text:
Is OSHA Falling Down on the Job?
Published: August 2, 1987
(Page 3 of 4)
A series of other recent studies indicates that OSHA never even finds out about many accidents that cause deaths on the job. Employers are required to report such accidents but OSHA has no organized program for assuring that the rule is followed and it rarely punishes companies that do not comply with it.
A new study by the National Institute for Occupational Safety and Health estimates that there may be several thousand more workplace deaths each year than employers report. In 1984, NIOSH said, there may have been 7,000 on-the-job deaths, 3,250 more than employers reported on their annual OSHA filings.
A separate survey this spring by the New Jersey Department of Health ''found it disturbing'' that as many as 35 percent of recent workplace deaths in that state were not reported to or investigated by OSHA.
Even when OSHA learns about workplace deaths, according to an internal Labor Department report, the agency's response is often passive. On March 10, the Labor Department investigators conducting the OSHA study wrote an urgent interim memo to Mr. Pendergrass. It called for ''immediate corrective action'' because, it said, OSHA was not moving properly against employers that have repeated on-the-job deaths. The National Safe Workplace Institute, a Chicago organization that works on occupational safety issues, gave the report to The New York Times. The group obtained the report through a Freedom of Information Act request.
The report said OSHA has no program for investigating employers who may have workplace deaths in different parts of the country. And when OSHA does learn of repeated job-site deaths, the report said, it rarely imposes the most severe fines.
One electrical contractor, which was not named by the report, had four employee deaths and one serious injury within the area covered by one OSHA office. The total penalties: $2,790. OSHA records showed, the Labor Department investigators said, that 19 other employees of the same company had been killed on the job over a nine-year period. The investigators said they uncovered evidence that at least 20 other companies have each had at least 10 employees killed on worksites in different parts of the country since 1980.
In a wide ranging two-hour interview in his Washington office last week, Mr. Pendergrass strongly defended OSHA's record. A genial former industrial health executive at the Minnesota Mining and Manufacturing Company, Mr. Pendergrass was appointed OSHA chief by Labor Secretary William E. Brock in May 1986. The Agency's View
In the interview, the 62-year-old Mr. Pendergrass criticized both the consultants' conclusions and the Inspector General's report. He stressed that the latter was only a draft and would not contain many of its harshest appraisals when it is released in final form this fall. He said the agency could not immediately correct what he acknowledged was a loophole pointed out by the Inspector General's staff concerning companies with repeated worker deaths. ''Unless all employers accept their responsibility to provide a safe workplace, we're going to have people slipping through loopholes,'' Mr. Pendergrass said.
Mr. Pendergrass insisted that the 1984 jump in the rate of worker injuries and illnesses was not a fair measure of the agency's effectiveness, pointing to the long-term improvements since the agency was born. But while injuries and illnesses may have declined since the early 70's, says Mr. Moran of NIOSH, statistics measuring lost work days indicate that injuries are more severe. And international statistics continue to show that more Americans die at work than do their counterparts in many other countries.
By general agreement, OSHA's most startling failure has been its inability to produce more rules to protect workers from scores of widely acknowledged dangerous substances. ''It's embarrassing to the agency,'' Mr. Pendergrass conceded. ''It's embarrassing to our compliance officers and, frankly, its embarrassing to the country.''
When OSHA was created, Congress declared a series of voluntary industry standards to be the law. Many of the industry associations have since updated their standards, while OSHA rules have lagged behind. One study estimates that one million workers are exposed to chemicals in amounts that exceed the levels now accepted as safe by the industry groups.
Mr. Pendergrass said there are tens of thousands of hazardous substances used in industry and that the enormity of the task of regulating them requires new approaches. Agency critics, including Franklin E. Mirer, the health and safety chief of the United Automobile Workers union, say the task is nowhere near as overwhelming as the agency makes it out to be because there is general agreement that a few dozen substances need special attention.
OSHA has repeatedly promised a handful of new regulations, then missed its own deadlines and been forced to defend itself in court. A recent series of sharply worded court decisions has expressed irritation with the delays. [ See box. ] In May, the Federal appeals court in Philadelphia lambasted OSHA for dragging its feet in giving all workers the protection of a new right-to-know rule. It guarantees employees access to information about chemicals they are exposed to at work. After years of delays, OSHA adopted the rule in 1983. But when it did finally act, the agency said that only manufacturing workers would be covered.


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These Days are NOT These Days, part 1 - Pastor David
Now, pay attention, THESE DAYS of Grace, THESE DAYS of the Body of Christ, are NOT THESE DAYS of Israel's hope, foretold by Moses, Samuel, and others. THESE DAYS (of Grace) were foreordained BEFORE the foundation of the world, but NOT, again I say NOT, foretold by Israel's prophets. To read this message go to http://lionandlambministry.com/index.php?option=com_content&id=693

subset: commoncrawl


text:
FileFriend is a small portable utility that offers 4 useful file management features.
You can split a large file into smaller pieces, later join pieces to reconstruct the original file. This can be useful if you need...
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Copyright 1997-2016 WebAttack, Inc. WEBATTACK and SNAPFILES are registered trademarks of WebAttack Inc. All rights reserved. All other trademarks are the sole property of their respective owners.


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Here the love story of Vivaan and Imli gets completed in Udaan, with the help of Suraj and Chakor. It is really sad for some to see Chakor getting dispatch with Vivaan. The story is taking a new turn now with the entry of Bhaiya Ji in the haveli which will bring the difficulties for Suraj. Now it is a fact to see that who will now save Suraj and defend him for his life. In the last episode, we had seen Chakor is decorating the house and even crying for Vivaan. She then says that we have to do this to complete their love and starts decorating again.
Ranjana goes to Chakor and appreciates her for her efforts. She then says that I am happy with your decision. Chakor thinks on at this. On the other side, Ragini talks to the jailor and ask him to free the man. First, the jailor neglects to do so but then agrees when he came to know that Ragini had kidnapped his wife. Imli and Vivaan come to the house and gets shocked seeing the decoration. Chakor welcomes both of them and they smile at her.
Then Vivaan and Imli perform the rituals and takes blessing from everyone. They then go to Suraj and thanks him for doing a lot. Suraj then leaves getting annoyed and Vivaan takes Imli to the room. Chakor comes to them and ask Vivaan to get ready as the groom. They both thanks Chakor and Chakor cries. Ragini calls Bhaiya Ji and tells that their plan is now successful. She also tells him about Imli and Vivaan's marriage and smiles. Bhaiya gets aimed at this and says, now I can go to haveli to make Suraj understand that who I am actually.
In today's episode, we are going to see Suraj gets on anger when Ranjana tells him about Imli and Vivaan's closeness intentionally. Suraj then goes to Imli and Vivaan and they get shocked seeing him there. Now it will be interesting to see that what is Suraj up to now. Stay tuned with us for more updates and gossips of Thapki Pyar Ki like this.


text: Theme Connection Project "Automatic" by Miranda LambertThis song, "Automatic", discuses how much people rely on technology. It shows how life was simpler without the technology innovations we have today. In the chorous, it tells of how technology has made people impatient in getting what they want and "waiting your turn". It also converses of how nothing is as valuable now as it was in the past, because tasks were done "all by hand". Without anyone investing any time and effort into their responsibilities, they don't receive credit or satisfaction. The song expresses how life was harder and slower before the technology we possess today, but everybody was more content and appreciative of what people did. Everyone had time to work out their problems and acknowledge what they had, instead of immediately wanting the next new thing.Technology As an Influence- Google Glasshttp://time.com/103510/google-glass-privacy-foregr...The article expounds upon technology's influence on revolutionizing the way individuals connect with others. Google Glass will change the way that we face everyday tasks, yet also poses some concerns related to the needs of society. While Google Glass will make our lives easier and information more accessible, there are concerns that have surfaced as the first few products have been sold. Some of the negative effects of Google Glass's innovations in society include: distracted driving and most importantly privacy issues. The concerns with Google Glass and the underlying ability to breach personal boundaries, in the form of taking photos without the consent of others, is similar to "The Pedestrian" and Automatic. Each of these sources reveal how technology has enabled efficiency but at a sacrifice of quality and community. Much like "The Pedestrian", individuals are isolated and don't communicate. Such is true as it relates to Google Glass because individuals seek the allure of technology above the direct connection with others. This article article also justifies how technology is a source that maintains ready access; however it leads to complicating the arena of public issues. In some cases negative consequences come from the use of technology, as is cited in the article. "Automatic" also demonstrates the role that technology plays in societal adaptation. Through displaying how technological advances have created negligence, there is much relevance to each new technology that comes to the market. Connection to Fahrenheit 451Fahrenheit 451's society is filled with many social concerns such as depression and isolation. The societal landscape is much like the setting described in "The Pedestrian." Similarly, the article that reviews the use of Google Glass cites that new technologies can surface underlying issues of the public. Each source warns about the evolution of communities, though the loss of knowledge and gain of innovations. "They want to know what I do with all my time. I tell them that sometimes I just sit and think," (Bradbury 20). In Automatic, just as in "The Pedestrian," Farenheit 451, and the article, the song lyrics reveal how evolution and technology has a major negative effect on the world around us. It shows some of the many problems that have been enlightened as the use of technology rises. Individuals are obsessed. "You play God to it. But who has ever torn himself from the claw that encloses you when you drop a seed in a TV parlour? It grows you any shape it wishes! It is an environment as real as the world. It becomes and is the truth. Books can be beaten down with reason" (Bradbury 138). Regardless of the context, technology is drastically changing the way that society interfaces with one another.


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Treat your tresses to bouncy, voluptuous volume with this black Cloud Nine 'The O' Gift Set. The perfect combination in achieving big, beautiful hair, 'The O' Gift Set contains the revolutionary O Pod, various O sized rollers, 12 x O clips and an amplify spray to finish off your look - all displayed in a luxury, black carry case for stylish and practical storage.
The Set Contains:
The O Pod
Boasting ground-breaking induction technology to heat each roller, this unique heat induction pod takes the soft grip rollers included in the Gift Set to optimum heat in less than four seconds. Rollers are heated from the inside out, so they're hot in the air and not in your hand.
- 4 x 30mm 'The O' rollers
- 4 x 40mm 'The O' rollers
- 4 x 50mm 'The O' rollers
- 12 x 'The O' clips
Various sizes of roller are provided as standard as part of this luxury gift set, meaning you can create a massive array of looks - from show-stopping volume to soft curls.
Amplify spray
Spritz onto your freshly styled hair for beautiful, long-lasting results.
Luxury black carry case
For stylish, safe storage of your Cloud Nine 'The O' Gift Set, when not in use or whilst travelling.
Please Note: Cloud Nine's 'O' heat induction pod can be operated with a mains voltage ranging from AC200-240V, 50-60Hz. The voltage in the US (120V) means that currently this is the only place you can't use your pod.
Not ideal for naturally straight, Asian hair types.


text:
About the Book
Wedding Album for Kurt & Krystle Daugherty
Features & Details
- Category Wedding
Large Format Landscape, 13×11 in, 33×28 cm
40 Pages
- Publish Date Oct 17, 2008
- Tags Janet Johnson, Kurt & Krystle, Wedding, Daugherty
See More
About the Creator
Janet Johnson
Forest, Virginia
College Teacher, Computer Scientist, Photographer, Playwright, Pianist - at various times. Devoted to God and family.
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text:
With First Tutors: Music you can search for a Bellshill flugel horn tutor. Flugel Horn lessons are the perfect method to start a stimulating new hobby, acquire a new talent and gain confidence with flugel horn. Use our site to search for Bellshill flugel horn tutors and select which best suits your requirements so you can begin your flugel horn lessons in Bellshill immediately!
First Tutors: Music is the fastest way to locate Bellshill flugel horn tutors in the UK, helping you discover Bellshill flugel horn tuition in your area and Bellshill teachers to assist with any instrument, for example, piano, violin or guitar. We can help you find Bellshill flugel horn tuition or a one-to-one Bellshill flugel horn teacher to prepare for flugel horn exams and improve your flugel horn skills! Our search will order Bellshill flugel horn teachers near you by their hourly charges and proximity to you.
If you are a flugel horn teacher and would like to offer flugel horn tuition services in Bellshill or throughout the UK, please register with First Tutors: Music or find out more about us here. Becoming a Bellshill flugel horn teacher is a brilliant way to improve your own skills by providing flugel horn tuition while helping others, all in your local Bellshill area!


text:
The location is the new pool site in the centre of Castlebar, next to Lough Lannagh. Full budget is €45,000.
This information including site maps is also available via http://www.mayococo.ie/en/Services/ArtsOffice/ArtsNews/StoryIfNoFilesToUpload ,29103,en.aspx
Site Visit: A site visit will take place on Tuesday 4th April at 11am, meeting at the playground car park in front of the development.
Attendance is advised, but not essential. If you are attending the site visit please let me know in advance firstname.lastname@example.com
SUBMISSION DEADLINE: 4.00pm Monday 8th May 2017
See for artist brief in English and artist brief in Irish


text:
Net Kritterz is an online-virtual pet game that Greg has been playing ever since he lost his doll Alfrendo.
Greg's pet's name is Gregory's Little Friend. In the game you have to do things to make your pet happy and it also costs real money to buy some items. However, Susan doesn't like it because Greg keeps asking her for money for his pet. One day, Greg's account got locked by Manny because Manny sneaked into his account and changed the password.
- Diary of a Wimpy Kid: Cabin Fever (First and only appearance)
Known In-game content
- Queasy
- Agitated
- Depressed
- Snazzy
- Gregory's Little Friend's Mansion
- Kritterz Kasino
- It is unknown how Manny changed the password without knowing the original one, as most sites require also the original password to make a new one.
- It is based on many virtual pet games on the web, most notably Neopets and Webkinz.
- The fact that buying Kritterz Kash with real money is vital is a reference to many video games that require real money to progress.


text:
- Hurries
- Hurries Hur"ries, n. A staith or framework from which coal is discharged from cars into vessels. [1913 Webster]
The Collaborative International Dictionary of English. 2000.
The Collaborative International Dictionary of English. 2000.
hurries — hur·ry || hÉœrɪ / hÊŒrɪ n. rush, haste; urgency v. make haste, rush; urge, prod … English contemporary dictionary
Adam Carriker — Infobox NFLactive currentteam=St. Louis Rams currentnumber=90 currentposition=Defensive tackle birthdate=birth date and age|1984|5|6 birthplace=Hastings, Nebraska heightft=6 heightin=6 weight=308 debutyear=2007 debutteam=St. Louis Rams highlights … Wikipedia
Dan Wilkinson — Dan Big Daddy Wilkinson No. 72, 95, 99 Defensive Tackle Personal information Date of birth: March 13, 1973 (1973 03 13) (age 38) Place of birth … Wikipedia
Chris Long — For other uses, see Chris Long (disambiguation). Chris Long Long at a game in Denver in November 2010. No. 91 St. Louis Rams Defensive … Wikipedia
Al Lucas — NFL player ImageWidth=220 DateOfBirth=birth date|1978|9|1|mf=y Birthplace=Macon, Georgia, U.S. DateOfDeath=April 10, 2005 (age age|1978|9|1|2005|4|10) Deathplace=Los Angeles, California, U.S. College=Troy State Position=Defensive tackle… … Wikipedia
Darryl Tapp — No. 55 Philadelphia Eagles Defensive end Personal information Date of birth: September 13, 1984 (1984 09 13) (age 27) … Wikipedia
C. J. Mosley — Infobox NFLactive currentteam=New York Jets currentnumber=69 currentposition=Defensive tackle birthdate=birth date and age|1983|8|6 birthplace=Waynesville, Missouri heightft=6 heightin=2 weight=314 debutyear=2005 debutteam=Minnesota Vikings… … Wikipedia
Dukart's Canal — Principal engineer Davis Dukart Date of act 1767 Date completed 1777 Date closed 1787 Start point Drumglass End point … Wikipedia
Chris Ellis (American football) — This article is about the American football player. For other people with the same name, see Chris Ellis. Chris Ellis Chris Ellis during his tenure at Virginia Tech. No. Free Agent Linebacker … Wikipedia
Charles Hill (American football) — Charles Hill Date of birth: November 1, 1980 (1980 11 01) (age 31) Place of birth: Palmer Park, Maryland Career information Position(s) … Wikipedia


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It is our priority to provide you with the exceptional service you have come to expect from Ralph Lauren. Your feedback is important to us.
For press inquiries, e-mail us at firstname.lastname@example.com
Phone: +44 (0) 203 450 7750
Please note that RalphLauren.co.uk does not use your email for promotional purposes or disclose it to a third party. Privacy Notice.


text:
Varicella-zoster virus (VZV) causes varicella, establishes a life-long latent infection of ganglia and reactivates to cause herpes zoster. The cell types that transport VZV from the respiratory tract to skin and ganglia during primary infection are unknown. Clinical, pathological, virological and immunological features of simian varicella virus (SVV) infection of non-human primates parallel those of primary VZV infection in humans. To identify the host cell types involved in virus dissemination and pathology, we infected African green monkeys intratracheally with recombinant SVV expressing enhanced green fluorescent protein (SVV-EGFP) and with wild-type SVV (SVV-wt) as a control. The SVV-infected cell types and virus kinetics were determined by flow cytometry and immunohistochemistry, and virus culture and SVV-specific real-time PCR, respectively. All monkeys developed fever and skin rash. Except for pneumonitis, pathology produced by SVV-EGFP was less compared to SVV-wt. In lungs, SVV infected alveolar myeloid cells and T-cells. During viremia the virus preferentially infected memory T-cells, initially central memory T-cells and subsequently effector memory T-cells. In early non-vesicular stages of varicella, SVV was seen mainly in perivascular skin infiltrates composed of macrophages, dendritic cells, dendrocytes and memory T-cells, implicating hematogenous spread. In ganglia, SVV was found primarily in neurons and occasionally in memory T-cells adjacent to neurons. In conclusion, the data suggest the role of memory T-cells in disseminating SVV to its target organs during primary infection of its natural and immunocompetent host.
Author Summary
Varicella-zoster virus (VZV) causes varicella, establishes life-long latent infection in ganglia and reactivates later in life to cause zoster. VZV is acquired via the respiratory route, with skin rash occurring up to 3 weeks after exposure. The cell types that transport VZV to skin and ganglia during primary infection are unknown. Simian varicella virus (SVV) infection of non-human primates mimics clinical, pathological and immunological features of human VZV infection. African green monkeys were infected with recombinant SVV expressing enhanced green fluorescent protein (SVV-EGFP) or wild-type SVV (SVV-wt) as a control. By visualizing SVV-EGFP−infected cells in the living animal and in tissue samples, we identified the virus-infected cell types in blood, lungs, skin and ganglia during primary infection. Our data demonstrate that during viremia, SVV predominantly infects peripheral blood memory T-cells. Detection of SVV-infected memory T-cells in lungs, in early varicella skin lesions and also, albeit to a lesser extent, in ganglia suggests a role for memory T-cells in transporting virus to these organs. Our study provides novel insights into the cell types involved in virus dissemination and the overall pathology of varicella in a non-human primate model.
Citation: Ouwendijk WJD, Mahalingam R, de Swart RL, Haagmans BL, van Amerongen G, Getu S, et al. (2013) T-Cell Tropism of Simian Varicella Virus during Primary Infection. PLoS Pathog 9(5): e1003368. https://doi.org/10.1371/journal.ppat.1003368
Editor: Paul Kinchington, University of Pittsburgh, United States of America
Received: January 24, 2013; Accepted: April 2, 2013; Published: May 9, 2013
Copyright: © 2013 Ouwendijk et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by Public Health Service grant AG032958 from the National Institutes of Health (WJDO, RM, DG, and GMGMV). RLdS was supported by the VIRGO Consortium, an innovative cluster approved by the Netherlands Genomics Initiative and partially funded by the Dutch Government (BSIK 031012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: Co-author ADMEO wishes to declare, for the avoidance of any misunderstanding on competing interests, that he co-founded and is chief scientific officer of Viroclinics Biosciences, a company set up in collaboration with Erasmus MC. However, for clarification, no materials or support were received from the company, and no agreements were in place concerning the execution or publication of this work. This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials. The authors have no additional financial interests.
Varicella-zoster virus (VZV) is a ubiquitous human neurotropic alphaherpesvirus that causes varicella (chickenpox) as a primary infection and herpes zoster (shingles) upon reactivation of latent virus [1]. Primary VZV infection is acquired via the respiratory route and varicella occurs 2–3 weeks after exposure [2], [3]. The pathogenesis of varicella is largely unknown, mostly due to the prolonged incubation period and restricted host range of the virus. VZV is detected in lymphocytes of varicella patients [4], suggesting that the virus spreads to susceptible organs including skin and ganglia via a cell-associated viremia [4]. However, the low number of VZV-infected lymphocytes has precluded their identification during natural infection in humans [5].
Most of the current understanding of VZV pathogenesis is based on experimental infection of human fetal tissue transplanted in severe combined immunodeficient mice (SCID-hu model) [6], [7]. In this model, VZV has a tropism for T-cells within thymus and liver xenografts [8]. It has been postulated that VZV initially replicates in respiratory epithelial cells and is transferred to T-cells within tonsilar lymphoid tissue contacting the upper respiratory tract [9], [10]. Virus transport to human fetal skin and ganglia explants in SCID-hu mice can be mediated by T-cells [11], [12], most likely activated memory CD4 T-cells expressing the skin homing markers C-C type chemokine receptor type 4 (CCR4) and cutaneous lymphocyte antigen (CLA) [10]. However, the VZV SCID-hu mouse model does not reproduce the complex and dynamic virus-host interactions involved in the dissemination of VZV to its target organs during primary infection in its natural and immunocompetent host [6], [7].
Simian varicella virus (SVV) produces a naturally occurring disease in non-human primates with clinical, pathological and immunological features that parallel human VZV infection [13], [14]. The prevalence of SVV in free-ranging non-human primates is largely unknown. However, SVV outbreaks in primate centers have been associated with the introduction of monkeys captured from the wild into the colony [15]. The genomes of SVV and VZV are similar in size, structure and genetic organization, with an estimated 70–75% DNA homology [16]. SVV causes varicella, becomes latent in ganglionic neurons and reactivates after stress and immunosuppression to cause herpes zoster [17], [18]. A cell-associated viremia is detected from 3 days post-infection (dpi), with the highest number of infected lymphocytes just before the onset of skin rash [14], [19]. SVV reaches the ganglia before skin rash [20], [21], indicating viremic spread to ganglia.
The aim of the present study was to characterize the kinetics of virus infection and the cell types involved in the dissemination of SVV during primary infection. We have previously shown that infection of macaques with recombinant measles virus expressing EGFP (rMV-EGFP) facilitated the identification of the cell types involved in MV pathogenesis with unprecedented sensitivity [22], [23], [24], [25]. To detect SVV-infected cells at the low frequencies expected in blood and lungs, we infected African green monkeys (AGMs) with recombinant SVV expressing EGFP (SVV-EGFP) and, as a control, wild-type SVV (SVV-wt) to study SVV pathogenesis at the whole organism, tissue and cellular levels in its natural and immunocompetent host. The data presented suggest a crucial role for memory T-cells in the dissemination of SVV during primary infection.
SVV-infected African green monkeys develop transient fever and skin rash
Five SVV-seronegative adult AGMs were infected intratracheally with SVV-wt (n = 2) or SVV-EGFP (n = 3). A transient increase in body temperature was seen between 6 and 11 dpi (Fig. 1A and B). SVV-wt−infected animals developed skin rash starting at 6 dpi, which increased in severity until 9 to 10 dpi and resolved thereafter (Fig. 1C). Macroscopic EGFP fluorescent lesions were detected on the skin and lips of all SVV-EGFP−infected animals starting at 7 dpi, which increased in severity until 9 dpi and resolved by 13 dpi (Fig. 1D and E). EGFP fluorescent lesions were also detected on the tongue of SVV-EGFP−infected monkeys, coinciding with appearance of skin rash (Fig. 1F). No lesions were observed on the lips and tongues of SVV-wt−infected animals, demonstrating the increased sensitivity of using SVV-EGFP to study varicella pathogenesis. Skin rash was more severe in SVV-wt− compared to SVV-EGFP−infected monkeys. Collectively, the findings indicate the close resemblance of the clinical signs associated with experimental SVV-EGFP infection of AGMs and those of primary VZV infection in humans.
(A, B) Fluctuations in body temperature after infection with SVV-wt and SVV-EGFP, respectively, were measured by intraperitoneally implanted temperature transponders during primary infection. Arrows indicate time of SVV inoculation; horizontal lines indicate normal range in body temperature before infection. (C) Vesicular skin rash at 8 dpi with SVV-wt. (D) Macroscopic detection of EGFP fluorescence on skin at 8 dpi with SVV-EGFP. (E) Macroscopic detection of EGFP fluorescence (arrows) on lips at 9 dpi with SVV-EGFP. (F) Macroscopic detection of EGFP-positive lesions (arrows) on tongue at 8 dpi with SVV-EGFP.
SVV infection of alveolar myeloid cells and T-cells in the lung
All SVV-infected animals became dyspneic at the time of skin rash. Macroscopic examination of lungs showed multifocal pulmonary consolidation and hemorrhage affecting at least one lobe in all animals euthanized 9 or 13 dpi (Fig. 2A). Diffuse EGFP fluorescence was detected in an SVV-EGFP−infected monkey at 9 dpi (Fig. 2B and C). Combined immunohistochemical (IHC) and immunofluorescence (IF) analyses for SVV antigens and EGFP on consecutive sections of lung showed that EGFP expression was restricted to SVV antigen-positive cells (Fig. 2D–G), demonstrating that EGFP is a valid marker to identify SVV-infected cells in the monkeys. To investigate SVV-infected cell types in situ, lung tissue sections were analyzed by dual-IF staining with SVV-specific antiserum and anti-keratin, -CD3, -CD68 and -CD11c mouse monoclonal antibodies (mAbs). SVV-infected cells were readily detected in lungs at 9 dpi, but not at later times (data not shown). At 9 dpi, abundant SVVposkeratinpos lung epithelial cells were observed (Fig. 2H), as well as SVVposCD3pos T-cells (Fig. 2I). In addition, SVV antigens were found in intra-alveolar cells that co-expressed CD68 and/or CD11c, consistent with alveolar macrophages (AM), some of which appeared to have phagocytosed SVV-infected cells (Fig. 2J). Occasionally, SVVposCD11cpos dendritic cell (DC)-like cells displaying multiple branched projections were observed adjacent to bronchi (Fig. 2K).
(A) Macroscopic appearance of consolidated dark-red lesions (black arrow) in the lung of an SVV-wt−infected monkey at 13 dpi. (B) Macroscopic detection of EGFP fluorescence in affected area of lung (white arrow) of an SVV-EGFP-infected monkey at 9 dpi. (C) Magnification of the affected area in panel B shows EGFP fluorescence. (D–G) Serial lung sections obtained from an SVV-EGFP−infected monkey at 9 dpi analyzed by immunohistochemistry (IHC) for SVV antigens (D) or by immunofluorescence (IF) for EGFP (E), with two sections analyzed by IHC (F) or IF (G) using normal rabbit serum (NRS) and isotype control antibodies, respectively. Lung sections obtained from an SVV-wt−infected monkey at 9 dpi were analyzed using dual IF for SVV (green) and: cytokeratin (red) (H), CD3 (red) (I), CD68 (red) (J), and CD11c (red) (K) antigens. Arrows indicate double-positive cells. Asterisks indicate autofluorescent erythrocytes. Dashed lines indicate alveolar septa. Br: bronchus. Nuclei were counterstained with DAPI. D–G: 100× magnification; H, J: 400× magnification; I, K: 400× magnification and 2× digital zoom.
To define the kinetics of virus replication and the cell types infected in the respiratory tract during primary SVV infection, bronchoalveolar lavage (BAL) cells were obtained at 5 dpi, 9 dpi and at necropsy (9, 13 or 20 dpi). SVV DNA load and infectious virus titers in BAL cells peaked at 5 dpi and declined rapidly thereafter (Fig. 3A and B). Infectious virus was not recovered from BAL cells at 13 and 20 dpi (Fig. 3B and data not shown). The viral DNA load and infectious SVV titer in BAL samples were similar in SVV-wt− and SVV-EGFP−infected monkeys at 5 dpi, indicating a similar level of replication of both viruses in lung.
(A) Bronchoalveolar lavage (BAL) cells obtained at 5, 9 and 13 dpi were analyzed for viral DNA by SVV-specific real-time qPCR. Data are expressed as genome equivalent copies (geq) per 105 BAL cells. (B) BAL cells were analyzed for infectious virus by co-cultivation with BSC-1 cells. (C–F) Percentage of EGFP-positive cells as assessed by flow cytometry in: all BAL cells (C); leukocytes (CD45pos cells), non-leukocytes (CD45neg cells) from BAL samples (D) and leukocyte subsets within BAL (E). Leukocyte subsets were identified based on the differential expression of the following markers: AM/DC = CD45posCD3negCD20negMHC-IIposCD14pos/dim, T-cells = CD45posCD3pos, B-cells = CD45posCD20posMHC-IIpos; and the indicated T-cell subsets (F). AM/DC are BAL-derived lymphocytes expressing markers shared by dendritic cells (DC) and alveolar macrophages (AM). Horizontal bars indicate median values. (D–F) BAL cells were obtained at 5 dpi and data are given as means ± SEM.
At 5 dpi, 7.2% of BAL cells from SVV-EGFP−infected monkeys were EGFPpos (Fig. 3C). In EGFPpos BAL cells, equal numbers of CD45pos (leukocytes) and CD45neg cells, most likely bronchial and alveolar epithelial cells, were detected (Fig. 3D and Fig. S1). CD45pos BAL cells could be categorized as T-cells, B-cells and alveolar myeloid cells, i.e. large granular cells expressing high levels of both CD14 and HLA-DR (Fig. S1). These myeloid cells could be alveolar macrophages (AM) and/or DC. At 5 dpi, 82% of CD45pos BAL cells were alveolar myeloid cells, 17% were T-cells and only 1% were B-cells (data not shown). Most EGFPposCD45pos BAL cells were alveolar myeloid cells and T-cells (Fig. 3E). CD4pos, CD8dim and CD8bright T-cells were infected at equal frequencies (Fig. 3F). The number of BAL-derived T-cells was too low to determine their differentiation status unequivocally (data not shown). At 9 dpi, frequencies of EGFPposCD45pos BAL cells were too low to conclusively identify the SVV-infected leukocyte subsets (Fig. 3C).
SVV infection of memory T-cells in blood during viremia
To determine the kinetics of virus infection and identify the blood lymphocyte subsets infected during the viremic phase of varicella, peripheral blood mononuclear cells (PBMC) isolated at multiple dpi from SVV-infected monkeys were analyzed. SVV DNA was detected in PBMC from 2 dpi until necropsy (Fig. 4A). Viral DNA load in PBMC peaked at 7 dpi and was higher in SVV-wt− compared to SVV-EGFP−infected monkeys (Fig. 4A). Infectious virus was isolated from PBMC of both SVV-EGFP− and SVV-wt−infected monkeys until 9 and 11 dpi (Fig. 4B). EGFPpos lymphocytes were detected from 5 to 11 dpi, peaking at 7 dpi (Fig. 4C). Together, the data indicate that the kinetics of viral DNA load and infectious virus titer represent the temporal change in the number of circulating SVV-infected lymphocytes, but not in the replication of SVV in blood lymphocytes during viremia. The rapid loss of SVV-infected lymphocytes from the circulation could be caused by virus-induced apoptosis [26] or, alternatively, infected lymphocytes may be cleared from the circulation by the SVV-specific adaptive immune response [14], [21], [27].
(A) Average SVV DNA load in PBMC of SVV-wt− (closed squares) and SVV-EGFP− (open squares) infected monkeys determined by SVV-specific real-time qPCR. (B) PBMC from SVV-wt− (closed squares) and SVV-EGFP− (open squares) infected monkeys were analyzed for infectious SVV by co-cultivation with BSC-1 cells. (C) PBMC from SVV-EGFP−infected monkeys were analyzed for EGFP expression by flow cytometry. (D) EGFP expression in PBMC subsets from SVV-EGFP−infected monkeys. Data are given as percentage of EGFPpos cells within each lymphocyte subset relative to the total number of PBMC, as determined by flow cytometry. Lymphocyte subsets were defined by differential expression of the following markers: T-cells = CD3posCD16neg cells, B-cells = CD20posMHC-IIposcells, natural killer (NK) cells = CD3negCD16pos cells, dendritic cells (DC) = CD3negCD14negCD16negCD20negCD14negMHC-IIpos cells, and monocytes = CD3negCD14posMHC-IIpos cells. (E and F) Percentage of EGFPpos cells among each T-cell subset relative to the number of CD8bright, CD8dim and CD4pos T-cells (E) and in naive, central memory and effector memory T-cells (F) from SVV-EGFP−infected monkeys as evaluated by flow cytometry. In all panels, data are means ± SEM.
At 5 dpi, EGFPpos cells were detected at similar frequencies in all major PBMC subsets (i.e., T-cells, B-cells, natural killer cells, monocytes and dendritic cells) (Fig. 4D and Fig. S2). However, given that most PBMC are T-cells (Fig. S2), T-cells were identified as the main SVV-infected lymphocyte subset in blood (Fig. 4D). Moreover, at the peak of viremia (7 dpi) T-cells were the only SVV-infected cells demonstrated in blood. Unlike humans and macaques, AGMs have three distinct T-cell subsets: CD8bright, CD8dim and CD4pos T-cells (Fig. S2) [28]. While CD8bright T-cells correspond to classical human CD8+ T-cells, CD4pos T-cells and CD8dim T-cells are considered dynamic populations of AGM T-helper cells functionally equivalent to human CD4+ T-cells [28]. Similar levels of CD8bright, CD8dim and CD4pos T-cells were SVV-infected, most of which were memory T-cells (Fig. 4E and F). Importantly, at 5 and 7 dpi, predominantly central memory (CM) T-cells and effector memory (EM) T-cells, respectively, were infected (Fig. 4F). The apparent dual phase of SVV-infected CM and EM T-cells may reflect the organ in which the T-cells have been infected. CM T-cells are preferentially found in lymphoid tissues, whereas EM T-cells are migratory memory T-cells that home to peripheral tissues to orchestrate local immune responses and may ultimately function as tissue-resident T-cells to sense the cognate antigen locally for extended periods of time [29], [30]. CM T-cells may have been infected in lymphoid tissues and EM T-cells in lungs. Alternatively, SVV infection might have altered the expression of membrane markers used herein to identify AGM-derived CM and EM T-cells. Finally, virus infection may have induced differentiation of CM T-cells into EM T-cells in vivo.
In vitro infection studies on human tonsil-derived lymphocytes showed that VZV preferentially infects T-cells expressing the activation marker CD69 and skin-homing markers CCR4 and CLA [10]. To address this issue in SVV-EGFP−infected monkeys, peripheral blood-derived EGFPpos T-cells obtained at 5 and 7 dpi were analyzed for expression of both CCR4 and the T-cell activation marker CD137, the latter marker is selectively expressed by T-cells early after recognition of their cognate antigen [31], [32]. No preference of SVV for memory T-cells expressing CCR4 or CD137 was seen in vivo (Fig. S3), suggesting that SVV did not infect virus-specific T-cells that recognized SVV-infected antigen presenting cells like macrophages or DCs.
To determine whether the predominant infection of memory T-cells in vivo reflects viral tropism for a specific lymphocyte subset, PBMC from SVV-naive AGMs were infected in vitro with SVV-EGFP. Expression of EGFP was restricted to lymphocytes that expressed SVV antigens (Fig. S4A), supporting the use of EGFP as a surrogate marker for SVV-infected cells in flow cytometry. While all major PBMC subsets appeared to be equally susceptible to SVV infection, T-cells were the prominent SVV-infected PBMC subset in vitro (Fig. S4B), with similar infection levels in CD4pos, CD8dim and CD8bright T-cells (Fig. S4C). In particular, significantly more memory T-cells were infected compared to naive T-cells (p<0.05; Mann-Whitney test) (Fig. S4D). Thus, SVV preferentially infects memory T-cells rather than naive T-cells both in vivo (Fig. 4) and in vitro (Fig. S4).
Detection of SVV in lymphoid organs
Alveolar macrophages and lung-resident DC transport antigens to lung-draining lymph nodes for presentation to T-cells [33], [34], and VZV-infected human DCs can transfer infectious virus to T-cells in vitro [35]. We hypothesized that SVV-infected alveolar myeloid cells transport SVV to draining lymph nodes for subsequent virus transfer to memory T-cells. High SVV DNA loads were detected in lymph nodes, tonsils and spleens of SVV-infected monkeys at 9 dpi, declining rapidly thereafter (Fig. 5A). Cells in lymph nodes and tonsils of SVV-infected monkeys contained intranuclear inclusions bodies and SVV antigen (Fig. 5B and C). Tracheobronchial lymph nodes showed more pronounced SVV-induced histopathology compared to peripheral lymph nodes (data not shown). However, SVV DNA loads were comparable in different lymph nodes collected at 9 dpi (Fig. 5A), emphasizing the need to investigate lymph nodes at earlier times after infection. In addition, detection of SVV-infected memory T-cells in blood may represent lung-resident T-cells involved in SVV dissemination. SVV infects alveolar epithelial cells leading to alveolar wall damage (data not shown) [19], [27], [36], which may result in egress of SVV-infected T-cells into the circulation.
(A) Real-time qPCR analysis of SVV DNA load in tonsil, lymph nodes and spleen from SVV-wt− (closed squares) and SVV-EGFP− (open squares) infected monkeys at 9, 13 and 20 dpi. Squares indicate individual tissues, i.e., tonsils (red), tracheobronchial lymph nodes (LN) (green), axillary LN (pink), mandibular LN (blue), inguinal LN (orange) and spleen (black). Horizontal bar indicates the median value. (B–D) Serial sections of tonsil from an SVV-wt−infected monkey stained with hematoxylin and eosin (inset shows a Cowdry type A intranuclear inclusion body) (B) or examined immunohistochemically using rabbit anti-SVV antibodies (C) or control normal rabbit serum (D). Magnification: 200×. The area of tonsils containing multiple intranuclear inclusion bodies contained numerous cells expressing SVV protein. **p<0.01 by Mann-Whitney test.
SVV-infected perivascular lymphocytes in early varicella lesions implicate hematogenous spread of SVV to the skin
Detailed in situ analysis was performed to identify the SVV-infected cell types in varicella skin lesions. Macroscopic detection of EGFP fluorescence corresponded to SVV infection of the skin, as demonstrated by the co-localization of SVV protein and EGFP in consecutive skin sections obtained from an SVV-EGFP–infected monkey (Fig. 6A and B). In vesicular skin lesions, SVV predominantly infected keratinocytes (Fig. 6C and D). In deeper skin layers, SVV protein was frequently detected in hair follicles (Fig. 6E and F) and sebaceous glands (Fig. 6G and H).
(A, B) Consecutive sections of skin obtained from an SVV-EGFP-infected monkey at 9 dpi and stained by immunofluorescence (IF) for EGFP (A) and by immunohistochemistry (IHC) for SVV antigens (B) show co-localization of SVV proteins and EGFP. Squares indicate the same area of tissue. (C–H) Consecutive sections of skin obtained from an SVV-wt−infected animal at 9 dpi and examined by staining with hematoxylin and eosin (H&E) or by IHC for SVV show virus-induced histopathology and viral proteins in epidermal blisters (C and D), dermal hair follicles (E and F) and dermal sebaceous glands (G and H). (I, J) Consecutive skin sections obtained from an SVV-EGFP-infected monkey at 9 dpi and stained with H&E (I) or by IHC for SVV antigens (J) show blood vessels (asterisks) surrounded by SVV protein-positive cells (arrows). Inset: magnification of the epidermis showing Cowdry type A intranuclear inclusion bodies in panel I (arrowheads) and SVV protein-positive cells in panel J (arrows). (K) Skin section from an SVV-EGFP-infected animal obtained at 9 dpi and double-stained for EGFP (green) and alpha-smooth muscle actin (SMA; red). Asterisks indicate SMA-positive sweat glands, arrowheads indicate SMA-positive blood vessels, and arrows indicate EGFP-positive cells. (L–N) Skin sections obtained at 9 dpi and double-stained for EGFP (green) and: CD68 (red) (L); CD11c (red) (M); and CD3 (red) (N). Arrows indicate dual-stained cells. (O) Skin section obtained at 9 dpi and double-stained for SVV (green) and CD3 (red). Arrows indicate dual-stained cells. A, B: 100× magnification; C–K: 200× magnification; L–O: 400× magnification and 2× digital zoom.
Analysis of skin biopsies from SVV-EGFP−infected monkeys allowed investigation of the early stages of varicella, as evidenced on the skin by the appearance of EGFP fluorescent areas in the absence of lesions visible to the naked eye. In these biopsies, SVV protein expression was consistently located within perivascular lymphocytes (Fig. 6I–K). Dual-IF staining for EGFP and specific lymphocyte markers identified SVV-infected perivascular cell subsets as CD68pos macrophages (Fig. 6L), CD11cpos DCs (Fig. 6M) and CD3pos T-cells (Fig. 6N). The remaining SVV-infected cells, which stained negative for lymphocyte markers, phenotypically resembled dendrocytes (data not shown) [37]. Interestingly, SVV-infected T-cells were also observed in the epidermis of SVV-wt infected monkeys at 9 dpi (Fig. 6O). Flow cytometric analysis of skin-resident T-cells showed exclusively memory T-cells, mostly EM T-cells (data not shown).
Collectively, these data suggest that SVV reaches the skin hematogenously. Since the skin vasculature is composed of an upper horizontal superficial vascular plexus just beneath the epidermal surface and a deep vascular plexus that supplies the hair bulbs and sweat glands [38], it seems likely that SVV-infected memory T-cells transfer the virus to skin-resident perivascular macrophages, DCs or dendrocytes, which in turn transfer SVV to adjacent epidermal or hair follicle keratinocytes via cell-to-cell spread. Alternatively, epidermal SVV-infected T-cells may transfer the virus directly to skin epithelial cells (Fig. 6O).
Neurons are the main SVV-infected cell types in ganglia
The hallmark of primary SVV and VZV infection is the capacity of virus to infect and establish latency in ganglionic neurons along the entire neuraxis [1], [13], [39]–[42]. Virus may reach ganglia hematogenously or by retrograde axonal transport along axons innervating varicella lesions [12], [20], [21], [43], [44]. We determined the kinetics of virus infection and the cell types infected in ganglia during primary SVV infection. The SVV DNA load in ganglia was significantly higher in SVV-wt− compared to SVV-EGFP−infected monkeys (p<0.01; Mann-Whitney test) (Fig. 7A), peaking at 9 dpi and decreasing thereafter (Fig. 7A), as might be expected during the establishment of latency. Despite high SVV DNA loads, no virus-mediated cytopathology was seen in ganglia (data not shown). Virus antigen was more abundant at 9 dpi than at 13 and 20 dpi (data not shown). SVV-infected cells in ganglia were detected in situ by IHC using SVV-specific antiserum (Fig. 7B–D). Dual-IF staining for SVV and the neuron-specific marker NCAM (neural cell adhesion molecule) showed that most SVVpos cells were neurons (Fig. 7E). Occasionally, SVV antigens were seen at the neuronal cell surface or potentially within satellite glial cells (SGC) (Fig. 7D). SGC form a sheet that completely enwraps neuronal cell bodies, providing physical and metabolic support to the neurons and contributing to regulation of the immune response in the peripheral nervous system [45], [46]. Virus-infected cells located in vicinity to neurons did not express the SGC-specific marker glial fibrillary acidic protein (GFAP) [45], implicating that SGC were not infected with SVV at 9, 13 and 20 dpi (Fig. 7F and data not shown). To address the possibility of T-cell–mediated transfer of SVV to neurons, ganglia were examined using dual-IF staining for SVV antigens and CD3. In an SVV-wt−infected monkey euthanized at 9 dpi, SVV-infected T-cells were detected in close proximity to neurons (Fig. 7G). Notably, this animal also had the highest SVV DNA load in blood and ganglia. Flow cytometric analysis of ganglion-derived single-cell suspensions demonstrated that ganglion-resident T-cells were memory T-cells, predominantly EM T-cells (Fig. 7H).
(A) Virus DNA load was determined in ganglia at 9, 13 and 20 dpi by SVV-specific real-time qPCR. Filled and open squares represent pooled ganglia from the same level of the neuraxis from animals infected with SVV-wt and SVV-EGFP, respectively. Colors indicate level of the neuraxis: trigeminal (black), cervical (red), thoracic (blue), lumbar (green) and sacral (pink) ganglia. Horizontal bars represent mean viral DNA load per animal. (B) Immunohistochemical detection of SVV proteins (arrowheads) in a cervical ganglion at 9 dpi. Squares indicate corresponding tissue areas shown at higher magnification in (C) and (D). (E) Dual-immunofluorescence (IF) staining of a thoracic ganglion at 9 dpi for SVV proteins (green) and neural cell adhesion molecule (NCAM; red). Arrows indicate SVV-positive neurons. (F) Dual-IF staining of a thoracic ganglion at 9 dpi for SVV protein (green) and glial fibrillary acidic protein (GFAP; red). Arrow indicates neuron-adjacent SVV-positive cell. (G) Dual-IF staining of a thoracic ganglion from a monkey at 9 dpi for SVV protein (green) and CD3 (red). Arrows indicate SVV-positive T-cells. Asterisks indicate autofluorescent lipofuscin and the borders of the neuronal cell bodies are indicated with dashed lines. (H) Ganglion-derived single-cell suspensions were analyzed by flow cytometry and T-cells were categorized as naive, central memory (CM) and effector memory (EM) T-cells. E–G: nuclei were counterstained with DAPI (blue). ** p<0.01 by Mann-Whitney test. B: 200× magnification; C, D: 400× magnification, 2× digital zoom; E: 400× magnification; F, G: 400× magnification, 2× digital zoom.
Our findings in ganglia contrast with the pronounced VZV-induced histopathology of both SGCs and neurons found in VZV-infected human fetal ganglia xenografts in the SCID-hu mouse model [12], [47]. Most likely, these differences are due to the use of fetal human ganglia and the lack of adaptive immune responses in the SCID-hu mouse model. The absence of SVV-induced histopathology in ganglia is consistent with previous studies [19], [27] and the inability to recover infectious virus from ganglia [15] at 10 dpi. Nonetheless, virus-induced cytopathology of ganglia may have occurred during the peak of viremia (5–7 dpi), which will be considered in future studies. The detection of SVV protein in the cytoplasm of neurons, but not in the interacting SGC (Fig. 6B–F), supports the notion of retrograde axonal route of virus entry into ganglia [43], [44], [48], [49]. In contrast with this hypothesis, the SVV DNA load did not differ among ganglia, including those that innervated the dermatomes showing varicella rash (Fig. 7A and data not shown). The alternative scenario is that virus traffics to ganglia during viremia within lymphocytes. Indeed, both SVV and VZV enter ganglia before the onset of rash, arguing for hematogenous virus spread [1], [20], [21]. VZV-infected T-cells infiltrate human ganglion xenografts and transmit VZV to neurons in the VZV SCID-hu mouse model [12]. The occasional detection of neuron-interacting, SVV-infected memory T-cells within ganglia (Fig. 7G) supports the role of memory T-cells in virus dissemination to ganglia. Further studies on ganglia from SVV-EGFP−infected monkeys euthanized at earlier times after primary infection are warranted to test this hypothesis.
The current study is the first to present experimental evidence (summarized in Fig. 8) that supports the role of memory T-cells in the inter-organ dissemination of varicella virus in its natural and immunocompetent host. Our current hypothesis on the pathogenesis of primary SVV infection is presented in Figure 9. We hypothesize that upon intratracheal inoculation, SVV replicates in the respiratory tract and infects epithelial cells, alveolar myeloid cells (AM and/or DC) and T-cells in the lungs. Subsequently, the virus enters the circulation as cell-associated virus predominantly within memory T-cells, first within CM and subsequently within EM T-cells. Most likely, virus-infected alveolar myeloid cells transport SVV to lung-draining lymph nodes, with subsequent transfer of SVV to memory T-cells. Peak viremia coincided with onset of fever and appearance of skin rash. SVV reached the skin by the hematogenous route, most likely via virus-infected memory T-cells. SVV may enter ganglia by retrograde axonal transport from the infected epithelia and/or by the hematogenous route. In addition to memory T-cells, other lymphocyte subsets may also contribute to the viremic spread of SVV. Virus-infected DC, NK cells, B-cells and monocytes were detected in peripheral blood at 5 dpi, albeit at low frequencies compared to memory T-cells. The contribution of each lymphocyte population in transfer of SVV to its target organs will be addressed in future studies by analyzing virus-infected lymphocytes in tissues of animals euthanized during peak viremia at 5–7 dpi.
Figure shows the kinetics of SVV infection and virus-infected cell types in African green monkeys during primary SVV infection. Horizontal lines indicate the time-frame covered by the sampling days. Width of the black bars indicates onset and severity of clinical signs, amount of SVV DNA detected in blood and the sampled organs, and the frequency of SVV-infected cells in peripheral blood during primary SVV infection. Note that BAL samples were obtained no earlier than 5 dpi and animals were euthanized no earlier than 9 dpi. BAL: bronchoalveolar lavage; NK cells: natural killer cells; DC: dendritic cell; TCM: central memory T-cells; TEM: effector memory T-cells.
Upon intratracheal inoculation of African green monkeys, SVV replicates in the lower respiratory tract and infects lung epithelial cells, alveolar macrophages (AM), dendritic cells (DC) and T-cells. SVV-infected AM and DC may transport the virus to draining lymph nodes and subsequently transfer SVV to local lymphocytes resulting in a cell-associated viremia. Memory T-cells are the predominant SVV-infected lymphocyte subset during viremia and may play a central role in dissemination of SVV to its target organs. SVV reaches the skin by the hematogenous route, presumable via virus-infected memory T-cells, which results in the infection of perivascular macrophages, DC and dendrocytes. Subsequently, SVV may infect epidermal and hair follicle keratinocytes via cell-to-cell spread and cause vesicular skin lesions. SVV may enter ganglia by (a) retrograde axonal transport and/or (b) by viremic spread via virus-infected lymphocytes.
Like VZV, SVV is considered to spread to naive monkeys via aerosols and therefore most likely targets mucosal epithelial cells of the upper respiratory tract, although – depending on the size of the aerosols – some virus may also directly reach the lower respiratory tract [1], [13], [14], [25]. In the current study, we have used intratracheal inoculation of monkeys with SVV, bypassing the putative initial site of local SVV replication in the upper respiratory tract or tonsils [1], [13], [14]. Primary VZV infections in adults are more severe than in children and frequently complicated by varicella pneumonia [1]. Consequently, the adult status of SVV-infected AGM may have enhanced disease severity, although pneumonia is a common feature in SVV-infected monkeys due to the intratracheal route of inoculation [19], [27]. Recombinant SVV-EGFP was attenuated in vivo compared to SVV-wt, possibly due to insertion of the EGFP gene between open reading frames (ORFs) 66 and ORF67 [50]. Recombinant VZV lacking ORF67 is severely impaired for growth in cell culture [51]. Although attenuated in severity, SVV-EGFP–induced disease resembled that of a SVV-wt infection and attenuation did not alter the cell tropism of SVV-EGFP. Both SVV-wt and SVV-EGFP infected the same cell types in lung, lymph nodes, ganglia and skin in vivo, and identical PBMC types in vitro. The recent cloning of the SVV-wt full-length genome in a bacterial artificial chromosome facilitates the generation of a potentially less attenuated recombinant EGFP-expressing SVV by inserting the EGFP gene adjacent to SVV genes dispensable for growth in vitro [51], [52].
Future studies on juvenile African green monkeys, infected with less-attenuated SVV-EGFP strains and via alternative inoculation routes (e.g., via the nose or throat), are warranted. Particularly, analysis of tissues obtained from infected animals euthanized shortly after primary infection are needed to unequivocally determine the early target cell types of SVV, their role in virus dissemination to the target organs affected during primary infection and the route of SVV entry into sensory ganglia [25]. Our current SVV-EGFP/AGM model, which largely covers the clinical and pathological features seen in both SVV-wt−infected monkeys and human varicella patients, provides novel opportunities to elucidate the virus-host cell interactions involved in varicella pathogenesis. This will open new avenues to develop and test new VZV vaccination and therapeutic interventions that limit viremic spread, while inducing long-lasting adaptive VZV-specific immunity.
Materials and Methods
Ethics statement
This study was performed in strict accordance with European guidelines (EU Directive on Animal Testing 86/609/EEC) and Dutch legislation (Experiments on Animals Act, 1997). The protocol was approved by the independent animal experimentation ethical review committee DCC in Driebergen, the Netherlands (Erasmus MC permit number EMC2374). Animals were housed in groups, received standard primate feed and fresh fruit daily, and had access to water ad libitum. Cages also contained sources of "environmental enrichment" such as hiding places and hanging ropes, tires and other toys. During infection, study animals were housed in HEPA-filtered, negatively pressurized BSL-3 isolator cages. Animal welfare was monitored daily and all animal handling was performed under light anesthesia (ketamine) or deep anesthesia (ketamine and medetomidine) to minimize animal discomfort. After deep anesthesia, atipamezole was administered to antagonize the effect of medetomidine. Animals were euthanized by sedation with ketamine and medetomidine followed by exsanguination.
Low-passage clinical isolates of the Delta herpesvirus strain of SVV-wt and SVV-EGFP were obtained from PBMC of acutely infected AGM and propagated less than 5 times in AGM- kidney epithelial cell line BSC-1 (American Tissue Type Culture no. CCL-26) to generate virus stocks as described [53]. Virus stocks were confirmed as Mycoplasma-free. SVV-EGFP was generated by insertion of the EGFP gene downstream from a Rous sarcoma virus promoter between SVV ORF66 and ORF67 [20], [50].
SVV infection of PBMC in vitro
PBMC from SVV-naive AGM were infected by co-cultivating PBMC (5×105) with SVV-EGFP–infected Vero cells (0.5–1×105), showing 70% virus-induced cytopathic effect (CPE), in 0.5 ml DMEM supplemented with antibiotics and 10% heat-inactivated fetal bovine serum (FBS) for 24 hr in 24-well plates at 37°C in a CO2-incubator. Mock-infected PBMC were similarly generated by co-cultivating PBMC with uninfected Vero cells. SVV-EGFP−infected PBMC were stained and analyzed by flow cytometry or spotted on microscope slides, fixed and stained by immunofluorescence for SVV as described below.
Experimental SVV infection of AGM, necropsy and collection of tissues
Five adult (10- to 12-year-old) SVV-seronegative AGMs (Cercopithecus aethiops) with intraperitoneal implanted temperature transponders were inoculated intratracheally with ∼106 plaque-forming units (pfu) of SVV-EGFP (n = 3 animals; 1 male and 2 females) or SVV-wt (n = 2 animals; 1 male and 1 female) diluted in 5 ml of phosphate-buffered saline [21]. Just before infection, animals were sedated with ketamine and medetomidine. The abdomen and back of the animals were shaved to allow careful examination for skin rash every other day until necropsy. Heparinized blood samples were collected under light ketamine sedation at 0, 2, 7, 11, 13, 17 and 20 dpi. Bronchoalveolar lavage (BAL) samples and peripheral blood (PB) samples were collected under deep anesthesia at 5 and 9 dpi. Three punch biopsies (3 mm) of varicella rashes and EGFP fluorescent skin tissue, while showing no characteristic varicella-like skin rash by the naked eye, were obtained from anesthetized animals at 9 dpi under anesthesia. SVV-EGFP−infected animals were checked for macroscopic EGFP fluorescence using a custom-made lamp containing 6 LEDs (peak emission 490–495 nm) mounted with D480/40 bandpass filters [22]. Fluorescence was detected by an amber cover of a UV transilluminator used for screening DNA gels [22]. Photographs were taken using a Nikon D80 SLR camera. SVV-infected animals were euthanized at 9 dpi (n = 2; one SVV-wt- and one SVV-EGFP−infected animal), 13 dpi (n = 2; one SVV-wt− and one SVV-EGFP−infected animal) and 20 dpi (one SVV-EGFP-infected animal). Multiple tissues including lung, lymph nodes, spleen, tonsils, skin and ganglia were collected at necropsy and either snap-frozen or fixed and paraffin-embedded.
Collection and processing of PB and BAL samples
PBMC were isolated by density-gradient centrifugation and used for virus isolation, DNA isolation and flow cytometry or cryopreserved as viable cells as described [19]. Cells recovered from BAL samples were centrifuged, dissolved in RPMI-1640 medium supplemented with 10% FBS plus antibiotics (R10F medium), and used for virus isolation, DNA isolation and flow cytometry as described [22].
Virus isolation from PB and BAL samples
Infectious SVV was isolated from PB and BAL cells by incubating 1–2×106 cells in 10-fold serial dilutions in R10F medium on confluent monolayers of BSC-1 cells in 6-well plates. Cells were monitored for SVV-induced CPE or EGFP expression after 7 days of co-cultivation and results were expressed as numbers of SVV-infected cells per 106 input PBMC and BAL cells.
Nucleic acid extraction and quantitative PCR (qPCR)
DNA was isolated from PBMC, BAL cells, pooled ganglia, pooled lymph nodes, tonsils and spleen using a QIAamp DNA Mini Kit (Qiagen). qPCR was performed in triplicate on a ABI Prism 7500 using Taqman 2× PCR Universal Master Mix (Applied Biosystems) with primers and probes specific for SVV open reading frame 21 (ORF21) and the pan-primate single-copy gene oncostatin-M (OSM) as described [14], [21], [54]. DNA dilutions obtained from uninfected PBMC were used to validate the OSM Taqman assay.
Flow cytometry
PBMC were either directly used for flow cytometry to detect EGFP+ cells or stained for indicated markers using fluorochrome-conjugated mAbs: CD3APC-Cy7 (clone SP34-2), CD4AmCyan (L200), CD8PerCp (SK1), CD14PE (M5E2), CD16AF647 (3G8), CD20PE-Cy7 (L27) and HLA-DRPacificBlue (L243) (all from BD Biosciences) to delineate SVV-infected PBMC subsets. To identify SVV-infected T-cell subtypes, PBMC from infected AGMs were stained with mAbs specific for CD3APC-Cy7 (SP34-2), CD4PacificBlue (L200), CD8MCyan (SK1), CD28APC (28.2), CD95PerCp (DX2), CCR4PE-Cy7 (1G1) (all from BD Biosciences) and CD137PE (4B4-1; Miltenyi biotec). T-cells were categorized into naive, central memory (CM) and effector memory (EM) T-cells based on differential expression of CD28 and CD95 (Fig. S2) [28]. In contrast to humans and macaque species, AGMs have three distinct CD3pos T-cell populations based on expression of CD4 and CD8α: CD4posCD8αneg (CD4pos), CD4neg CD8αdim (CD8dim) and CD4negCD8αbright (CD8bright) (Fig. S2)[28]. BAL cells were stained as described for PBMC, except for inclusion of anti-CD45APC (MB4-6D6; Miltenyi biotec) instead of anti-CD16 mAb. Fluorescence was detected on a FACS Canto II and analyzed using FACS Diva software (BD Biosciences). At least 106 viable cells were measured to accurately identify EGFPpos cells.
In situ analyses
Immunohistochemical and immunofluorescence staining was performed using predefined optimal dilutions of primary mAbs directed against: CD3 (clone F7.2.38; Dako), CD11c (NCL-L-CD11c-563; Novocastra), CD20 (L26; Dako), CD68 (KP1; Dako), NCAM (123C3.D5; Thermo Fischer Scientific), GFAP (4A11; BD Biosciences), keratin (AE1/AE3; Thermo Fischer Scientific), α-smooth muscle actin (1A4; Sigma-Aldrich) and rabbit polyclonal antibodies directed against GFP (IgG fraction; Invitrogen) and SVV nucleocapsid proteins [19]. As isotype controls, sections were incubated with mouse IgG1, IgG2a and IgG2b and rabbit immunoglobulins (Dako). Paraformaldehyde-fixed (4%), paraffin-embedded tissue sections were deparaffinized, rehydrated, subjected to heat-induced antigen retrieval in citrate buffer (10 mM, pH = 6.0), blocked and incubated with primary antibodies overnight at 4°C as described [46], [55]. Immunohistochemical staining was visualized using the avidin-biotin system (Dako) in combination with 3-amino-9-ethylcarbazole (AEC) (Sigma-Aldrich) and sections were counterstained with hematoxylin (Sigma-Aldrich) as described [46], [55].
For immunofluorescence staining, sections were incubated with secondary Alexa Fluor 488 (AF488)- or AF594-conjugated goat-anti-mouse and/or goat-anti-rabbit antibodies and mounted in Prolong Gold Antifade reagent with 4′,6-diamidino-2-phenylindole (Invitrogen) [56]. Sections were analyzed on a Zeiss LSM 700 confocal laser scanning microscope fitted on an Axio Observer Z1 inverted microscope (Zeiss). Images were obtained using 2–4× frame averaging and the pinhole adjusted to 1 airy unit. ZEN 2010 software (Zeiss) was used to adjust brightness and contrast.
Supporting Information
Figure S1.
Gating strategy for flow cytometric differentiation of bronchoalveolar lavage (BAL) cells of African green monkeys. BAL cells were gated on viable cells based on forward scatter (FSC) and sideward scatter (SSC) properties and defined as CD45neg cells or CD45pos leukocytes. CD45pos BAL leukocyte subsets were defined as follows: CD3negCD20negMHC-IIposCD14pos/dim = alveolar macrophages (AM) or dendritic cells (DC); CD20posMHC-IIpos = B-cells; CD3posT-cells; CD4negCD8αhigh = CD8bright T-cells, CD4negCD8αdim = CD8dim T-cells, and CD4posCD8αneg = CD4pos T-cells.
Figure S2.
Gating strategy for flow cytometric differentiation of PBMC subsets from African green monkeys. (A) Viable lymphocytes were selected based on forward scatter (FSC) and sideward scatter (SSC) properties and PBMC subsets were defined as follows: CD3posCD16neg = T-cells; CD3negCD16pos = natural killer (NK) cells; CD3negCD14posMHC-IIpos = monocytes; CD20posMHC-IIpos = B-cells; CD3negCD20negCD14negCD16negMHC-IIpos = dendritic cells (DC). (B) AGM-specific T-cell subsets were categorized based on the expression of CD8α and CD4: CD4negCD8αhigh = CD8bright T-cells, CD4negCD8αdim = CD8dim T-cells, and CD4posCD8αneg = CD4pos T-cells. (C) Based on the differential expression of CD28 and CD95, T-cells were categorized as naive (CD28posCD95neg), central memory (CM; CD28posCD95pos) and effector memory (EM; CD28negCD95pos) T-cells.
Figure S3.
Peripheral blood CCR4pos and CD137pos T-cells were not preferentially infected in African green monkeys. Flow cytometric detection of EGFP expression in central memory (CM) and effector memory (EM) T-cells at 5 dpi (A) and 7 dpi (B). Gating strategy was according to Figure S2. Data are given as means ± SEM.
Figure S4.
Memory T-cells were preferentially infected in vitro. (A) SVV-naive African green monkey peripheral blood mononuclear cells (PBMC) were infected with SVV-EGFP in vitro and stained 24 hr later for SVV proteins to show that EGFP fluorescence (green) co-localized with SVV proteins (red). Nuclei were counterstained with DAPI (blue). Magnification: 400×. (B) African green monkey PBMC were infected with SVV-EGFP in vitro and analyzed 24 hr later by flow cytometry for EGFP expression in the indicated lymphocyte subsets. Data are plotted as the frequency of EGFPpos cells within individual PBMC subsets (within subset) or as the percentage of EGFPpos cells within each lymphocyte subset relative to the total number of PBMC (absolute). (C, D) Percentage of EGFPpos cells in the indicated T-cell subsets as assessed by flow cytometry. The lymphocyte subsets were defined as described in Figure S2. Data represent means ± SEM of three independent experiments performed on PBMC from three animals. * p<0.05 by Mann-Whitney test.
We thank Rory D. de Vries, Monique van Velzen, Freek B. van Loenen and Gijsbert P. van Nierop for technical assistance.
Author Contributions
Conceived and designed the experiments: WJDO RM BLH ADMEO GMGMV. Performed the experiments: WJDO GvA SG. Analyzed the data: WJDO RLdS BLH SG GMGMV. Wrote the paper: WJDO RM RLdS BLH DG ADMEO GMGMV.
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text:
- Trending Now: More Trending:
- Svengali syzygy hegemon repugnant white nationalist, white supremacist
make an appearance
Definition of make an appearance
: to go to an event, gathering, etc., usually for a short period of time The candidate made an appearance at the rally.
Word by Word Definitions
: behave, act
: to begin or seem to begin (an action)
: to cause to happen to or be experienced by someone
: the manner or style in which a thing is constructed
: brand
: the physical, mental, or moral constitution of a person
: external show : semblance
: outward aspect : look
: outward indication
Seen and Heard
What made you want to look up make an appearance? Please tell us where you read or heard it (including the quote, if possible).


text:
I though it was extremely rare.
We broke up almost 2 weeks ago because of the distance but we still talk and we get along well.
Yesterday we were talking and he told me "You need to sleep with someone and tell me if he was better than me"
It means he never really cared about me?
Most Helpful Guy
He knows your done, but still thinks about you sexually. Thinking about you having sex with someone is making him horny. You telling him if he was better validates that you did it, making his fantasy more real and also helps him because he's self conscious about his performance sexually.
If he is the more kinky type maybe he's hoping to work up to a 3 way? My girlfriend and I like to include other women in sex. But that is outside social norms so its hard for a guy to ask straight up. You have to ease into it. Maybe this is a start?
Whatever the case, I agree with him. Your apart now, move on and do it. Sure tell him about it, it will help him to, because whatever his thoughts were I don't think it will hurt. It will either help him move on also, or maybe help with his how thoughts and fantasies.00|00|0
Most Helpful Girl
He is Being Sarcastic and wants your Reaction to an Action that he Feels he Needs to know and that Is... Would you dare?
Of course he cares, this is why he said it. He still has feelings for you and is being territorial in his own way and Curious to see if you may have even have Done the Dirty.
Good luck. xx10|10|0
mho. paris knows her shit.


text:
Yale has four $25,000 entrepreneurship prizes that share a single application. These prizes are designed to generate world-changing ideas from Yale students around health, education, environment, equality, sustainability and technology.
DEADLINE: Thurs., March 30, midnight
The PRIZES:
- Thorne Prize for Social Innovation in Health or Education: This $25,000 cash prize is awarded by InnovateHealth Yale to the best student-led venture focused on social innovation in health or education.
- Sabin Sustainable Venture Prize: The Sabin Prize, managed by the Yale Center for Business and the Environment, awards cash prizes totaling $25,000 to students and/or faculty with the best ideas for products, services, projects, or programs that advance a more sustainable way of life.
- Miller Prize: The Miller Prize is a $25,000 entrepreneurship prize from the Yale Entrepreneurial Institute for a student venture with a tech or tech-enabled service startup.
- Aetna Foundation Prize for Health Equity Innovation: The Aetna Foundation Prize for Health Equity Innovation is a $25,000 cash prize awarded by InnovateHealth Yale with the support of the Aetna Foundation to the best student-led venture focused on developing a technology solution to address a health disparity challenge.
Finalists for all four prizes will pitch before panels of expert judges at Startup Yale, a major entrepreneurship event held April 21 at Yale School of Management. Winners of three prizes–the Thorne, Sabin and Miller Prize–all receive automatic acceptance into the YEI Fellowship, an 8-week summer bootcamp for accelerating ventures at the Yale Entrepreneurial Institute that includes $15,000, mentorship, pitching oppotunities, education and breakfast & lunch.
QUESTIONS? CHECK OUT OUR YALE PRIZES FAQs.
WANT MORE INSIGHTS? COME TO AN INFO SESSION! Find out more about the application process, hear from past winners, and get advice on your application. We will be holding three sessions across the campus, so sign up for the one that works best for you.
Yale Prizes Info Sessions – REGISTER HERE
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Questions? Contact YEI Program Director Kassie Tucker, firstname.lastname@example.com


text:
Some things that are seen cannot be unseen:
This is still less disturbing than finding your mom reading the book, I suppose. But only a little.
Some things that are seen cannot be unseen:
This is still less disturbing than finding your mom reading the book, I suppose. But only a little.
Padres first baseman Wil Myers hit an RBI single off of Nick Pivetta in the bottom of the fourth inning of Wednesday afternoon's game, giving his team a 1-0 lead. He then proceeded to steal second base, then third base, and finally home on a double-steal, scoring the Padres' second run.
Per CSN Philly's Marshall Harris, it's the first time a player has stolen all three bases in the same inning since Marlins second baseman Dee Gordon in 2011. Indeed, on July 1 that year, Gordon stole all three bases against Angels pitcher Bobby Cassevah.
Myers is currently batting .238/.322/.459 with 24 home runs, 59 RBI, 61 runs scored, and 14 stolen bases in 491 plate appearances this season.
Jon Morosi hears that the Marlins are "willing to engage with other teams" on a possible Giancarlo Stanton trade.
As we noted yesterday, Stanton has cleared revocable waivers, so he's eligible to be dealt to any club. The price for Stanton is likely to be high given that he's enjoying a career year, batting .285/.376/.646 with a league-leading 44 home runs and 94 RBI in 116 games this season. He's also, obviously, the cornerstone of the franchise.
You also have to assume that anyone looking to acquire Stanton would want the Marlins to chip in money on his $285 million contract. If not, someone might've simply claimed him on waivers with the hope that the Marlins would simply let him walk, right? Which suggests that any negotiation over Stanton would be a long and difficult one. It might also involve Stanton agreeing to restructure his deal, which currently gives him an opt-out after the 2020 season. That would likely involve the MLBPA as well, which just makes it all the more complicated.
I think it's a long shot that the Marlins would trade Stanton in-season, but it's not hard to imagine him being traded this winter.


text:
1. This is a method the player uses to control the ball by using their head. A player can pass and even score a goal by deflecting the ball off their head.
1. Beckham launches the corner kick and bends it precisely in front of the goal where Donovan is able to execute the perfect header that sends the ball over the goalkeeper's reach and into the top net.
Sport The Term Is Used
1. Soccer
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text:
Do you listen to FM radio on your phone?
0. phoneArena posted on 17 Apr 2013, 04:44
It has probably come to your attention that Samsung decided to release its newest flagship smartphone, the Galaxy S4, without a built-in FM radio. While the company tried to explain this decision with the fact that its customers tend to use more digital services nowadays, this kind of sounds strange, because Samsung is used to providing its customers with options, even though many may not need them...
This is a discussion for a news. To read the whole news, click here
1. nobelset (Posts: 270; Member since: 17 Oct 2012)
No harm in adding fm option, what if there was an alien invasion by aliens who like eating submarine communications cables and the only way to communicate is through radio? Yea, that's a stupid example but you get the general idea.
28. akki20892 (Posts: 3902; Member since: 04 Feb 2013)
35. EXkurogane (Posts: 863; Member since: 07 Mar 2013)
I want a FM transmitter, not receiver though. I need it for my car audio system. And, only my old Symbian phones have it.
39. weinerslav (Posts: 126; Member since: 31 Jul 2012)
I want both, but have to agree that the transmitter is the one that I want (and use) more
2. TreyTreyTaylor (Posts: 709; Member since: 21 Dec 2010)
Last phone I had with FM Radio was my DROID X. Kinda forgot about it after my nexus and note 2. It'd be nice to have though....
3. tiara6918 (Posts: 2111; Member since: 26 Apr 2012)
I use the fm radio when I already get sick of the songs on my phone
4. abdane (Posts: 494; Member since: 07 Oct 2011)
hahaha same comment i was about to write ! ^_^
25. Ohrules (Posts: 327; Member since: 11 Jun 2012)
and when you are without tv and want to listen to cricket commentary on the radio
5. windroid (unregistered)
Just didn't bought a WP already because the FM radio is still to be released by MS.
32. snowgator (Posts: 3604; Member since: 19 Jan 2011)
Which is beyond stupid, and proves the trend, because my Samsung WP 7 has the radio, and I have used it from time to time. Would miss the option.
42. windroid (unregistered)
Because the base of WP8 is completely different from WP7, it wasn't just a case to migrate the radio functionality to the new platform.
MS assumed it just hadn't time to develop the radio in WP8.
6. TechBizJP08 (Posts: 495; Member since: 25 Mar 2013)
Radio simply gives you the option to listen on the trending music. The most listened, the top 10,20,100. New singles, new release, new albums. Pretty much any news from your favorite celebrities and artist. It has been in our life ever since it was created. And we can listen for free without using data or wifi. I guess it shouldn't hurt anyone to include in the package. :)
7. hipnotika (Posts: 353; Member since: 06 Mar 2013)
poor s4 :) it hasnt got any fm radio :( ooo my littleeee sweetieee
9. eliasafram7 (Posts: 83; Member since: 18 Mar 2013)
poor iphone it hadn't got any FM radio since the first iphone ;)
11. hipnotika (Posts: 353; Member since: 06 Mar 2013)
poor i-fan :) it hadn't got any brain :)
53. ZeroCide (Posts: 785; Member since: 09 Jan 2013)
LOL! Preach on preacher!
8. MrJerry (Posts: 403; Member since: 05 Oct 2012)
I do but i use Apps for that
10. wendygarett (unregistered)
Nope, I don't need to
22. recond9 (Posts: 50; Member since: 11 Jan 2013)
Dude,don't say your opinion here anymore...the trolls will make you 'bleed' from these thumbs.. lol
43. windroid (unregistered)
Now we all know what's the deal with samsung:
I'm interested to know how much samsung is paying you to troll WP topics.
12. InspectorGadget80 (unregistered)
I basicall dont listen to the radio. Now and days todays music kinda sucks. So i listen too old tracks from the 90s-2003 & the late 80s. Only download a new song if it have a good beat
13. Nathan_ingx (Posts: 4319; Member since: 07 Mar 2012)
I agree with the music part... Music these days are spoiled by the remix and all that party beats and stuff! I don't call that music...
And i'm quite surprised these polls came out after the S4 has been released!
Call it co-incidence but i don't like it. First the uni-body versus the removable battery cover and now devices with the FM versus devices without FM.
14. scriptwriter (Posts: 396; Member since: 13 Nov 2012)
I have samsung galaxy nexus which has no fm radio but if it did, i'd use it from time to time.
15. xperiaDROID (banned) (Posts: 5629; Member since: 08 Mar 2013)
Yes, I do, they play many latest trending songs! FM radio rocks! Everything you want to listen is all inside the radio, American pop, K-pop etc.
FM radio rocks! :D
16. scriptwriter (Posts: 396; Member since: 13 Nov 2012)
I say turn off fm radio all together and sell the bandwidth to better 4G services or in preperation of 5G services
17. karas (Posts: 82; Member since: 08 Jul 2011)
Why dont these manufactures read phone arena polls?
18. pookiewood (Posts: 631; Member since: 05 Mar 2012)
I don't but my brother in law would. He got a L920 also and was hoping for iHeart Radio. Not sure why they ditched us. I told him the phone has built in FMand MS is going to enable it in an update.
19. Edison64 (Posts: 1; Member since: 17 Apr 2013)
Is a big mistake to think that people only listen to the radio for music...!
20. Tsepz_GP (Posts: 1042; Member since: 12 Apr 2012)
Nah, I don't listen Radio except when in my car and even then 90% of the time I play music from my phone through my Jeep's audio system, and only listen to FM Radio now and then, still though, I like to have my options!
21. recond9 (Posts: 50; Member since: 11 Jan 2013)
If they remove it just because ''it's old and no one use it'' they've done a mistake.There are lots of people who use Fm everyday,especially on the third world countries (some will buy it there,eventually).Even I use it, because my WIFI isn't strong enough in the kitchen and i hook my s3 to the audio system everyday,for some radio love...
But then again... if they remove it for the space and other hardware things,i am fine. :)


text:
Workers at one company are looking forward to the next Game of Thrones premier more than most after bosses promised them the following morning off.
A school in Washington state raised a few eyebrows last week when it turned the old cliché of days off for bad weather on its head, offering a "sun day" to its staff and students
That's one way to hang on to employees: a New York realtor is giving his employees a financial incentive to get the company logo tattooed on their bodies.
In an excerpt from the July 1943 edition of a publication called 'Transportation Magazine', male supervisors received some useful advice about managing women in the workplace.
As far as job specifications go, it's not your typical list: must have loud voice and be willing to brave the elements for three weeks.
Just in case any evidence was needed that employees take their coffee extremely seriously, an email outlining in fastidious detail the last known whereabouts of a worker's favourite mug has gone viral.
A.J. Clemente's first words as an anchor on a live news show were a bit blue – and there was no time for beeping.
It's an important HR rule that the guidelines and policies apply equally to management as front line staff. One US judge took that to heart, finding himself in contempt of court.
A recent communications graduate has launched a Kickstarter-inspired resume that offers rewards to those who share it across social media platforms.
A year two teacher has been fired after the state learned he urinated in class and had his students clean up.
Resignations can illicit feelings of relief or panic depending on the individual, but it's hard to stay mad if the consolation is a piece of cake.


text:
Lots And Land
- $25,000 price
- 789326 mls id
- Lot Size: 0.23 acres
- Neighborhood: 343 - SE Palm Bay
- Status: Active
Are you looking for a Palm Bay lot with city water and sewer? This beautiful lot on Brocksmith St has both city water and sewer. Build your home here and enjoy the Florida lifestyle. Possible owner financing available. Call David Moallem at 321-626-3590!
- Site Link
- Property Sub Type Single Family Detached
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Listing Agent: M David Moallem
Courtesy of: M. David Moallem, Inc
Office Phone: 321-917-6596
Updated: 22nd August, 2017 11:21 PM.
979 Brocksmith Street Palm Bay, FL


text:
Country Wise Production and Distribution of Sugarcane around the World! Sugarcane is grown in a very wide range of tropical and sub-tropical countries but the largest producers are from Latin America and Southern and Eastern Asia.
Tag Archives | Sugarcane
Geographical Conditions Required for the Growth of Sugarcane
Geographical Conditions Required for the Growth of Sugarcane! The most important commercial crop of tropical countries is sugarcane. Sugarcane is a member of the grass family, and is a tall tropical variety, with a hard, thick stem and sugar is stored in them.
Sugarcane Cultivation in India: Conditions, Production and Distribution
Sugarcane Cultivation in India: Conditions, Production and Distribution! Sugarcane belongs to bamboo family of plants and is indigenous to India. It is the main source of sugar, gur and khandsari. About two-thirds of the total sugarcane produced in India is consumed for making gur and khandsari and only one third of it goes to sugar […]
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