It’s Over and You Did Nothing
by Daniel J Towsey
While you slept on your couch and your Pacifier HD Wide screen television spewed out lies and deceptions.
Liberties ended, freedom ended, Your rights ended, your constitution all gone. Crowds of armed Illegals invaded your country.
All the while the warning signs were all around you. I think your digital surround sound was too loud so You ignored them all.
your immediate selfish wants were more important than everything else.
You absolutely refused to believe that anything was really wrong.
For you to even consider that anything was wrong, would of meant that you had to go where insanity dwells.
Every-time you may of heard a neighbor or stranger express to you that something is very wrong, you choose to ignored it. And you thought the messenger was insane.
Will that’s where you were deadly wrong. It’s not the messenger that’s insane. It’s the ones that control the money that are insane.
The messenger was just trying to tell you about the insanity going on all around you. BuT the closed minded like you know everything, But see nothing.
Now it’s really over, for the world is now run and controlled by the insane.
The money masters have gone completely mad and out of their minds.
There was nothing to stop them. No one said anything, No one Spoke up. Except a few very brave and honorable freedom fighters.
And many more.
President John F Kennedy in his last speech warned us all about secret societies. They killed him for his efforts.
But you sat there getting fatter, lazier and dumber in front of your television, while your civilization was being covertly and secretly destroyed. But you did not care. It had nothing to do with you.
Will soon after the NWO masters take full control. all is going to go to hell. They are actively now finalizing their plans to de-populate the world. [ Don’t stop reading now and running to your tv ]
It has now been proven that the Anthrax came from Fort Detricks biological warfare lab. It has also been proven that the HIV Aids virus was created from a virus gathered from Icelandic sheep in 1932 and then they used monkey serum to manufacture it. They then gave out HIV Infected Hepetitis Shots to gays in NY and L.A. Then they put the HIV Aids in Polio vaccines for Africa.
Want some proof. Here’s the Patent for HIv US Patent For Aids # 4647773
[ See Attached Document below ] that the US Government holds. And the 1997 Cure for HIV Patent USA Patent 5676977 CURE FOR AIDS (Tetrasil/Imusil)
Everything is in place. They, like the Nazi’s have created a concentration prison population to run their factories. They have over 800 concentration camps scattered all around the country, that they are just beginning to fill.
Have you noticed that the prisoners in US jails has gone from about 1.6 million in 2000 to about 9 million now?They are using the exact same plans as they used in Germany.
THe signs are all around you. But still you watch that stupid propaganda television. While you had the most powerful mechanism ever created for you to learn what has been happening. You have had access to thousands of independent Internet media, writers, researchers, investigators etc etc … All free.
The information was available 24 hours a day..
But still you would not get off your lazy fat ass and go use your computer and actually start using your mind and visit sites like www.rense.com http://mathaba.net www.alternet.org www.globalresearch.ca www.informationclearinghouse.info www.commondreams.org and thousands more. The list is endless. THey have all been putting the warnings out for years. But now all these websites are going to be made criminal with this new H.R 1955 “THought Crimes” act that is about to be made law. Soon they won’t be able to tell you the truth about anything the terrorist NWO is doing.
After that it’s all over…..
THe NWO government will then have free reign to do to anything it wants with your Widescreen HD Television. And under the provisions in the Patriot act you will disappear if you tell anyone.
hey don’t worry about closing your curtains. With the invasion of privacy laws they have already passed. They have access to all your private information. THey know everything that everyone is buying. They know where you live. THey can listen in anytime they want through your cell phones and vehicles because they have listening systems and GPS built in.
You have been totally f__cked while your head was buried in the dirt and you had your but up in the air. That really attracted them to violate you. Even if your curtains oh I mean britchises were on.
You don’t have to be guilty of anything….
Hey if a black opts team needs some extra funds for a HD Widescreen Television for their club house and they drive by while your window is open and see it, will humm …They can just take it.
All that violence from movies, television, video games has done a really good job of creating a really dumbed down, sick and violent society.
The violence you have done to the rest of the world is now going to be turned on you….
9/11 should of really woke you up…..but you probably thought it was no different than the rest of the violence you watched on your Widescreen HD Television. Soon you’ll be schocked to see how truly realistic that HD Television is With surround sound.
You are soon going to experience anarchy and martial law, where the insane will really show you how they don’t think about you.
They have disarmed all the populations of the world. Your country is the last democracy that is still armed….
But again if you had tuned in to the alternative media on the net. You would be aware of your NWO government starting to disarm your population. THe first group they are taking guns from is ex- military. As they are truly afraid of people who know how to use their weapons….
You can’t run for the hills as all Public Lands in the USA has been secretly handed over to the UN…
Regrettably one day you will remember when you bullied geeks and nerds like me….
The nerds are now your only chance to learn what is happening.
You will soon regret bullying and attacking the vulnerable and defenseless nerds of the world….
Nerds know how to count. And your number is up…..THe NWO Masters are deliberately destroying your currency and economy.
You have been completely set up. Most of your manufacturing and most of your good paying jobs have been shipped out. Even Fort Knox is empty.
Your food is being genetically altered so farmers can not reuse their seeds. Monsanto has created what is called suicide seeds. They do not germinate.
The farmers have to get their seeds from Monsanto every year or you starve…
I have barely touched on the insanity that is going on…. Please read my “REcipe for insanity” if you want to better understand.
The NWO Masters have completely dumbed down and disarmed you. Through lies and deceptions. While you slept in front of that Widescreen HD Television. It’s amazing the subliminal effects a Digital Sound television can have on you while you are resting, not to mention what it does while your awake.
It’s ok this is all an illussion, just change the channel and everything will be alright. CLICK!
They have taken the most powerful weapon from you, and that is the knowledge of truth. For true words are mightier than a bomb.
That’s why the truth can blow your mind.
Signed: The Literary Bomber
loaded with thousands of resources,videos,links,articles,documents,photos
Recipe for insanity
Patent To MAke Aids
USA Patent # 5676977 CURE FOR AIDS
Michael C Rupert
From the Wilderness – Mike Rupert
The whole picture – 2 1/2 hours – the Rockefeller family – CFC – Bush – 9/11 – Bin Laden – Carlyle Group – the CIA – investment banks – patriot act – internment camps – financial fraud – iran contra – cocaine – heroin – Wall Street – the drug trade – S & L crisis – the DEA – Iraq – Iran – Pakistan – nuclear weapons – looting social security – smallpox vaccine – Citibank and moreIt’s all connected and Ruppert shows how.After having his life threatened numerous time, Michael Ruppert has left the United States and says he will no longer engage in investigative reporting.
US politics has crawled through the gutter for so long, it’s easy to forget that the country was founded on ideals of freedom and human dignity. Or that in the past it has been led by people of character and intelligence. Here’s an important message from the past that speaks directly to where the US is today and what it must do to regain what it has lost
Aron Russo’s “America from Freedom to Fascism”
HERE IT IS…..YOU HAVE NO EXCUSE NOW…..YOU CAN’T SAY YOUR NOT INFORMED…WATCH THIS ONE
The U.S. Cure for AIDS-Patent #5676977 (Tetrasil/Imusil)
By Boyd E. Graves
The U.S. Cure for AIDS-Patent # 5676977 (Tetrasil/Imusil)
Date : Mon, 29 Jul 2002 21:27:06 -0700 (PDT)
United States Patent 5,676,977 Antelman October 14, 1997
Method of curing AIDS with tetrasilver tetroxide molecular crystal devices Abstract The diamagnetic semiconducting molecular crystal tetrasilver tetroxide (Ag.sub.4 O.sub.4) is utilized for destroying the AIDS virus, destroying AIDS synergistic pathogens and immunity suppressing moieties (ISM) in humans. A single intravenous injection of the devices is all that is required for efficacy at levels of about 40 PPM of human blood. The device molecular crystal contains two mono and two trivalent silver ions capable of "firing" electrons capable of electrocuting the AIDS virus, pathogens and ISM. When administered into the bloodstream, the device electrons will be triggered by pathogens, a proliferating virus and ISM, and when fired will simultaneously trigger a redox chelation mechanism resulting in divalent silver moieties which chelate and bind active sites of the entities destroying them. The devices are completely non-toxic. However, they put stress on the liver causing hepatomegaly, but there is no loss of liver function. ——————————————————————————– Inventors: Antelman; Marvin S. (Rehovot, IL) Assignee: Antelman Technologies Ltd. (Providence, RI) Appl. No.: 658955 Filed: May 31, 1996 Boyd E. Graves, J.D. US Supreme Court Case No. 00-9587 http://www.supremecourtus.gov/docket/00-9587.htm Cell – 619-204-5683 Fax – 785-263-1568 Email – email@example.com Web – http://www.boydgraves.com http://www.boydgraves.com
By Boyd E. Graves firstname.lastname@example.org Fax – 785-263-1568
(c) Independent Media Center. All content is free for reprint and rebroadcast, on the net and elsewhere, for non-commercial use, unless otherwise noted by author. IMC not for content (expand this). more…
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Method of continuous production of retroviruses (HTLV-III)
from patients with AIDS and pre-AIDS
4647773 Method of continuous production of retroviruses (HTLV-III) from patients with AIDS and pre-AIDS
Inventor: Gallo, et al.
Date Issued: March 3, 1987
Filed: April 23, 1984
Inventors: Gallo; Robert C. (Bethesda, MD)
Popovic; Mikulas (Bethesda, MD)
Assignee: The United States of America as represented by the Department of Health (Washington, DC)
Primary Examiner: Warren; Charles F.
Assistant Examiner: Tarcza; John Edward
Attorney Or Agent: Roberts, Jr.; John S.
U.S. Class: 424/208.1; 435/235.1; 435/239; 435/372.3; 435/948
Field Of Search: 435/235; 435/239; 435/240; 435/948; 435/5; 435/29; 424/89; 128/1T
U.S Patent Documents: 4464465; 4520113
Foreign Patent Documents:
Other References: Popovic et al, Science, 224(4648):497-500, May 4, 1984..
Gallo et al, "Isolation of Human T-Cell Leukemia Virus in Acquired Immune Deficiency Syndrome (AIDS)", Science, 220, pp. 865-867 (5-1983)..
Barre-Sinoussi et al, "Isolation of a T-Lymphotropic Retrovirus from a Patient at Risk for AIDS", Science, 220, pp. 868-871 (5-1983)..
Marx, "Strong New Candidate for AIDS Agent", Science, 224, pp. 475-477 (5-1984)..
Abstract: A cell system is disclosed for the reproducible detection and isolation of human T-lymphotropic retroviruses (HTLV-family) with cytopathic effects (HTLV-III) from patients with the acquired immune deficiency syndrome (AIDS), pre-AIDS and in healthy carriers. One neoplastic aneuploid T-cell line derived from an adult with lymphoid leukemia, and termed HT, was susceptible to infection with the new variants of HTLV, which are transformed and providing T-cell populations which are highly susceptible and permissive from HTLV-III, and convenience for large scale production, isolation and biological detection of the virus.
Claim: We claim:
1. A method for continuous production of HTLV-III virus which comprises infecting in cell culture highly susceptible, permissive cells consisting of a neoplastic aneuploid HT-cell linewith said virus, said cells preserve the capacity for permanent growth after the infection with said virus, growing said cells under conditions suitable for cell growth and recovering said virus produced by said cells.
2. The method of claim 1, wherein said virus consists of cytopathic variants of HTLV.
3. The method of claim 1 wherein said infecting comprises cocultivating said virus with said cells to produce a cell line and recovering said cell line.
4. The method of claim 1 wherein said infecting comprises cell-free infection of said cells with said virus.
5. The method of claim 1 wherein said cells are neoplastic aneuploid T-cells derived from an adult with lymphoid leukemia.
6. A process for the continuous production of HTLV-III virus which comprises cocultivating said virus with a target HT-cell to produce an infected cell line, growing said cell line and recovering said virus from supernatants produced by saidcell line.
7. The process of claim 6 wherein said target T-cell is a neoplastic aneuploid T-cell susceptible to infection with HTLV-III.
8. A process for the continual production of HTLV-III by infecting T-cells in cultures which comprises cocultivating HTLV-III virus with an HT-clone to produce an infected cell line, said clone being an aneuploid T-cell line derived fromlymphoid leukemia, growing said cell line and recovering said virus from supernatants produced by said cell line.
9. The process in claim 8 wherein said clone is a mature T-cell phenotype of OKT3.sup.+ (62%), OKT4.sup.+ (39%) and OKT8.sup.-.
10. A method of producing a cell line containing an antigen of HTLV-III which comprises infecting an HT-cell line derived from lymphoid leukemia and susceptible to infection with HTLV-III, said cell line is capable of continuous large-scaleproduction of HTLV-III, and growing the infected cell line under conditions suitable for growth.
11. The method of claim 10 wherein said cell line is a neoplastic aneuploid T-cell line.
12. The method of claim 10 wherein said HTLV-III are variants of human T-lymphotropic retrovirus, exhibit cytopathic effects and are non-transforming.
13. A cell line containing HTLV-III designated H9/HTLV-III.sub.B, ATCC Accession CRL 8543.
14. A process for producing a cell line H9/HTLV-III.sub.B which comprises infecting a target T-cell with HTLV-III virus to produce a cell line and recovering same, said infecting process overcomes the normal cytopathic effect of HTLV-III andpreserves the immortal growth capacity of the target cell.
15. An HT cell line permanently infected with HTLV-III virus, wherein said cell line continually produces said virus.
Description: The present invention describes a cell system for the reproducibledetection and isolation of human T-lymphotropic retroviruses (HTLV-family) with cytopathic or cell killing effects (HTLV-III) from patients with the acquired immune deficiency syndrome (AIDS), pre-AIDS and in healthy carriers. One neoplastic aneuploidT-cell line derived from an adult with lymphoid leukemia, and termed HT, was susceptible to infection with the new variants of HTLV, providing T-cell populations which are highly susceptible and permissive for HTLV-III, and convenience for large scaleproduction, isolation, and biological detection of the virus.
BACKGROUND OF THE INVENTION
The disclosure of this invention is contained in the following journal articles: Gallo et al., "Detection, Isolation, and Continuous Production of Cytopathic Human T-Lymphotropic Retroviruses (HTLV-III) from Patients with AIDS and pre-AIDS,"Science, in press; and Gallo et al., "Human T-Lymphotropic Retrovirus, HTLV-III, Isolated from AIDS Patients and Donors at Risk for AIDS," in press.
Epidemiologic data strongly suggest that acquired immune deficiency syndrome (AIDS) is caused by an infectious agent which is apparently horizontally transmitted by intimate contact or blood products. Though the disease is manifested byopportunistic infections, predominantly Pneumocystis carcinii pneumonia and Kaposi’s sarcoma, the underlying disorder affects the patient’s cell-mediated immunity with absolute lymphopenia and reduced helper T-lymphocyte (OKT4.sup.+) subpopulation(s). Moreover, before a complete clinical manifestation of the disease occurs, its prodrome, pre-AIDS, is frequently characterized by unexplained chronical lymphadenopathy and/or leukopenia involving a helper T cell subset. This leads to the severe immunedeficiency of the patient, suggesting that a specific subset of T-cells is the primary target for an infectious agent. Although patients with AIDS or pre-AIDS are often chronically infected with cytomegalovirus or hepatitis B virus, for various reasonsthese appear to be opportunistic or coincidental infections apparently not linked to the immunological response deficiency. It is believed that the cause of AIDS may be a virus from the family of human T-cell lymphotropic retroviruses (HTLV) which,prior to the present invention, comprised two major well characterized subgroups of human retroviruses, called human T-cell leukemia/lymphoma viruses, HTLV-I and HTLV-II. The most common isolate, HTLV-I, is mainly obtained from patients with matureT-cell malignancies. Seroepidemiological studies, in vitro biological effects, and nucleic acid hybridization data indicate that HTLV-I is etiologically associated with these malignancies, affecting adults primarily in the south of Japan, the Caribbeanand Africa. HTLV of subgroup II (HTLV-II) was first isolated from a patient with a T-cell variant of hairy cell leukemia. To date, this is the only reported isolate of HTLV-II from a patient with a neoplastic disease. Virus isolation andseroepidemiological data show that HTLV of both subgroups can sometimes be found in patients with AIDS.
Evidence suggests that the retrovirus(es) of the HTLV family is an etiological agent of AIDS based on the following: (1) there is precedence for an animal retrovirus cause of immune deficiency (feline leukemia virus in cats); (2) retroviruses ofthe HTLV family are T-cell tropic; (3) they preferentially infect "helper" T-cells (OKT4.sup.+); (4) they have cytopathic effects on various human and mammalian cells as demonstrated by their induction of cell syncytia formation; (5) they can alter someT-cell functions; (6) in some cases infection may result in selective T-cell killing; and (7) they are transmitted by intimate contact or through blood products. The presence of antibodies directed to cell membrane antigens of HTLV infected cells hasbeen shown in sera of more than 40% of patients with AIDS [Essex et al., Science, 220:859 (1983)]. This antigen has since been defined as part of the envelope of HTLV [Schupbach, et al., Science, in press; and Lee, et al., Proc. Nat. Acad. Sci. U.S.A., in press].
The original detection and isolation of the various HTLV isolates were made possible by two earlier developments: the discovery of T-cell growth factor (TCGF), also called Interleukin 2 (Il-2), which enabled the routine selective growth ofdifferent subsets of normal and neoplastic mature T-cells [Ruscetti, et al., J. Immunol., 119:131 (1977); and Poiesz, et al., Proc. Nat. Acad. Sci. U.S.A, 77:6134 (1980)] and the development of sensitive assays for detection of retroviruses based onreverse transcriptase assays. The methods of HTLV isolation and transmission involved a cocultivation procedure using permissive T-cells for the virus. The use of normal human T-cells in cocultivation experiments preferentially yielded HTLV of bothsubgroups with immortalizing (transforming) capability for some of the target T-cells.
However, HTLV variants (now termed HTLV-III), lack immortalizing properties for normal T-cells and mainly exhibit cytopathic effects on the T-cells and is now believed to be the cause of AIDS. In fact, such variants were frequently but onlytransiently detected using these normal T-cells as targets in cocultivation or cell-free transmission experiments. The cytopathic effect was overcome by finding a highly susceptible, permissive cell for cytopathic variants of HTLV, thus preserving thecapacity for permanent growth after infection with the virus. The present invention discloses the identification and characterization of this new immortalized T-cell population and its use in the isolation and continuous high- level production of suchviruses from patients with AIDS and pre-AIDS.
Early experiments identified one neoplastic aneuploid T-cell line, termed HT, derived from an adult with lymphoid leukemia, that was susceptible to infection with the new cytopathic virus isolates.
This cell line is a sensitive target for transmission of these virus isolates (HTLV-III) and it allows continuous large-scale virus production and development of specific immunologic reagents and nucleic acid probes useful for comparison of thesenew isolates among themselves and with HTLV-I and HTLV-II. In addition to their differences in biological effects that distinguish them from HTLV-I and HTLV-II, HTLV-III also differs from these known HTLV subgroups in several immunological assays and inmorphology. However, these new retroviruses are T4 lymphotropic and exhibit many properties similar to HTLV-I and II, including similar properties of the reverse transcriptase, cross reactivity of structural proteins as determined by heterologouscompetition radioimmune assays with patients’ sera and with animal hyperimmune sera, and induction of syncytia. These new retrovirus isolates are collectively designated HTLV-III, together with detectable differences in some of their proteins andgenetic information, HTLV-III’s ability to kill T-cells clearly separates these variants from other members of the HTLV family.
STATEMENT OF DEPOSIT
A cell line corresponding to the present invention, and denoted H9/HTLV-III.sub.B, has been deposited in the ATCC (under ATCC No. CRL 8543) on April 19, 1984, prior to the filing of this patent application. This deposit assures permanence of thedeposit and ready accessibility thereto by the public. H9 is a representative and preferred cell line in accordance with the invention.
The cell line which is a product of the present invention (H9/HTLV-III.sub.B) is presently useful for the production of vaccines for the relief of AIDS and for the detection of antibodies to the virus in blood samples.
A susceptible cell line HT was tested for HTLV before in vitro infection and it was negative by all criteria, including lack of proviral sequences. Continuous production of HTLV-III is obtained after repeated exposure of parental HT cells(3.times.10.sup.6 cells pretreated with polybrene) to concentrated culture fluids containing HTLV-III harvested from short term cultured T-cells (grown with TCGF) which originated from patients with pre-AIDS or AIDS. The concentrated fluids were firstshown to contain particle associated reverse transcriptase (RT). When cell proliferation declined, usually 10 to 20 days after exposure to the culture fluids, the fresh (uninfected) HT parental cells are added to cultures. Culture fluids from theinfected parental cell line was positive for particulate RT activity and about 20% of the infected cell population was positive in an indirect immune fluorescent assay (IFA) using serum from a hemophilia patient with pre-AIDS (patient E.T.). Serum fromE.T. also contained antibodies to proteins of disrupted HTLV-III but did not react with proteins of HTLV-I or HTLV-II infected cells.
As has been mentioned above, an aneuploid HT-cell line exhibited the desired prerequisites for the continuous propagation of HTLV-III. This cell line is a neoplastic aneuploid T-cell line derived from an adult patient with lymphoid leukemia,selected for its mature T-cell phenotype [OKT3.sup.+ (62%), OKT.sup.4 + (39%) and OKT8.sup.- ], as determined by cytofluorometry using a fluorescence-activated cell sorter. Cultures of these cells are routinely maintained in RPMI/1640 with 20% fetalcalf serum and antibiotics. These cultures are shown in Example 1, Table 1. Clone H9 is preferred, with Clone H4 being secondarily preferred.
HTLV-III culture fluids are isolated from cultured cells of patients with acquired immune deficiency syndrome (AIDS). Peripheral blood leukocytes from these patients are banded in Ficoll-Hypaque, incubated in growth media (RPMI 1640, 20% fetalbovine serum 0.29 mg/ml glutamine) containing 5 .mu.g/ml phytohemagglutinin (PHA-P) for 48 hours, at 37.degree. C. in a 5% CO.sub.2 atmosphere. The leukocytes are then refed with growth medium containing 10% purified T cell growth factor (TCGF);optionally, some of the cells also received rabbit antibody to alpha interferon. Cells and growth media from these lymphocytes are then assayed for the presence of HTLV subgroups I-III. Samples exhibiting more than one of the following were consideredpositive: repeated detection of a Mg.sup.++ dependent reverse transcriptase activity in supernatant fluids; virus observed by electron microscopy; intracellular expression of virus-related antigens detected with antibodies from sero-positive donors orwith hyperimmune serum; or transmission of particles, detected by reverse transcriptase assays or by electron microscopic observation, to fresh human cord blood, bone marrow, or peripheral blood T-lymphocytes. All isolates not classified as eitherHTLV-I or HTLV-II by immunological or nucleic acid analysis were classified as HTLV-III. The cells in the HTLV-III producing cell cultures, characterized using established immunological procedures, are predominantly T-lymphocytes (E rosette receptor,OKT/3 and Leu/1 positive, with a T4 phenotype (OKT4, leu 3a positive). This process is also described by Gallo, et al., in Science, 220:865-867 (1983).
The infection of parental HT cells as well as other cloned cell populations occurs by exposure of these cells to concentrated or nonconcentrated culture fluids (cell-free infection) from T-cell cultures from AIDS or pre-AIDS patients, or bycocultivation; that is, HT cells are infected by exposure to HTLV-III containing cultures. The usual cell-free infection procedure is as follows: 2 to 5.times.10.sup.6 cells are treated with polybrene (2 .mu.g/ml) or DEAE dextran for 30 minutes inCO.sub.2 incubator at 37.degree. C., and then exposed to the virus inoculum (0.1 to 1 ml) for one hour in the incubator (CO.sub.2 /37.degree. C.). The cells are kept in suspension by shaking at regular intervals. After one hour of incubation aregular growth medium is added. The positivity of infected cultures for HTLV-III is assessed after one, two, and three weeks of cultivation.
The infection of HT cells (clones) is also obtained by cocultivation procedure–HT cells are mixed in various proportions (usually 1:5) with short- term cultured T-cells (about 5 to 20 days) from AIDS or pre-AIDS patients. The positivity forHTLV-III was scored by the detection of viral antigens or viral nucleic acid sequences in the infected recipient cells at various intervals (7, 14, 21 days, etc.) after cocultivation. The mixed cultures are maintained in growth medium for severalmonths.
As shown in Table 1 below, single cell HT clones were isolated as described by Popovic, et al., in Neoplasma, 18:257 (1971), and Bach, et al., Immunol. Rev., 54:5 (1981) from a long-term cultured aneuploid HT-cell line exhibiting mature T-cellphenotype (OKT3.sup.+ [62%], OKT4.sup.+ [39%] and OKT8.sup.-) as determined by cytofluorometry using a fluorescence-activated cell sorter. The cultures were routinely maintained in RPMI/1640 with 20% fetal calf serum and antibiotics. The terminal celldensity of the parental cell culture, seeded at a concentration of 2.times.10.sup.5 cells/milliliter of culture media, was in the range 10.sup.6 -1.5.times.10.sup.6 cells/ml after 5 days of culture.
For detection of multinucleated cells, cell smears were prepared from cultures 6 and 14 days after infection and stained with Wright-Giemsa. Cells having more than 5 nuclei were considered as multi-nucleated cells. Cloned cells from uninfectedcultures also contained some multi-nucleated giant cells as well; however, the arrangement of multiple nuclei in a characteristic ring formation was lacking and the number of these cells was much less (0.7% to 10%).
Immunofluorescence positive cells were washed with phosphate-buffered saline (PBS) and resuspended in the same buffer at concentration 10.sup.6 cells per milliliter. Approximately 50 .lambda. of cell suspension were spotted on slides, airdried, and fixed in acetone for 10 min. at room temperature. Slides were stored at -20.degree. C. until use. Twenty microliters of either hyperimmune rabbit antiserum to HTLV-III (diluted 1/2000 in PBS) or serum from the patient (E.T.) diluted 1/8 inPBS was applied to cells and incubated for 50 min. at 37.degree. C. The fluorescein-conjugated antiserum to rabbit or human immunogloblin G was diluted and applied to the fixed cells for 30 min. at room temperature. Slides then were washed extensivelybefore microscopic examinations. The uninfected parental cell line as well as the clones were consistently negative in these assays.
To determine reverse transcriptase activity, virus particles were precipitated from cell-free supernatant as follows: 0.4 ml of 4M NaCl and 3.6 ml of 30% (wt/vol.) polyethylene glycol (Carbowax 6000) were added to 8 ml of harvested culture fluidsand the suspension was placed on ice overnight. The suspension was centrifuged in a Sorvall RC-3 centrifuge at 2000 rpm at 4.degree. C. for 30 min. The precipitate was resuspended in 300 .mu.l at 50% (vol/vol) glycerol (25 mM Tris-HCl, pH 7.5/5 mMdithiothreitol/150 mM KCl/0.025% Triton X-100. Particles were disrupted by addition of 100 .mu.l of 0.9% Triton X-100/1.5M KCl. Reverse transcriptase (RT) assays were performed as described by Poiesz, et al., Proc Nat. Acad. Sci. U.S.A., 77:7415(1980) and expressed in cpm per milliliter culture medium.
TABLE 1 __________________________________________________________________________ Response of Cloned T-Cell Populations to HTLV-III Infection Clones Characteristics H3 H4 H6 H9 H17 H31 H35 H38 __________________________________________________________________________ Total cell number (.times. 10.sup.6) 6 days after infection 1 1.5 1.5 0.3 0.4 0.3 0.5 1.8 14 days after infection 2.2 7.3 7.5 10.0 4.7 5.0 4.5 3.2 Multinucleatedcells (%) 6 days after infection 24 42 32 7 13 14 30 45 14 days after infection 45 48 45 30 22 45 60 60 Immunofluorescence positive cells (%) 6 days after infection Rabbit antiserum to HTLV-III 55 56 32 32 39 21 10 87 Patient serum (E.T.) 56 2921 ND ND ND ND 73 14 days after infection Rabbit antiserum to HTLV-III 50 74 60 97 71 40 20 80 Patient serum 45 47 56 78 61 43 22 89 Reverse transcriptase activity (.times. 10.sup.4 cpm/ml) 6 days after infection 2.4 1.8 2.1 4.1 2.6 1.4 1.7 2.5 14 days after infection 16.2 18.1 16.1 20.2 17.1 13.4 15.1 18.2 __________________________________________________________________________ ND = not done
As shown in Table 2 below, cocultivation with H4 recipient T-cell clone was performed with fresh mononuclear cells from peripheral blood of patients RF and SN, respectively. In the case of patients BK and LS cocultivation was performed withT-cells grown in the presence of exogenous TCGF (10% V/V) for 10 days. The ratio recipient/donor (patients’) cells was 1:5. The mixed cultures were maintained in RPMI/1640 (20% FCS and antibiotics) in the absence of exogenous TCGF. Cell-free infectionof H9 T-cell clone was performed using concentrated culture fluids harvested from T-cell cultures of the patient WT. The T-cell cultures were grown in the presence of exogenous TCGF for two weeks before the culture fluids were harvested and concentrated. Cells of H9 clones were pretreated with polybrene (2 .mu.g/ml) for 20 min. and 2.times.10.sup.6 cells were exposed for one hr. to 0.5 ml of 100-fold concentrated culture fluids positive for particulate RT activity.
HTLV-III virus expression in both cocultured and cell-free infected cell cultures were assayed approximately one month after in vitro cultivation. There was considerable fluctuation in HTLV-III expression (see Table 2). For details of bothreverse transcriptase (RT) assays and indirect immunofluorescence assays (IFA) see Example 1.
TABLE 2 __________________________________________________________________________ Isolation of HTLV-III from Patients with Pre-AIDS and AIDS Virus Expression IFA with RT Activity Rabbit Serum Human Serum (ET) Patient Diagnosis Origin (.times. 10 cpm) (% Positive) (% Positive) EM __________________________________________________________________________ RF AIDS Haiti 0.25 80 33 ND (heterosexual) SN Hemophiliac U.S. 6.3 10 ND + (lymphadenopathy) BK AIDS U.S. 0.24 44 5 + (homosexual) LS AIDS U.S. 0.13 64 19 + (homosexual) WT Hemophiliac U.S. 3.2 69 ND ND (lymphadenopathy) __________________________________________________________________________ RT = reverse transcriptase IFA = immunofluorescence assays EM =electron microscopy ND = not done
To select for high permissiveness for HTLV-III and to preserve permanent growth and continuous production of virus, extensive cloning of the HT parental T-cell population was performed. A total of 51 single-cell clones was obtained by bothcapillary and limited dilution techniques using irradiated mononuclear cells from peripheral blood of a healthy donor as a feeder. The growth of these cell clones was compared after HTLV-III infection. A representative example of response to virusinfection of 8 T-cell clones which are susceptible to and permissive for HTLV-III is shown in Table 1. In parallel experiments, 2.times.10.sup.6 cells of each T-cell clone were exposed to 0.1 ml of concentrated virus. Then cell growth and morphology,expression of cellular viral antigen(s), and RT activity in culture fluids were assessed 6 and 14 days after infection. Although all 8 clones were susceptible to and permissive for the virus, there were considerable differences in their ability toproliferate after infection. The cell number decreased by 10% to 90% from the initial cell count within 6 days after infection, and a high proportion of multinucleated (giant) cells were consistently found in all 8 infected clones. The percentage ofT-cells positive for viral antigen(s) determined by immunofluorescent assays with serum from AIDS patient (E.T.) and with hyperimmune rabbit serum raised against the whole disrupted HTLV-III ranged from 10% to over 80%. Fourteen days after infection,the total cell number and the proportion of HTLV-III positive cells increased in all 8 clones. The virus positive cultures consistently showed round giant cells which contained numerous nuclei. These multinucleataed giant cells are similar to thoseinduced by HTLV-I and HTLV-II except that the nuclei exhibit a characteristic ring formation. Electron microscopic examinations showed that the cells released considerable amounts of virus.
To determine whether HTLV-III is continuously produced by the infected T-cells in long-term cultures, both virus production and cell viability of the infected clone, H4, were followed for several months. Although the virus production fluctuated,culture fluids harvested from the H4/HTLV-III cell cultures at approximately 14-day intervals consistently exhibited particulate RT activity which has been followed for over 5 months. The viability of the cells ranged from 65% to 85% and doubling timeof the cell population, which is called H4/HTLV-III, was approximately 30-40 hours. Thus, this permanently growing T-cell population can continuously produce HTLV-III.
The yield of virus produced by H4/HTLV-III cells was assessed by purification of concentrated culture fluids through a sucrose density gradient and assays of particulate RT activity in each fraction collected from the gradient. The highest RTactivity was found at density 1.16 g/ml, similar to other retroviruses.
* * * * *
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