shown in numerous studies to increase prostaglandin output by cultures of human amnion cells. This is due to an increase in the expression of type-2 prostaglandin H synthase (PGHS-2), the inducible form of the enzyme, in these cultures. Amnion consists of an epithelial layer of cells and a subepithelial mesenchymal layer of cells. The purpose of the present study was to determine the cell-type(s) responsible for the IL-1beta-induced PGHS-2 expression in amnion cultures. Amnion was obtained at term after elective Cesarean section or vaginal delivery. Tissues were dispersed with collagenase, and cells were plated in multichamber culture slides and cultured for 7 days in media supplemented with 10% fetal bovine serum. Cell types were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Cultures contained both cell types, and the proportion of these varied considerably from one culture to another. Cells were treated with various concentrations of IL-1beta for 6 or 24 h and were then fixed in 4% paraformaldehyde. The fixed cells were permeabilized with Triton and examined by immunohistochemistry for PGHS-2 protein using specific antisera, and PGHS-2 mRNA was localized by in situ hybridization using a specific oligonucleotide probe. The cell type(s) expressing PGHS-2 was characterized using double labeling with antisera to keratin (epithelial cell marker) and vimentin (mesenchymal cell marker). IL-1beta was found to increase expression of immunoreactive PGHS-2 and PGHS-2 mRNA. This increased expression was found to occur only in the vimentin-positive cells and not the epithelial cells. These results highlight the potential importance of the subepithelial cells in the mesenchymal layer of amnion in the formation of prostaglandins during pregnancy and possibly in preterm labor with infection.

Regulation of prostaglandin H 2 synthase-2 expression in primary human amnion cells by tyrosine kinase dependent mechanisms

Prostaglandin H 2 synthase (PGHS)-1 and PGHS-2 expression was examined in primary cultures of human amnion cells, an in vitro model of amnion tissue. Epidermal growth factor (EGF), the protein kinase C (PKC) activating phorbol ester TPA, and the protein phosphatase inhibitor, okadaic acid (OA), stimulated PGHS activity and the level of PGHS-2 mRNA, but did not affect the level of PGHS-1 mRNA. In situ hybridization suggested that the same population of cells responded to EGF, TPA and OA. Okadaic acid promoted PGHS activity independently of PKC. EGF stimulated the activity of extracellular signal-regulated protein kinase (Erk) and N-terminal c-Jun kinase (Jnk). OA increased Jnk activity but had no effect on Erk activity, while TPA had no influence on either Erk or Jnk activity. PD098059, a selective inhibitor of the Erk-activating kinase MEK, blocked the stimulation of PGHS expression by EGF, but did not decrease stimulation in response to OA. Herbimycin A, a tyrosine kinase inhibitor, suppressed the stimulation of PGHS activity and PGHS-2 mRNA abundance by all three stimulants, and blocked signalling via the Erk and Jnk mitogen-activated protein kinase pathways. Thus, growth factor stimulation, PKC activation and protein phosphatase inhibition induced the expression of PGHS-2 in primary amnion cells by distinct regulatory mechanisms involving tyrosine kinase(s). Tyrosine kinase inhibitors may constitute a new category of PGHS-2 inhibitors that act by blocking the expression of the enzyme.

Glucocorticoids stimulate the expression of prostaglandin endoperoxide H synthase-2 in amnion cells.

Corticosteroids increase the production of prostaglandin E2 (PGE2) and the activity of prostaglandin endoperoxide H synthase (PGHS) in cultured amnion cells, although they inhibit prostanoid biosynthesis in numerous other cell types. This suggests that glucocorticoids control the level of PGHS in amnion cells by a hitherto unexplored, positive regulatory mechanism. We have tested the possibility that corticosteroids act by stimulating the expression of messenger RNAs (mRNAs) encoding one or both isoforms of PGHS. Ribonuclease protection assays were used to determine the levels of PGHS-1 and -2 mRNAs and, for reference, gamma-actin mRNA levels in confluent primary cultures of human amnion cells. In untreated cultures, PGHS-1 and -2 mRNA levels were low, often not reaching the level of detection. Dexamethasone (DEX) treatment for 4 h resulted in a measurable level of PGHS-2 mRNA, which increased further 10-fold and 20-fold after incubation with the glucocorticoid for 8 h and 16 h, respectively. The stimulation was dependent on DEX concentration, and was concomitant with an increase in the capacity of the cells to metabolize arachidonic acid to PGE2. PGHS-1 mRNA levels remained low in DEX-treated cells, while the gamma-actin message level showed no change. Estradiol and progesterone had no influence on PGHS-2 mRNA expression, but cortisol increased the PGHS-2 mRNA abundance. The glucocorticoid antagonist RU486 blocked the effect of DEX. Conditioned media of DEX-treated cells did not contain steroid-induced factor(s) stimulating PGE2 production. Inhibition of protein synthesis by cycloheximide potentiated the effect of DEX, and raised the abundance of PGHS-1, PGHS-2, and gamma-actin mRNAs in untreated cells. DEX did not affect the stability of the PGHS-2 mRNA. These results show that glucocorticoids promote PGE2 synthesis by amnion cells by stimulating the expression of PGHS-2 mRNA in a receptor-dependent, selective, and immediate fashion.

Transforming growth factor-β potentiates epidermal growth factor-induced prostaglandin E2 production in amnion cells

Primary cultures of human amnion cells and the amnion-derived cell line WISH were used to evaluate the hypothesis that transforming growth factor-β (TGF-β) can modulate epidermal growth factor (EGF)-induced prostaglandin E 2 (PGE 2) production. Cells were preincubated for 1 hr with TGF-β (0.0001–10 ng/ml) and then incubated in the presence or absence of EGF (10 ng/ml) for 4 hrs. TGF-β alone did not stimulate PGE 2 synthesis at any dose examined. However, when primary cultures of amnion cells or WISH cells were preincubated with TGF-β and then challenged with EGF, there was a potentiation of PGE 2 production that was much greater than the additive values of TGF-β or EGF alone. These data suggest that EGF-induced PGE 2 production by amnion cells can be modulated by low concentrations of TGF-β.

Effects of fetal urinary corticosteroids, catecholamines and kallikrein on PGE 2 synthesis in monolayer cultures of human amnion and chorion cells

We have studied the effects of several compounds isolated from fetal urine on the production of PGE 2 by amnion and chorion cells which were maintained in primary monolayer culture. We conclude that desoxycorticosterone and vanillylmandelic acid stimulate PGE 2 synthesis by amnion, but adrenaline and kallikrein increase the biosynthesis of PGE 2 by chorion. These data suggest that human fetal urine could play a major role in events of parturition.

Typing of herpes simplex virus with type-specific human immunoglobulin M in an indirect immunofluorescence assay.

Sera from nine individuals with suspected primary herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) infection were screened to identify those containing HSV type-specific immunoglobulin M (IgM). Selected sera were then utilized in an IgM-specific indirect immunofluorescent-antibody HSV-typing assay (patent pending). To evaluate the procedure, 29 HSV isolates were grown in cultures of continuous human amnion cells, fixed, and used as substrates for indirect immunofluorescence. Determination of virus type was based on intensity of fluorescence of the substrate with HSV-1- and HSV-2-specific antisera after staining with fluorescein-conjugated anti-human IgM. Typing of the isolates by restriction endonuclease digestion showed that of 29, 18 were HSV-2 and 11 were HSV-1. Results by IgM-specific indirect immunofluorescent-antibody assay were identical to those by restriction endonuclease digestion for 27 of the isolates; 2 isolates failed to replicate adequately in the test cells. The IgM-specific indirect immunofluorescent-antibody procedure appears to be a simple, rapid, and accurate technique which could be of use to clinical virology laboratories.

Laboratoriumspraxis bei Adenoviren

Six prototype strains of human adenoviruses (Ad1, 4, 7, 8, 12, 19) were passed and titrated comparatively in four strains of human diploid fibroblast cultures. The viruses were readily passed 5 times with cytopathic effect; no signs of adaptation or diminution of CPE were apparent. The virus yield was about equal in fibroblasts and in HeLa cells. High and low passage fibroblasts were of equal susceptibility. In endpoint titrations, fibroblast cultures showed an about 10-fold lower susceptibility towards Adl and Ad7; however, they compared favorably for Ad4, 8, 12, and 19 with HeLa or (for Ad 8) with human amnion cell cultures. In some instances differences in the susceptibility between fibroblast strains were observed. Fibroblast cultures and human amnion cells showed equal susceptibility for Ad8 from 9 original conjunctival specimens, while results with other types varied. For adenovirus isolation, fibroblast cultures are suitable as an adjunct to HeLa cell cultures, particularly for Ad8. However, the long duration till the appearance of CPE which sometimes exceeds 50 days is a disadvantage. Sechs Prototypstämme menschlicher Adenoviren (Ad1, 4, 7, 8, 12, 19) wurden vergleichend in 4 Stämmen von humanen diploiden Fibroblasten passagiert und titriert. Eine Passagierung mit cytopathischem Effekt über 5 Passagen war in allen Fällen möglich; Zeichen einer Adaptation oder Abschwächung des CPE fanden sich nicht. Der Virusertrag in Fibroblastenkulturen war etwa gleich wie in HeLa-Zellkulturen. Fibroblasten aus niedriger und hoher Passage zeigten gleiche Empfänglichkeit. In Endpunkttitrationen zeigten Fibroblastenkulturen bei Ad1 und Ad7 eine um etwa den Faktor 10 geringere Empfänglichkeit; sie waren dagegen bezüglich Ad4, Ad8, Ad 12 und Ad19 den Vergleichskulturen (HeLa- bzw. humane Amnionzellen für Ad 8) mindestens ebenbürtig. In einigen Fällen zeigten sich Unterschiede in der Empfänglichkeit zwischen den Fibroblastenstämmen. Fibroblastenkulturen erwiesen sich gegenüber Ad 8 in 9 Konjunktivalabstrichen als gleich empfänglich wie humane Amnionzellen, während Proben mit anderen Typen sich unterschiedlich verhielten. Fibroblastenkulturen sind als Ergänzungskultur für die Anzüchtung von Adenoviren (z.B. neben HeLa-Zellen) geeignet, insbesondere auch zum Nachweis von Ad8. Nachteilig ist die oft sehr lange Dauer bis zum Auftreten des CPE, die gelegentlich 50 Tage übersteigt.

A new candidate type of human enterovirus (Drilon strain) isolated in the Philippines.

A cytopathogenic agent was isolated in monkey kidney (MK) cell cultures from the stool specimen of a 3-month-old Filipina hospitalized with lower respiratory disease. The agent was designated the Drilon strain. It was characterized as an enterovirus on the basis of electron microscopic morphology, nucleic acid type (RNA), resistance to ether and acid (pH 3.0) treatments, stabilization by molar MgCl2 against heat inactivation, and buoyant density in CsCl. The strain caused mild febrile illness in experimentally inoculated cynomolgus monkeys, but not in suckling mice. In addition to its effect on primary MK cells, the virus was cytopathogenic in primary and secondary human amnion or embryonic lung cell cultures and in WI-38 or HEp-2 cell lines, but not in primary bovine kidney, primary porcine kidney, primary embryonic mouse or primary embryonic chick cell cultures. The Drilon strain was not neutralized by reference antisera against the known enterovirus serotypes, and the antiserum prepared with the Drilon strain did not neutralize any of the recognized prototype enterovirus strains. Although the patient's sera were not available, antibodies against the Drilon strain were prevalent in normal Filipinos and Indonesians, but not in Japanese people. The Drilon strain fulfilled the criteria of human enterovirus and is considered a candidate for designation as a new type.

Inhibition of measles virus replication by cyclic AMP

The replication of measles virus was examined in cells treated with adenosine 3′,5′-monophosphate (cyclic AMP). Addition of 1 m M exogenous cyclic AMP to human amnion cell cultures (AV 3) productively infected with the Edmonston strain of measles virus completely inhibited the formation of multinucleated giant cells and reduced the production of infectious virus particles by greater than 99%. While infected cyclic AMP-treated cells demonstrated measles virus antigens, discrete intracellular viral inclusion bodies were smaller and were observed much less frequently than in infected control cultures. Electron microscopy of the nucleotide-inhibited, infected cells showed that virus capsids of the “smooth” type were present in the cellular cytoplasm, but “granular” or “fuzzy” capsids were not. In addition, the association of virus nucleocapsids with the cell membrane and the budding of complete virion particles were not seen in the cyclic nucleotide-treated cultures. Analysis of infected cell immunoprecipitates by sodium dodecyl sulfate -polyacrylamide gel electrophoresis showed that the synthesis of the P and M polypeptides of the virus was significantly reduced in the nucleotide-treated infections. These data suggest that exogenous cyclic AMP inhibits the assembly of measles virions in infected AV 3 cells by specifically blocking the synthesis of certain viral proteins.

Aryl hydrocarbon (benzo[a]pyrene) mono-oxygenase activity in human primary amnion cell cultures

Amnion cells were isolated from placenta at term of healthy women and grown in monolayer cultures. The amnion cells formed dense sheets of cells of epithelial appearance after several days of growth. To evaluate these cells as targets for xenobiotics which have to be metabolically activated we have investigated their level and inducibility of the primary drug-metabolizing enzyme, aryl hydrocarbon (benzo[a]pyrene) mono-oxygenase (AHM). Amnion cells were found to contain very low levels of AHM which were highly inducible by addition of benz[a]anthracence to the growth medium. The inducibility of the enzyme was greatest during the first days of culture and declined by about two-thirds during the 5th and 8th day. After addition of the inducer, AHM activity increased several fold within 3 hr and reached a maximum at 16–24 hr. The culture age had no significant effect on the time course of induction. Constitutive and benz[a]anthracene-induced AHM were strongly inhibited by 7,8-benzoflavone suggesting that amnion cells in culture contain predominantly a cytochrome P-448-dependent mono-oxygenase form. Induced AHM activities in cultured amnion cells from 13 healthy donors ranged from 0.6 to 8.0 pmole of phenolic benzo[a]pyrene products/ min/ mg protein. The results indicate that primary amnion monolayer cultures offer a valuable model to study in human epithelial cells the activation and inactivation of cytotoxic and mutagenic substances which are metabolized by cytochrome P-448-dependent mono-oxygenase(s).

Immunodiffusion and neutralization studies on porcine adenoviruses

Precipitating antigens were prepared from porcine kidney cell cultures infected with the four approved serotypes of porcine adenoviruses (PAV) and from human amnion cell cultures infected with serotype 5 human adenovirus. For extracellular precipitating antigens (EPA) concentration by ammonium sulphate precipitation from cell culture fluids was used. Cell-associated precipitating antigens (CAPA) were extracted from cell sediments by repeated freezing and thawing. All antigens reacted alike and formed a single coalescent precipitin line of identity when tested against sera collected from a sow infected in the field or from weaners intranasally infected with serotypes 3 or 4 of PAV. In order to determine the optimal time of harvesting cell culture materials for the preparation of precipitating antigens, the kinetics of production and release of infectious virus and precipitating activity of PAV serotype 3 in porcine kidney cell cultures were studied. Precipitating activity first appeared with CAPA 24 h p.i. and 12 h later with EPA. Infectivity titers did not correlate with precipitating activities of EPA or CAPA beyond that stage. At the time the infectivity of EPA was decreasing, its precipitating titer continued to increase. The peaks of precipitating activities of CAPA and EPA were demonstrated at 96 and 144 h p.i., respectively. Three 7-week-old weaners with serum neutralizing antibodies against the four serotypes of PAV, but without detectable precipitating antibodies, were inoculated intranasally with serotype 3 of PAV. Serum samples collected 1 week p.i. showed precipitating activities and steep increases in neutralizing antibody titers against the homologous serotype 3 and the heterologous serotypes 1, 2 and 4 of PAV. The serum neutralizing antibody titers remained nearly constant over a period of 18 weeks p.i. while the intensity of the precipitating reaction decreased. Intranasal infection of the pigs with serotype 4 PAV induced heterotypic anamnestic neutralizing antibody response as well as an increase of the precipitating antibodies.

Activation of adenovirus type 5 latent infections of tissue culture.

To study the in vitro activation of latent adenovirus type 5 infection and enhancement of virus production, primary human amnion and HEp-2 cell cultures were inoculated with low doses of virus and subsequently treated with steroid hormones, carcinogens, or a pyrogen drug 'Pyrago' containing killed cells of bacteria and fungi. Immunofluorescence and CPE revealed that only pyrogen induced conversion from latency to activation and that the drug also enhanced virus production. Although prednisolone failed to induce activation, it did have an enhancing effect on slowly multiplying virus. Presumably, activation and enhancement of virus replication do not occur as the result of the same mechanism but the latter may be influenced by hormones. Microbial products may produce both effects separately or simultaneously.

Cell-protective effect of human saliva specific for herpes simplex virus

Saliva from normal human adults pre-incubated with human amnion cell cultures protected these cells against destruction by herpes simplex virus. In two-thirds of subjects tested, protection amounted to plaque reduction of 55–94 per cent. Protection seemed to be achieved by direct action on cells rather than inactivation of virus. Evidence indicates that the active material was not antibody, although it was more active against herpes simplex virus than vaccinia or vesicular stomatitis virus. This cell-protective activity correlated with recurrent herpes labialis and may be significant in the varying resistance of individuals to symptomatic recurrences and may explain the tendency of recurrent lesions to involve the lips and circumoral skin although sparing the oral mucous membrane.

795 ECHO 6. A CAUSE OF SHINGLES

A skin infection resembling bullous Herpes zoster in a boy of 7 years was investigated by tissue culture and neutralizing antibody titration. The unexpected recovery of ECHO 6 is the reason for this report.Vesiculo-bullous lesions involving 3 adjacent dermatomes on the neck, right shoulder and chest had been present for two days with temperatures of 101°F when he was first seen. Tissue culture using WI-38 cells was done. The lesions were denuded and crusting on a red base after two weeks. After two months vesicles and bullae had healed leaving an irregular red, raised skin eruption where they had been.ECHO-6 grew on human amnion and kidney cell cultures causing cytopathic changes within 24 hours when subcultured.Serum obtained 2 weeks after onset neutralized cytopathic effects of 100 TCD inoculum of ECHO 6 at a dilution of 1:32. The neutralizing antibody titer rose to 1:640 after 2 months. No antibody versus Herpes zoster, vaccinia or other entero-viruses was present.The recovery of ECHO 6 from vesiculo bullous lesions and the demonstration of a 5 fold rise in neutralizing serum antibody support the implication that this enterovirus was responsible for a systemic disease closely resembling Herpes zoster.

Changes in mitotic behavior of a cell culture caused by sensitins

A study of the effect of three types of lyophilized preparations of sensitins on human amnion cell cultures showed an increase in the number of pathological mitoses, arrest of division in metaphase, and agglutination of chromosomes which were not present in the control. These changes indicate a harmful action of these preparations on chromosomes and on the achromatic spindle and, in the author's opinion, they characterize the toxic properties of sensitins.