Coming soon to a store near you?

"Here in the U.S., we've not found any high pathogenicity H5N1 influenza viruses, be it in our wild waterfowl, our domestic poultry, or our human population. But a recent story shows how quickly (and quietly) it could enter our country"

Tara, why are you, as an assistant professor, spreading shit like this over computer screens all over the world?

I agree that the H5N1 is very dangerous indeed because one day I will certainly get badly hurt falling of my seat with roaring laughter at the sight of so much nonsense, but that in itself has not much to do with the infectious diseases you've studied and earn your living with. Might that be the reason you promote the importance of the killer virus H5N1 joke, financial dependancy?

What if H5N1 indeniably turned out to be nonsense? Would your world fall apart?

Think it over, Tara.

JS
----
http://www.nightsofarmour.com

Right on JS. Twice in one day you have shown your high ethics and wonderful wit and intelligence. The other instance that I am referring to is your wonderful note to JPee Moore and his refusal to any type of discussion on paradigms that threaten his pocketbook.

"It's very important to know that this virus can be killed by plain old soap and water, any type of disinfectant or household bleach. If it's in food that is cooked to the proper temperatures, it will die."

I've heard the Disney studios are interested in this script. They are planning a movie entitled "The little virus that wanted to be bacterial"

JS, you have a delightfully original mind. Thanks for the respite from the choking earnestness SEED seems to adore. (Though I can tell you some of their own staff are closeted dissidents.)

Fascinating words: "Seized and destroyed," "ethnic grocery stores," (on the one hand) "mainstream stores and restaurants" on the other.

And my least favorite word in all of reportorial language: "could."

used to be that we understood anything "could" happen but didn't report breathlessly as all that on that which only "could," happen.

The single word "could" is a media portal to mass panic, which is what Bird Flu of course is, except nobody's really buying it. The reason is it reminds them of something that already wore out their deepest nervous system starting in the mid 1980s.

Remember the deadly virus that "could" and maybe even "would" kill up to 70 million Americans by 1990? (Oprah Winfrey, Tony Fauci etc.)

Remember Laurie Garrett's embarasing drivel in SEED about SARS? Vietnam? Everything officials feared "could" happen?

She's not on staff there anymore is she? Was she eventually thanked and released from her services as chaser of imaginary "emerging" viruses and pandemics?

She has a good word for what she imagines happened between myself and Harper's, namely that I "hornswaddled" them with my story about a murdered pregnant woman, a trial in Uganda so bad it spun the minds of Westat, and a scientist who is so "wrong" about HIV one can't even report on the climate of hatred against him without eliciting a 35 page manifesto from "treatment activists" calling for all involved in the formation of the text to quit the profession and "apologize" to the "community."

No I understand Tara Smith has claimed that the catastrophe at ICC in which orphans were hurt and in some cases killed by life-saving drugs tubally cut straight into their stomachs was a fabricated tale? (Liam Scheff's story.) I worked on the documentary that aired on BBC and across Europe on this. I stood on the mass grave and collected each name, and checked the death records, and got two death certificates, which repudiate the NYT lie that "no children died." Those two were only the ones I was able to secure, after months of work.

Are you really not embarassed to take truth and break it in your bare hands Tara? Does nothing inspire you to look, seek, question, investigate?

No I understand Tara Smith has claimed that the catastrophe at ICC in which orphans were hurt and in some cases killed by life-saving drugs tubally cut straight into their stomachs was a fabricated tale?

Once again, Celia, putting words in my mouth. I'm doing exactly what you suggest--being skeptical and questioning.

Naw Tara,

you're not being skeptical, you're being a corporate whore.

And you've libeled me, the NY Press, A&U Magazine, Hustler magazine, the BBC, the Associated Press, GNN, CruxMag, the NY Post, the UK Observer, Jonathan Fishbein, Vera Sharav, the producer and director of the BBC piece, and more.

You're not skeptical, you're a bloody coward. You refuse to answer the very clear question - would you do what has been done to these children, to your own.

If you doubt the veracity of the research, princess, come with your best fucking shot. My books are open.

Liam Scheff

Tara,

"Being skeptical and questioning" means nothing as long as there exists a borderline beyond which questionning becomes absurd.

I asked you:

"What if H5N1 indeniably turned out to be nonsense? Would your world fall apart?"

It was not meant to be an embarrassing question, or a mean one. What I meant to say is that as long as your world would fall apart if something you believe in turned out to be wrong, you cannot really question. Simply because the questions beyond the borderline have only one dogmatic answer and every other answer is unthinkable. Literally unthinkable: you cannot even think of it.

The more you fight against a different solution of the same problem, the less it becomes accessible. The more angrily you become when calling people denialists, the bigger your personnal catastrophe will be the day you have to admit they were right.
Nobody knows the truth about anything and science is not about knowing or not knowing. Science is about people questionning and searching the univers for answers they might never find.

The Gallos and the Wainbergs of the world condemn people who question and call them Denialists. But not the people who question are to be condemned, but those who try to silence. Who silence others for the money and the glory. And in doing so they sell their soul to the devil. This is why Gallo, Chermann, Moore, Wainberg etc. behave so often completely hysterical and go beserk when they have to face what they call denialists.

Think it over, Tara.

JS
----
www.nightsofarmour.com

I am again unable to post my response. I will try it in later, and post it at Barnes world and send it around.
http://barnesworld.blogs.com/barnes_world/2006/07/banned_at_aetio.html#…

------

Just to let everyone in on the joke.

I asked Smith many months ago to answer the 'would you do to your children what is done at ICC'

She did not answer. I asked and re-asked. She deflects, as is her skill and wont, and I asked again.

She then decides to imagine that the story is not true:
http://scienceblogs.com/aetiology/2006/07/against_gates_adn_buffet.php#…

"But it's not that simple. Liam is suggesting that all children given antiretrovirals are treated in an abusive manner when the drugs are administered, based on an exposé written by none other than...himself. He then extrapolates that to ask if I'd treat my children in that manner, when he's the source of the story that any children are being treated in this manner. He can pout, throw insults and make ridiculous accusations about me, but I'll not play his game, thank you.

Tara then shuts down the thread, perhaps knowing that she just put her entire blog at risk for suit.

Tara, I'll offer you some schooling, dummy, First, I'm not the source of the story. I'm the reporter who broke the story, and, as Celia illustrates with the heartbreak that dogged and scarred every fucking person who had to cover this goddamn monstrosity born out of your beloved paradigm, I am but one of the people who brought it to public attention, who interacted with the children and the moms, the docs and the ACS, the city and the state, the NIH and the CDC.

You can find the sources of the stories and the reference information in any number of the stories from various authors and published in various outlets.

You can trace back through the grizzly files of clinicaltrials.gov to look up each and every one of the trials that was conducted -

The trials that are still being conducted, under your obscene enforced death-paradigm.

That's www.clinicaltrials.gov , you illiterate corporate shill.

And if you find that you haven't been robbed of your ability to read, along with your ability to think before you act, you can find reference after bloody reference, personal story, interview, etc throughout.

---->

We're 'waiting for blog owner to approve second half of this comment,' you can read it here, for your edification:

http://barnesworld.blogs.com/barnes_world/2006/07/banned_at_aetio.html

That's it for me, unless you have anything useful to say in terms of backup for you libel.

I'm sure we're all waiting with baited breath, to see you unravel the mysteries of gastric tubes and NIH orphanages for us. Won't you just show us the way, Tara? Cleanse us of what we've seen, and make it all better?

Or maybe just answer the question, you fucking coward.

Would you do what they do there, to your own children?

You already gave the answer -

You wouldn't even think about it. You couldn't even consider it. It's just too goddamn barbaric and inhumane.

You bloody hypocrite. Stick your aids,incorporated, up your ass.

Liam writes It's just too goddamn barbaric and inhumane.
Inhumane I suppose as opposed to the 'humanity' that would be exhibited by just leaving these children untreated and allowing 60- 75% of them to die? Check the CDC statistics for years before any drugs were approved for treating pediatric AIDS. And then maybe you'd like to do some research into some of the treatments that infants born prematurely or seriously ill children with cancer or birth defects are subjected to. But perhaps doctors trying to save lives doesn't draw quite the level of righteous indignation that you are aiming to inspire?

Liam writes It's just too goddamn barbaric and inhumane.
Inhumane I suppose as opposed to the 'humanity' that would be exhibited by just leaving these children untreated and allowing 60- 75% of them to die? Check the CDC statistics for years before any drugs were approved for treating pediatric AIDS. And then maybe you'd like to do some research into some of the treatments that infants born prematurely or seriously ill children with cancer or birth defects are subjected to. But perhaps doctors trying to save lives doesn't draw quite the level of righteous indignation that you are aiming to inspire?

Posting the same thing twice doesn't make it twice as credible, Dale. CDC statistics for pediatric AIDS? Are you sure we're not talking about crack babies instead of AIDS babies? Sure, seriously ill children are being born. But a doctor with even a modicum of competence and conscience should go all out to treat the child for what it's ailing from. Not finish the poor thing off with toxic antiretrovirals.
I wonder if most women know how dangerous it is to bear a child in America, or any American-dominated country.

Ostrea Jr. EM (1997) Pediatrics volume 100 p79 Results.  A total of 2964 infants was studied. At birth, 44% of the infants tested positive for drugs: 30.5% positive for cocaine, 20.2% for opiate, and 11.4% for cannabinoids. Compared to the drug negative group, a significantly higher percentage (P < .05) of the drug positive infants had lower weight and smaller head circumference and length at birth and a higher percent of their mothers were single, multigravid, multiparous, and had little to no prenatal care. Within the first 2 years of life, 44 infants died: 26 were drug negative (15.7 deaths per 1000 live births) and 18 were drug positive (13.7 deaths per 1000 live births). The mortality rate among cocaine, opiate, or cannabinoid positive infants were 17.7, 18.4, and 8.9 per 1000 live births, respectively.

Nope, mortality in crack babies is nowhere near 50%. Want to try another possibility?

Cocaine: 30.5%
opiate: 20.2%
Morphine analogs(ie Dilaudid, other methohoxylated heroin derivatives, etc): ?
This looks to me as if 50% are the unfortunate offspring
of serious drug abusers.
Not included also, or not tested for, are other street drugs notorious for their toxicities,(pcp, meth,lithium , glue, gasoline, nitroethane,etc(although women are less often abusers of the latter)) owing to the fact that they were produced in undergound laboratories, where the arts of crystallization, distillation, chromatographic separation are but hindrances to be dispensed with , rather than practiced means of getting rid of contaminating poisons.

This says nothing about the "cut". Sorry , I don't have a scientific paper to tell you about cutting drugs in the basement of a rat infested crack house. But the "rat" part should give you some clue to an all-time favourite cutting agent.

That's at least 50%.
I'd say the other 50% die simply because they somehow know that nobody cares for them.

Everybody here wants these babies to live healthy lives with good parents.

A little temper control and civility, might .....might ....be a small step somehow towards achieving that.

S

J Pediatr. 1984 Nov;105(5):731-7.

Transfusion-associated acquired immune deficiency syndrome in infants.

Church JA, Isaacs H.

Two preterm infant boys not known to be at risk developed clinical, laboratory, and pathologic features of acquired immune deficiency syndrome (AIDS) after receiving multiple blood transfusions in the neonatal period. Their clinical courses were characterized by failure to thrive, recurrent otitis media, hepatomegaly, and fatal interstitial pneumonia. Laboratory evaluation revealed progressive lymphopenia, reversed T helper/suppressor ratios, increased percentages of B-lymphocytes, decreased lymphoproliferative responses to mitogens, hyperimmunoglobulinemia, and high levels of circulating immune complexes. At postmortem examination thymic involution, lymphocyte depletion in spleen and lymph nodes, and micronodular mineralization in the central nervous system were seen. The findings were not specific for other known congenital immune deficiencies and were most indicative of AIDS. The lack of other risk factors suggests transmission of AIDS via blood transfusions in the neonatal period.

N Engl J Med. 1984 Jan 12;310(2):76-81.

Acquired immunodeficiency syndrome in infants.

Scott GB, Buck BE, Leterman JG, Bloom FL, Parks WP.

Fourteen infants with clinical and laboratory features of an acquired immunodeficiency syndrome were identified in a single metropolitan area from November 1980 to July 1983. Patients were predominantly of Haitian parentage, although two cases occurred in offspring of non-Haitian intravenous drug abusers. Only one patient had received a blood transfusion before the development of clinical findings. The predominant clinical findings included failure to thrive, persistent infection of the oral mucosa by Candida albicans, chronic pulmonary infiltrates, hepatosplenomegaly, lymphadenopathy, and diarrhea. Immunologic studies showed most of the infants to have inverted ratios of T-cell subsets, greatly increased immunoglobulin levels, and circulating immune complexes. Lymphopenia was not common, as it is in adult patients. Infectious agents responsible for opportunistic infections in this series included Pneumocystis carinii, herpesviruses, particularly cytomegalovirus, and C. albicans. Bacterial infections were common, and gram-negative sepsis was the major cause of death in the seven infants who have died. At autopsy, two infants had disseminated lymphadenopathic Kaposi's sarcoma. These observations suggest the likelihood of transplacental, perinatal, or postnatal transmission of an as yet unidentified infectious agent that causes this disease.

Those two studies were before HIV was even identified. If you want to see how often kids - irregardless of the parent's drug use - developed diseases like this prior to 1980, use PubMed. I'm beginning to wonder if some of these people are so young that they actually just swallow the idea that something like disseminated CMV is just your run-of-the-mill consequence of mild immune suppression (or parental drug use!), because they weren't around when these things were vanishingly rare. And it's often not just one opportunistic infection; when cellular immune responses were massively compromised by HIV people often had multiple simultaneous opportunistic infections, and I don't think this happened much in the other rare settings where opportunistic infections were sometimes seen prior to 1980.

As regards Liam Scheff (truly one of the vilest creatures I have ever encountered on the internet), it is notable that a standard tactic of propaganda is to try and provoke an emotional response that precludes rational evaluation of whether the propaganda is based on fact (anyone remember some of the US propaganda during the first Gulf War?). Liam chose to interview David Rasnick for his BBC piece. Rasnick is Duesberg's pal and longtime AIDS denialist who has no expertise related to pediatric clinical trials. He is not a clinician of any sort. His take on ethical conduct of clinical trials can be ascertained from his recent activities as an employee of the Rath Foundation in South Africa (although I have heard he may soon be leaving, which, if true, will be a cause for much rejoicing) To their evelasting shame, the BBC billed Rasnick as an "expert" on drug toxicity, without any disclosure of who he actually is. They will find out soon enough.

The use of Rasnick tells you all you need to know about Liams's motives. He rails about the exploitation of children, then uses them as a propaganda tool to further his denialist agenda. Class act all the way around...

Richard,

"It is notable that a standard tactic of propaganda is to try and provoke an emotional response that precludes rational evaluation of whether the propaganda is based on fact"

What a fantastic phrase! And immediately applicable to the "HIV, the virus that causes AIDS" propaganda too.

I am confident that this is exactly your conclusion too.

Isn't it, Richard Jefferys?

Well, maybe it isn't.

Why do you use the term denialist? That's propaganda too, my friend. If somebody is wrong, you demonstrate why he's wrong and there's no need for the term denialist, a word used to pin down people who are said to deny reality.

But, unless someone is really mentally disturbed, nobody on the whole wide world denies reality. For example, nobody will ever deny there's a tree in my garden after he/she has seen and touched it.

"HIV, the virus that causes AIDS" is NOT reality. It is a theory, at best, a lie and propaganda, at worst. And people who question theories and denounce lies and propaganda are NOT denialists but critics.

Think it over, Richard.

JS

http://www.nightsofarmour.com

To: R.Jeffries
in Re: Church and Isaacs

What does it mean, "not known to be at risk". Does that mean the parent was not known to be a drug addict? I will presume that. Also , is it safe to presume that tranfusions are neccessary in preterm delivery? I know nothing about it. Duesberg states that(and I understand you do not favour the man) ,---and I don't have the reference here, but the mnemonic I use is Snibits>SNBTS> Scottish National Bureau of Tranfusion Services---, anyway , he points out that one of the benign effects of blood tranfusion in post-tranplant therapy is immunosupression. He cites numerous cased to support this (and related,(ie hemophelia) phenomena). So , based upon his method of analyzing this(Church), one would logically ask:
What led to the premature birth? How do we know that the mother was not abusing drugs, or that she was otherwise excercising good prenatal care ,and ,in the absence of other information, did not the tranfusions in and of themselves bring about the immunodefieciency.? Also, there is a stated viral latency periond , stipulated by the conventional ...pathology, if that is the correct word...of method of action for this virus, wherein it integrates into the genome and does nothing for several years(there is now an integrase inhibitor on the market which prevents such genomic insertion). The abstract does not reveal how young these children were when they died, but throughout they are referred to as infants.

These are my observations on Church, admittedly derived from Duesberg's methodology.
Now, if the main article answers these questions , in full or in part, I will attempt to access it. But for the moment, I am going to presume the same puzzle exists: how can a virus , present in such miniscule amounts, cause such a different pattern of death in the infant than it does in an adult.
I recognize you have a prima facie case for microbial attack , but I would expect to find (yes , its the way I've been convinced by Duesberg) lots of this microbe somehere in the body of these unfortunate children.

I appreciate the detail you have offered in your advancing your argument. It is unlikely, because of circumstance, that I will ever be able to respond in kind. Just letting you know that from the outset.

May you have a pleasant day,
Must rush

S

Just sent a short response - 'pending for review by blog owner'

Our own Richard Jeffries:

"vile creature" - very good! A little Elizabethan?

but, Richard, "irregardless"... well, that's you. Right where you live.

We force-feed poisonous drugs to children that make them violently ill,

* but 'irregardless',

those who oppose the practice are 'vile creatures'

We institutionalize our self-aggrandizing historical racism via the false-positive eugenics-thermometer African-round-up we hysterically call "hiv testing"

* but, 'irregardless,'

those who would prefer to feed, clothe and provide clean water and protection from the tropical elements to the starving millions are 'denialists'

Yes, you may have held the beaten child victims of our brutal paradigm in your arms, spent hours, weeks, months, years and decades talking to the victims of our drug poisoning, reported on it dutifully, against the wishes and permission of the culpable and collusive press,

* but, 'irregardless,'

You must be making it all up, when it throws chaotic our work into question.

Clever stuff, Rich. but regardless, you are a very cold hearted bastard.

Oh! says the historian: Look at history! At Africa, at the slave nation we have made it.

Is it any wonder that Africa remains the despot of the medical world? Unyielding to the advances of the West, our great contributions to the world: The sewage treatment plants and refined food, the waste management and the petrol-culture, the obesity and diabetes, the mental illness and cancer? Is it any wonder that Africa stubbornly mires itself in all of the diseases we've so well outsmarted?

Is it any wonder that the most pillaged, land-locked, ferociously distemperate, equitorial continent on earth has not invited more than the rape of its people, (rather than the colonization of its cruel land? (as in the Americas, where the land was not so cruel, where the land was inviting?)

Colonization - that unsubtle and eternal tribal war, that makes 'Them' into Us, and the Inhospitable Land into 'Home.'

Is it any wonder that Africa, never yielding, never inviting easy settlement or conquest, has remained stuck in the cruelty of equitorial illness? Would Rekyavik be any more hospitable if we had not learned to protect ourselves from its mortal, icy grip?

---> will be continued, depending on the blog software!

But Let's think only with our smallest selves, and imagine that it is a sex problem that drives Africa to tears. Said Malthus, said Sanger, said H.G. Welles, and G.B. Shaw.

And as our prejudices fly to Africa, so they fly to Africans, mired in the pit of our racism, culturally and legally enforced, with chains and picks and shovels, and nooses thrown around the branches of trees.

You are practicing eugenics, you see, focusing on the irreproducible at the level of useless, pointless, extravagant microscopy, while the world ticks on around you, as it always has done.

And there would be no problem with any of this, if you weren't, through the puckish proclivity of history, positioned as the pretenders to the throne of the enlightenment, now in charge for a time, after the excesses of the papacy.

You are not, of course, the heirs of the enlightenment (or the renaissance - they valued a broad world-view, after all); No, yours is the role of the tight-focus and the a priori confession, out of the a priori heretical. Call it the microscopal inquisition. Or something to that effect.

But then, 'irregardless', you will say, what about our precious studies?

Well, not to worry, Richard. When - what is it Wilhelm calls it? Oh, yes.

When "hatchet day" arrives, you will be pleased to note that we, the non-reductionist/non-eugenicists, are a much kinder group of beings. We will not force you out of your labs in shame.

We will let you continue your mostly masturbatory work, lying, as it does, on the very outer fringe, the illucid filligree of reality.

Because, after all, you may, from time to time, come up with something that has some value,

When placed in its proper context. A trick you cruel bastards should learn.

After 25 years of keeping track of AIDS cases, all the while assuming that AIDS cases have something to do with sexual activities, I was struck by the shockingly small number of alleged "hetersexual" AIDS cases among women in San Francisco.

There have been over 26,000 cases of AIDS in SF since 1981 but only 263, around ten per year, are attributed to heterosexual activities. How is this ot be explained, especially since my friend and I live near SF and can assure you that heterosexual women engage lustily in all sorts of sexual behaviors, usually without condoms wince they take charge of their own birth contyrol devices.

Also, I was amazed to discover how few cases of AIDS or even HIV-positives have ever been found at the sexually active campuses of universities in California. How is that to be explained?

Thanks

Many of the "aids" defining diseases are not caused by "aquired immune defiency"/"low "cd4" ", that includes Kaposi Sarcoma the original signal disease of "aids", wasting, TB, "pcp" ect.

Malnutrition, antioxidant, protein/amino acid, vitamin, selenium, micro nutient defiencies all predict disease ,death or survival, TB particularlar glutathion -GSH .

One would have to ask what is the bilogical/antibody/biochemicle/immune responce to blood transfusion/s? And what where the medical reasons for the transfusion ? And further details. Read Sir John Maddox editor nature "aids research turned upside down" ?.

I would srongly urge urgent long term , double blind plecebo clinnical trails into NAC,pro GSH agents, antioxidants, minerals ,selenium,amino acids, l glutamine, vitamins, vicro nutrients coupled with the elimination/reducing exposer to poverty, malnutrition, drugs-oxidizers, over crowding ,dirty water, stress's.

We could improve and prolong life cheaply with non toxic agents that would reduce the POSSIBLE need for the hell of alot more expensive and toxic but in some ways successfull combination drugs. New England editoral and study on vitamins shows reduction in "viral-load", reduction of "progression"/disease by 30 using very simple, very cheap , very non toxic primative multivitamin. Why isnt bill and bill and bono and the aids lobby demanding access, research, clinnical trials into these stratergies that I am 100% could help improve, save, prolong millions of lives and billions of dollars.

Best Wishes

Hugs

James
PS oxidative stresses increase "viral-replication"/"viral-load", antioxidants inhibit reduce both. Oxidative stimuli induces apoptosis/cell death/th1/th2 balances/mitrocondria, antioxidants inhib cell death and a th2 dominance and protect mitocondria of oxidative stimuli.

Glutathione and growth inhibition of Mycobacterium tuberculosis in healthy and HIV infected subjects
Vishwanath Venketaraman1 ,2 ,3 ,4 ,5 ,6 , Tatanisha Rodgers1 ,4 ,6 , Rafael Linares6 , Nancy Reilly1 ,4 ,6 , Shobha Swaminathan1 ,4 ,6 , David Hom2 ,6 , Ariel C Millman1 ,4 ,6 , Robert Wallis1 ,4 ,6 ,7 and Nancy D Connell1 ,2 ,3 ,4 ,5 ,6
1Division of Infectious Diseases, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA
2Center for Emerging and Re-emerging Pathogens, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA
3National Tuberculosis Center, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA
4Department of Medicine, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA
5Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA
6New Jersey Medical School, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA
7PPD, 1213 N Street NW, Apt. A, Washington DC 20005, USA

AIDS Research and Therapy 2006, 3:5 doi:10.1186/1742-6405-3-5

The electronic version of this article is the complete one and can be found online at: http://www.aidsrestherapy.com/content/3/1/5

Received 29 December 2005
Accepted 20 February 2006
Published 20 February 2006

© 2006 Venketaraman et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
--------------------------------------------------------------------------------

Outline Abstract

Abstract
Introduction
Experimental methods
Results
Discussion
Acknowledgements
References

Intracellular levels of glutathione are depleted in patients with acquired immunodeficiency syndrome in whom the risk of tuberculosis, particularly disseminated disease is many times that of healthy individuals. In this study, we examined the role of glutathione in immunity against tuberculosis infection in samples derived from healthy and human immunodeficiency virus infected subjects. Our studies confirm that glutathione levels are reduced in peripheral blood mononuclear cells and in red blood cells isolated from human immunodeficiency virus-infected subjects (CD4>400/cumm). Furthermore, treatment of blood cultures from human immunodeficiency virus infected subjects with N-acetyl cysteine, a glutathione precursor, caused improved control of intracellular M. tuberculosis infection. N-acetyl cysteine treatment decreased the levels of IL-1, TNF-α, and IL-6, and increased the levels of IFN-γ in blood cultures derived from human immunodeficiency virus-infected subjects, promoting the host immune responses to contain M. tuberculosis infection successfully.

Outline Introduction

Abstract
Introduction
Experimental methods
Results
Discussion
Acknowledgements
References

Tuberculosis (TB) is a major global health problem [7]. Approximately one-third of the world's population is latently infected with Mycobacterium tuberculosis (LTBI). Individuals with LTBI have a 5-10% lifetime risk of developing active disease [7]. Human immunodeficiency virus (HIV) infected subjects with LTBI are at very high risk of developing active tuberculosis. Development of active TB in HIV patients is due not only to reactivation of latent M. tuberculosis infection but also due to increased susceptibility to primary progressive M. tuberculosis infection [7].

Innate and adaptive immune responses are required for successful control of M. tuberculosis infection. Macrophages provide first line defense against M. tuberculosis infection. Murine macrophages can be activated to kill intracellular M. tuberculosis by treatment with LPS (a stimulus for TNF-α expression, via triggering of toll-like receptors) and IFN-γ (a product of activated lymphocytes). Nitric oxide (NO) produced by infected macrophages is the main mediator (effector molecule) in this process. Like those of mice, human macrophages also acquire antimycobacterial activity through IFN-dependent interactions with lymphocytes [12]. However, exogenous IFN-γ does not enhance the mycobactericidal activity of isolated human macrophages as it does those of mice. Several studies indicate instead that direct cellular contact is required for the induction of antimycobacterial activity in human macrophages [6,33], and that this activity reflects caspase-mediated induction of apoptosis, triggering of toll-like receptors, the release of antibiotic peptides (e.g., granulysin), or unknown mechanisms [4,36].

Glutathione (GSH) is an antioxidant and plays a vital role in cellular detoxification and enhancement of immune functions [10]. Interestingly, HIV-infected people have subnormal GSH levels in their plasma, lung epithelial lining fluid, peripheral blood mononuclear cells (PBMC), and other blood cells [5,11,14,23]. It has been recently reported that the decreased GSH levels in PBMC of HIV-infected individuals is associated with a poorer prognosis [24]. Immunodeficiency due to HIV-1 represents the greatest recognized threat to successful containment of latent M. tuberculosis infection. The aim of this study was to examine the role of GSH in immunity against TB in samples derived from healthy and HIV infected subjects.

In our previous studies using macrophages from different sources, we have demonstrated that GSH plays a vital role in innate immunity against TB infection [40,41]. In our recent studies we have shown that GSH has static effect on H37Rv growth in vitro [41]. The mechanism of toxicity of GSH to mycobacteria is not yet known. One possibility is that the presence of high concentrations of GSH may result in an imbalance in a bacterial cell already containing an alternative thiol for regulating reduction/oxidation activity (e.g., mycothiol).

In the present study, we reexamined the extent to which GSH levels are decreased in HIV positive subjects. We also examined the relationship between GSH levels and the ability to kill intracellular M. tuberculosis, in association with other immune functions, such as cytokine production. GSH levels were modulated by treating blood samples with N-acetyl cysteine (NAC) to increase or buthionine sulphoximine (BSO) to decrease intracellular GSH pools. Our results suggest that the inability of immune cells from healthy and HIV subjects to contain TB growth may be a consequence of the inability of their macrophages to maintain adequate GSH levels during in vitro infection.

Outline Experimental methods

Abstract
Introduction
Experimental methods
Results
Discussion
Acknowledgements
References

Subjects

A total of 20 subjects (10 healthy volunteer controls and 10 patients with HIV infection) were enrolled at UMDNJ-University Hospital of Newark and the NJ Medical School, in Newark, NJ. Subjects with HIV infection without tuberculosis (n = 10) were recruited at the Infectious Disease Clinic of UMDNJ-University Hospital. The Clinic is the site of several ongoing studies of HIV treatment; these studies provide anti-retroviral treatment (ART) to enrolled subjects without charge. Patient care was not altered by participation in this study. Patients were defined as being HIV-positive on the basis of a positive ELISA with a confirmatory Western Blot performed as part of their routine care in the clinic. The average CD4 numbers for HIV patients in this study was 423 ± 83/cumm. Only one patient had CD4 counts below 200/cumm. Seven patients were on ART and three patients were not on any treatment at the time of blood draw. Healthy subjects without HIV infection or a history of TB were recruited from the hospital and the university faculty and staff (n = 10). Healthy and HIV-positive subjects with a history of a positive tuberculin test (TST) were excluded from the study so as to maintain strict study definitions. This is according to the CDC recommendation that recognizes that a positive TST reflects latent TB infection.

Safety precautions for handling M. tuberculosis

All experiments with M. tuberculosis H37Rv were performed inside the bio safety level 3 (BSL-3) facility. The protocols for all experiments were approved by the UMDNJ Institutional Review Board, and the New Jersey Medical School Institutional Biosafety Committee. All experimental procedures were performed inside the biosafety cabinets in the BSL-3. All liquid and solid wastes from the experiments were treated with a disinfectant solution and then autoclaved.

Processing of H37Rv for infection

M. tuberculosis H37Rv was grown in 7H9 with albumin-dextrose complex (ADC). Static cultures of mycobacteria at peak logarithmic phase of growth (between 0.5 and 0.8 at A600) were used for infection. The bacterial suspension was washed and resuspended in RPMI containing AB serum. Bacterial clumps were disaggregated by vortexing five times with 3-mm sterile glass beads. The bacterial suspension was passed through a 5 μm filter to remove any further clumps. The total number of organisms in the suspension was determined by plating. Processed mycobacteria were frozen as stocks at -80°C. At the time of infection, frozen stocks of processed mycobacteria were thawed and used for macrophage infection.

Separation of monocytes from human blood

Human monocyte-derived macrophages (HMDM) were used to study the effects of IFN-γ and GSH in inducing intracellular killing of H37Rv. These experiments were performed only in blood samples from healthy subjects due to the non-availability of sufficient blood volume from HIV patients. Forty ml of blood from healthy subjects were used for monocyte isolation. PBMC were isolated by ficoll hypaque density centrifugation. PBMC were washed with PBS and resuspended in RPMI containing 5% AB serum. PBMC (10 à 106/ well) were distributed into Poly-DL-lysine coated 12 well plates and incubated overnight at 37°C, 5% CO2 in a humidified atmosphere, to allow monocytes to adhere to the plate. Non-adherent cells were removed by gentle washing and the adherent monocytes were cultured in RPMI containing 5% AB serum for 7 days before being used for infection experiments to allow differentiation to macrophages. The total number of macrophages per well (on day seven) was quantitated by detaching the macrophages from a single well by the addition of ice-cold accutase (Sigma). Viable detached macrophages were counted in a Neubauer counting chamber by trypan blue dye exclusion. The average number of macrophages per well on day 7 is approximately 5 à 105.

Macrophage infection

HMDM from healthy subjects were maintained in vitro as described above. Macrophages were infected with processed H37Rv at moi of 10:1. Macrophages were incubated with H37Rv for 2 h (for phagocytosis), after which extracellular organisms were removed by washing with PBS. Infected macrophages were maintained in RPMI containing 5% AB serum. Infected macrophage cultures were terminated at 4 h and 7 days after infection and treatment, to measure the intracellular viability of H37Rv. Cell free supernatants from infected macrophage cultures were diluted and plated for extracellular bacterial growth. Intracellular viability of H37Rv was determined by lysing the infected macrophages with sterile distilled water and plating the lysate on 7H11 enriched with ADC, to enumerate mycobacterial colonies.

Survival of H37Rv in IFN-γ, LPS treated HMDM

IFN-γ is considered a predominant activator of microbicidal functions in macrophages and is essential for prevention of uncontrolled progression of M. tuberculosis infection [2,18,27]. We therefore studied the survival of H37Rv in IFN-γ, LPS treated HMDM. HMDM were maintained in vitro and infected with H37Rv, as described previously. H37Rv-infected HMDM were treated with IFN-γ (100 U/ml) and LPS (1 μg/ml), the cultures were terminated at 4 h and 7 days after infection and treatment, to determine the intracellular viability of H37Rv inside unstimulated and IFN-γ, LPS-stimulated macrophages.

Survival of H37Rv inside NAC treated HMDM

We determined the effects of GSH in human macrophage mediated growth inhibition of intracellular H37Rv. HMDM were treated with different concentrations of NAC. Cysteine uptake is considered as rate-limiting step for synthesis of GSH. The most efficient way to increase the levels of cysteine in cells grown in vitro is to supply the culture medium with NAC. NAC is easily taken up by the cells and is non-toxic. Intracellularly, NAC is de-acetylated and cysteine is utilized for GSH synthesis. H37Rv infected HMDM were treated with 5, 10, 15, and 20 mM NAC, and intracellular growth of H37Rv was studied. Infected macrophage cultures were terminated at 4 h and 7 days, after infection and treatment. Infected macrophages were lysed and plated for mycobacterial colonies.

Whole blood mycobactericidal assay

Mycobacteria added to heparinized blood (after dilution with tissue culture medium), are rapidly ingested by monocytes and other phagocytic cells such as neutrophils. This model differs from other intracellular infection models in that all blood elements are represented. Interactions of infected monocytes with natural killer cells and antigen-specific T cells result in control of intracellular growth. In contrast to the studies with isolated macrophages, the whole blood assay requires a low volume of blood. Blood was diluted at the following proportion: 300 μl of blood from healthy subjects and patients were diluted to 1 ml with RPMI. Blood cultures were infected with 105 CFU of H37Rv. GSH levels in blood cultures were altered using agents such as NAC (10 mM) and BSO (500 μM) that specifically increase and decrease intracellular GSH. The effect of altered GSH levels on M. tuberculosis survival was studied. Treatment of cells with BSO causes inhibition of GSH synthesis. BSO specifically inhibits the activity of γ-glutamyl-cysteinyl synthetase enzyme, that catalyses the first step reaction in the synthesis of GSH. Blood cultures were treated with either NAC or combination of NAC and BSO for 24 h prior to infection. H37Rv infected whole blood cultures were incubated at 37°C and harvested at selected intervals (2 h and, 48 h) by sedimentation at 2000 rpm for 10 min. Supernatants were used to determine cytokine levels and extracellular mycobacterial growth. Host cells were disrupted by addition of sterile water. The lysates were plated on 7H11 medium enriched with ADC for mycobacterial colonies.

Assay of GSH

Intracellular GSH levels in PBMC, red blood cells (RBC), and plasma, from healthy individuals and HIV positive subjects were assayed by spectrophotometry, using a GSH assay kit procured from Calbiochem. This approach is used to determine whether GSH levels are decreased in all blood components or just in some specific components. Plasma and cell lysates of RBC and PBMC, derived from healthy and HIV positive subjects, were mixed with equal volume of ice cold 5% metaphosphoric acid (MPA) and centrifuged at 3000 rpm for 15 minutes. Supernatants were used for GSH assay, as per the manufacturer's instruction. Plasma, RBC, and PBMC were separated from whole blood by density gradient centrifugation using ficoll hypaque. Samples were also used for protein assay by Bradford's method using Bio Rad reagent.

Cytokine assay

Blood cultures were prepared by afore mentioned methods. Blood cultures from healthy subjects and HIV patients were treated as follows: no treatment, infection with H37Rv, and infection with H37Rv and treatment with NAC. Cultures were terminated at 2 h and 48 h, after infection. Uninfected cultures were terminated at the same time points. Cultures were centrifuged at 2000 rpm for 10 min. Cell free supernatants from healthy and HIV patients were used for the cytokine assay, which was performed using a Beadlyte kit procured from Upstate. This is a highly sensitive kit that can be used to detect multiple cytokines in tissue culture samples. A monoclonal antibody specific for a cytokine is covalently linked to a fluorescent bead set, which captures the cytokine. A complementary biotinylated monoclonal cytokine antibody then completes the immunological sandwich and the reaction is detected with streptavidin-phycoerythrin using a Luminex. The assay was performed as per the manufacturer's protocol.

Statistical analysis

Statistical analysis of the data was carried out using Statview program and the statistical significance was determined using unpaired t test. Data from cytokine assays was analyzed by non-parametric test (Kruskal-wallis). Differences were considered significant at a level of p < 0.05.

Outline Results

Abstract
Introduction
Experimental methods
Results
Discussion
Acknowledgements
References

Figures

Figure 1
Growth of H37Rv in unstimulated (Fig 1a), IFN-γ, LPS (Fig 1a), and NAC treated HMDM (Fig 1b)

Figure 2
Spectrophotometric assay of GSH in PBMC (Fig 2a) and RBC (Fig 2b), derived from healthy and HIV positive subjects

Figure 3
Growth of H37Rv in whole blood cultures of HIV patients

Figure 4
IL-1, TNF-α, IL-6 and IFN-γ assays in blood culture supernatants

Figure 5
IL-10 assay in blood culture supernatants

Figure 6
Model describing direct and indirect effects of GSH in growth control of H37Rv in blood cultures derived from healthy and HIV-infected subjects

Survival of H37Rv in HMDM

We studied the survival of H37Rv in HMDM from healthy subjects. H37Rv-infected HMDM were treated with IFN-γ (100 U/ml) and LPS (1 μg/ml), and the intracellular viability of H37Rv inside unstimulated and IFN-γ, LPS-stimulated macrophages was compared. Figure 1a shows results from six different subjects performed in triplicate. We observed significant growth of H37Rv inside unstimulated HMDM between 1 h and 7 days (Fig 1a). The increase was almost four fold. Stimulation of HMDM cells with IFN-γ, LPS also resulted in significant growth of intracellular H37Rv (Fig 1a). However, the increase in H37Rv growth was less than three-fold (Fig 1a). To examine whether GSH plays a major role in human macrophage killing of H37Rv, HMDM from healthy volunteers were treated with 5, 10, 15, and 20 mM NAC, and intracellular growth of H37Rv was measured. Experiments performed in six different subjects show that treatment of HMDM with 10 mM NAC resulted in stasis in H37Rv growth in three out of six subjects (Fig 1b). Treatment of HMDM with NAC at 5 mM and 15 mM induced growth inhibition of H37Rv, in one out of six, and two out of six subjects, respectively (data not shown). Treatment with 20 mM NAC had no effect on growth inhibition of H37Rv (data not shown). Therefore, NAC at 10 mM is more effective in inducing growth control of M. tuberculosis as compared to IFN-γ, LPS, or other concentrations of NAC (Fig 1b) in isolated HMDM.

Whole blood model

Several studies indicate that direct cell contact is required for induction of antimycobacterial activity in human macrophages [6,33], and that this activity reflects caspase-mediated induction of apoptosis, triggering of toll-like receptors, the release of antibiotic peptides (e.g., granulysin), or unknown mechanisms [4,36]. Mycobacteria are rapidly ingested by phagocytic cells when added to heparinized blood (after dilution with tissue culture medium). This model differs from other intracellular infection models in that all blood elements are represented.

We therefore tested whether interaction of monocytes with other immune cells will lead to growth inhibition of intracellular H37Rv using whole blood cultures, which provides a micro-environment that is conducive for cellular interactions.

Whole blood mycobactericidal assay in healthy subjects

Blood from healthy volunteers was diluted as described and treated with none or 10 mM NAC. The blood cultures were then infected with 5 Ã 105 CFU of processed H37Rv. Infected blood cultures were terminated at 2 h and 48 h after infection to determine the intracellular viability of H37Rv. Cell suspensions were centrifuged to separate the cell free supernatants and pellets. Supernatants were diluted and plated for extracellular bacterial growth. Intracellular viability of H37Rv was determined by plating the diluted blood cell lysates on 7H11. Infection of blood cultures with H37Rv resulted in almost two-fold increases in the intracellular growth of H37Rv (Fig 1c). The increase in H37Rv growth was statistically significant. Treatment of blood cultures with NAC (10 mM), caused growth inhibition of H37Rv in all seven individuals tested (Fig 1c). The data in Fig 1c are averages from seven healthy subjects. Treatment of cultures with BSO abrogated the growth inhibition effect of NAC (Fig 1c). These results indicate that growth inhibition of H37Rv in NAC treated blood cultures is due to combination of direct antimycobacterial effects of GSH and activation of immune cells induced by GSH.

Levels of GSH in blood samples from healthy and HIV-positive subjects

Intracellular GSH levels in PBMC and RBC were assayed by spectrophotometry as described. We observed a significant and more than 50% decrease in intracellular GSH levels in PBMC (Fig 2a) and RBC (Fig 2b) from HIV patients compared to healthy subjects. Data shown in Fig 2 are averages from six healthy and six HIV-infected subjects. We observed no difference in the plasma GSH levels between healthy and HIV patients.

Growth control of H37Rv by NAC-treated blood cultures from HIV patients

Intracellular growth of H37Rv was monitored in blood cultures of HIV-positive subjects. We observed a significant growth of H37Rv in unstimulated blood cultures (Fig 3a). NAC treatment induced growth inhibition of intracellular of H37Rv. Data in Fig 3b are averages from data obtained from eight different HIV-positive subjects. BSO treatment abrogated the inhibitory effect brought about by NAC treatment (Fig 3c).

Assay of cytokines in blood culture supernatants from healthy and HIV-positive subjects

Cytokines were measured in blood culture supernatants from healthy and HIV-infected subjects. Interestingly, in HIV subjects, H37Rv infection induced the blood cultures to produce increased levels of pro-inflammatory cytokines such as IL-1, TNF-α, and IL-6 (Fig 4a, 4b, 4c). H37Rv infection induced almost three fold increases in IL-1 production, compared to uninfected controls (Fig 4a). NAC treatment of H37Rv infected cultures down-regulated IL-1 levels (Fig 4a). Compared to uninfected controls, H37Rv infection induced seven fold increases in TNF-α levels in two patients tested (Fig 4b). NAC treatment of H37Rv infected cultures caused reduction in TNF-α levels (Fig 4b). H37Rv infection induced almost ten fold increases in IL-6 production, in three patients tested (Fig 4c). NAC treatment reduced the levels of IL-6 to those found in the uninfected control. We also observed that infection of HIV blood cultures with H37Rv caused six fold increases in IFN-γ production in two patients and three fold increases in one patient (Fig 4d). In comparison to untreated controls, NAC treatment of H37Rv infected cultures induced ten fold increases in IFN-γ production in two patients and almost four fold increases in one patient (Fig 4d). In summary, our studies show that NAC treatment down-regulated the synthesis of IL-1, IL-6, and TNF-α and increased the levels of IFN-γ (Fig 4a, 4b, 4c, 4d).

With the exception of IL-10, all other cytokines produced by healthy subjects showed no clear trend. The regulation of IL-10 synthesis in response to H37Rv infection and NAC treatment was similar in healthy subjects and HIV patients. H37Rv induced almost ten-fold increases in IL-10 levels in both healthy and HIV-infected subjects (Fig 5a, 5b). Furthermore, NAC treatment reduced the levels of IL-10 to those found in uninfected controls, in both healthy subjects and HIV patients (Fig 5a, 5b).

Outline Discussion

Abstract
Introduction
Experimental methods
Results
Discussion
Acknowledgements
References

Development of TB in HIV infected patients is based on a predisposition to reactivation of latent M. tuberculosis infection and to susceptibility to primary progressive M. tuberculosis infection [9]. However, the relationship of host immune responses to the development of TB during different stages of HIV disease is not clear. The opportunistic behavior of M. tuberculosis during human HIV infection can be explained by suppression of type-1 responses at the level of antigen-presenting cells, CD4 T cells and effector macrophages.

In vitro studies have shown that lowering of intracellular GSH levels decreases cell survival, alters T cell functions and increases HIV replication, NF-kB activation, and sensitivity to TNF-α induced cell death [10,11,19]. A role has also been proposed for GSH as a carrier molecule for NO. Nitric oxide also reacts with GSH to form GSNO, an NO donor with greater stability [34,35].

We first reported that GSH facilitates the control of intracellular M. bovis BCG in NO-deficient macrophages derived from iNOS knock out mice, and in HMDM [40]. These studies indicated that GSH has direct antimycobacterial activity distinct from its role as an NO carrier. Furthermore, in our recent studies we demonstrated that GSH is vital for growth control of intracellular H37Rv in J744.1 macrophages [41].

It has been reported that production of IFN-γ is crucial to the control of M. tuberculosis infection [18]. Impaired production of IFN-γ correlates with progression of immunodeficiency and is likely related to abnormalities in the IL-12-IFN-γ axis [8,31]. We therefore tested the growth of H37Rv in HMDM from healthy subjects that are unstimulated or stimulated in vitro with IFN-γ, LPS. We observed a significant, four-fold increase in growth of H37Rv inside unstimulated HMDM, between 1 h and 7 days (Fig 1a). Stimulation of H37Rv-infected HMDM cells with IFN-γ, LPS also resulted in a three-fold increase in growth of intracellular H37Rv (Fig 1a). Since our earlier studies suggested a role for GSH in innate immunity against M. tuberculosis, we tested whether NAC treatment would induce HMDM to inhibit the growth of H37Rv. We observed that NAC at 10 mM concentration induced growth inhibition of H37Rv in three out of six healthy individuals tested (Fig 1b). Although normal levels of GSH are present in cells derived from healthy subjects, those levels might decrease during oxidative and nitrosative stress generated during TB infection. Therefore, addition of NAC to HMDM caused growth inhibition of M. tuberculosis by augmenting intracellular GSH levels. These results suggest that growth inhibition of H37Rv in NAC treated HMDM is due to the direct antimycobacterial effects of GSH. Furthermore, the inability of HMDM from some healthy individuals to inhibit M. tuberculosis growth is probably due to the inability of macrophages to maintain adequate GSH levels, despite NAC treatment.

As described before, innate and adaptive immunity are essential for successful elimination of M. tuberculosis. Macrophages interact with other immune cells in vivo, for successful growth retardation of M. tuberculosis. The whole blood model of infection resembles an in vivo system in promoting cellular interactions. This model differs from other intracellular infection models in that all blood elements are represented. Infection of blood cultures from healthy volunteers with H37Rv resulted in an almost two-fold increase in H37Rv growth (Fig 1c). The increase in H37Rv growth was statistically significant. In contrast to HMDM, treatment of blood cultures with NAC (10 mM) caused growth inhibition of H37Rv, in all seven individuals tested (Fig 1c). Our results suggest that growth inhibition of H37Rv in NAC treated blood cultures is due to direct antimycobacterial effects of GSH and due to activation of blood cells induced by GSH.

We have confirmed the work of others that GSH levels are decreased in patients with HIV-1 infection [5,11,14,23], and then hypothesized that this decrease would be associated with reduced capacity of monocytes to kill intracellular M. tuberculosis. We further proposed that NAC treatment would improve the killing of M. tuberculosis. We tested our hypothesis by determining GSH levels in healthy and HIV positive subjects. We observed a significant and more than 50% decrease in GSH levels in PBMC and RBC from HIV patients compared to healthy subjects (Fig 2a, 2b). Since GSH enhances innate and adaptive immune functions, GSH deficiency in PBMC may contribute to the progressive immune dysfunction of HIV infection. Macrophages play a central role in HIV and TB infection because they are among the first cells to be infected [19]. Moreover, macrophages serve as an important reservoir for both HIV and M. tuberculosis. The major obstacle to eradication of HIV is latent virus in these reservoirs which has prompted the search for new drugs and strategies to protect this cell compartment. Erythrocytes have been used as a carrier system to deliver antiretroviral molecules to macrophages selectively. Fraternale et al [19] have reported that treatment of mice with AZT+DD1+GSH-loaded RBC significantly reduces the proviral DNA content, compared to mice treated with AZT+DD1. This result is consistent with our hypothesis and suggests that low levels of GSH in RBC, as observed in this and other studies, will affect the GSH carrier functions of RBC, compromising GSH delivery to macrophages.

In order to determine the effects of NAC treatment on PBMC and RBC in reducing the growth of intracellular H37Rv, whole blood cultures from HIV patients were treated in vitro with NAC and infected with H37Rv. We observed significant growth of H37Rv in unstimulated blood cultures from HIV patients (Fig 3a). In vitro NAC treatment to blood cultures derived from HIV subjects caused inhibition in growth of intracellular H37Rv (Fig 3b). Furthermore, BSO treatment abrogated the inhibitory effect brought about by NAC treatment (Fig 3c). This suggests that restoration of GSH levels in HIV subjects caused enhancement in immune cell functions to contain M. tuberculosis growth.

The decreased GSH content in immune cells of HIV-positive individuals was atleast in part attributed to the decreased in plasma cysteine and increased plasma glutamate (an inhibitor of cysteine permeation via the Xc- transport system), as observed during early infection. The decreased intracellular GSH and plasma cysteine observed in HIV patients is due to chronic oxidative stress, which may lead to the progression of the disease. The decreased availability of cysteine can be overcome to some extent by the cysteine precursor NAC [13]. A recent report of a carefully conducted clinical trial indicates that NAC treatment improves the clinical situation and delays the HIV disease progression [24]. This study showed that long-term administration of NAC to AIDS patients improves their hematological profile, GSH content and life expectancy [24].

We measured cytokine levels in whole blood culture supernatants from healthy and HIV infected subjects. No clear trend in cytokine profile was observed in healthy subjects. Interestingly, we observed that in vitro infection with H37Rv induced the whole blood cultures from HIV patients to synthesize increased levels of cytokines such as IL-1, TNF-α, IL-6 and IL-10 (Fig 4, 5). IL-1, TNF-α, IL-6 are the early pro-inflammatory cytokines produced by monocytes after various bacterial infections and share a wide array of biological activities [4,5]. In vitro studies have shown that mycobacterial preparations, including lipoarabinomannan, can cause the release of TNF-α and IL-1 from human PBMC [25,42,44].

The release of pro-inflammatory cytokines after mycobacterial infection is a host immune response that may be propitious or deleterious to the host. Newman et al. reported that increased survival of M. avium intracellulare (MAI) in isolated macrophages is correlated with the efficiency with which TNF-α and IL-6 are produced in response to MAI infection [28]. Nevertheless, increased levels of these pro-inflammatory cytokines may be disadvantageous to the host because they not only cause acute-phase events, such as fever, but also mediate cachexia, hemorrhagic necrosis and lethal shock [29,30,37]. TNF-α by classical cascade is known to up-regulate the levels of IL-1 and IL-6.

Elevated levels of IL-6 are present in plasma of patients with TB [15]. Studies by Van Heyningen et al [39] indicate that macrophages infected with M. bovis BCG released copious amounts of IL-6 which in turn inhibited the macrophage capacity to induce proliferation of CD4 T cell hybridoma. Nagabhushanam et al. [26] reported a novel function of IL-6 in inhibiting cellular immune response to eradicate M. tuberculosis infection. Their studies show that IL-6 produced by M. tuberculosis-infected macrophages selectively inhibited macrophage responses to IFN-γ. In other words, secretion of IL-6 by M. tuberculosis-infected macrophages may contribute to the inability of IFN-γ to eradicate M. tuberculosis infection [26].

The high levels of IL-6 released by infected macrophages have implications for co-infection with HIV [32]. Mycobacterial infections are one of the most common AIDS-defining illnesses and may even accelerate progression to AIDS [17]. The two infections seem to synergize, causing a shift of the host-pathogen balance in favor of the pathogen, which cannot be reversed by treatment with antimycobacterial agents [43].

TNF-α and IL-6, as well as IL-1, can increase HIV replication [3,21]. Thus, decreasing the pro-inflammatory cytokine production in vivo may enhance the control of viral replication. Elevated levels of IL-6, TNF-α and IL-10 have been described previously in cases of advanced HIV disease [1,20,22]. Therefore, increases in the levels of pro-inflammatory cytokines will cause a positive feedback loop in which the two infections complement one another, leading to accelerated progression of both diseases.

In our studies, we observed that NAC treatment caused down-regulation of the synthesis of IL-1, IL-6, and TNF-α (Fig 4a, 4b, 4c), and up-regulation of the synthesis of IFN-γ (Fig 4d). These results suggest that GSH might have a crucial role in vivo in reducing the levels of pro-inflammatory cytokines thereby protecting the host against disease progression.

Active TB is associated with suppression of T cell responses [17] and enhanced production and activity of immunosuppressive such as IL-10. IL-10 has been shown to be produced by macrophages infected with mycobacteria. IL-10 and TGF-β overlap with each other in many of their biological effects including, inhibition of T cell proliferation and IFN-γ production [21]. Elevated levels of IL-10 in serum during advanced HIV infection may enhance immune suppression, allowing opportunistic infections [21]. In our studies, we observed that NAC treatment decreased the levels of IL-10 favoring immune activation (Fig 5b).

We demonstrate growth inhibition of intracellular H37Rv in our in vitro studies using NAC-treated blood cultures from HIV patients. Furthermore, treatment of blood cultures with NAC modulated the production of cytokines in favor of the host. As described in the model (Fig 6a), our results strongly indicate that the immune cell enhancing and antimycobacterial functions of GSH are important for growth control of H37Rv in blood cultures from healthy and HIV-infected subjects (Fig 6a). Additionally, NAC treatment down-regulated the synthesis of IL-10 and pro-inflammatory cytokines in blood cultures from HIV-infected subjects favoring immune activation (Fig 6b). Current interventions to prevent tuberculosis in areas where TB and HIV are endemic, such as sub-Saharan Africa, have serious limitations. ART is limited by its cost and by its requirement for a sophisticated health care delivery system. Isoniazid chemoprophylaxis has limited efficacy in regions of high TB transmission, particularly in highly susceptible individuals with advanced HIV infection. In addition, isoniazid is ineffective against INH-resistant TB strains, which may account for 10-20% of all cases in some areas. NAC is inexpensive and non-toxic (it is considered a food supplement in the US, and is available without prescription in health food stores). The findings from this study may lead to long-term research that will be of potential importance for control of TB worldwide.

Acknowledgements

This work is supported by UMDNJ Foundation Grant (V.V), and American Heart Association-Scientist Development Grant 0335370T (V.V). The authors acknowledge Infectious Diseases division of UMDNJ and NIH AI34436 for partial support. We acknowledge Dr. Jerrold Ellner for helpful discussions. We thank Yaswant Kumar Dayaram for technical assistance and for reading the manuscript. We thank all patients, healthy volunteers, and the Blood Center of NJ, for providing us with samples for this study.

Outline References

Abstract
Introduction
Experimental methods
Results
Discussion
Acknowledgements
References

1. Aukrust P, Liabakk NB, Muller F, Lien E, Espevik T, Froland SS: Serum levels of tumor necrosis factor-alpha (TNF alpha) and soluble TNF receptors in human immunodeficiency virus type 1 infection - correlations to clinical, immunologic, and virologic parameters.
J Infect Dis 1994, 169:420-4. [PubMed Abstract]

Return to citation in text: [1]

2. Barnes PF, Abrams JS, Lu S, Sieling PA, Rea TH, Modlin RL: Patterns of cytokine production by mycobacterium-reactive human T-cell clones.
Infect Immun 1993, 61:197-203. [PubMed Abstract][PubMed Central Full Text]

Return to citation in text: [1]

3. Breen EC, Rezai AR, Nakajima K, et al.: Infection with HIV is associated with elevated IL-6 levels and production.
J Immunol 1990, 144:480-4. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1]

4. Brill KJ, Li Q, Larkin R, Canaday DH, Kaplan DR, Boom WH, Silver RF: Human natural killer cells mediate killing of intracellular Mycobacterium tuberculosis H37Rv via granule-independent mechanisms.
Infect Immun 2001, 69(3):1755-65. [PubMed Abstract] [Publisher Full Text] [PubMed Central Full Text]

Return to citation in text: [1] [2] [3]

5. Buhl R, Jaffe HA, Holroyd KJ, Wells FB, Mastrangeli A, Saltini C, Cantin AM, Crystal RG: Systemic glutathione deficiency in symptom-free HIV-seropositive individuals.
Lancet 1989, 2(8675):1294-8. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1] [2] [3]

6. Canaday DH, Wilkinson RJ, Li Q, Harding CV, Silver RF, Boom WH: CD4(+) and CD8(+) T cells kill intracellular Mycobacterium tuberculosis by a perforin and Fas/Fas ligand-independent mechanism.
J Immunol 2001, 167(5):2734-42. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1] [2]

7. Cohn DL, El-Sadr WM: Treatment of latent tuberculosis infection.
In Tuberculosis: A comprehensive international approach. 2nd edition. Edited by: Reichman LB, Hershfield E. Marcel Dekker, New York; 2000:471-502.

Return to citation in text: [1] [2] [3]

8. Clerici M, Lucey DR, Berzofsky JA, et al.: Restoration of HIV-specific cell-mediated immune responses by interleukin-12 in vitro.
Science 1993, 262:1721-4. [PubMed Abstract]

Return to citation in text: [1]

9. Daley CL, Small PM, Schecter GF, et al.: An outbreak of tuberculosis with accelerated progression among persons infected with the human immunodeficiency virus. An analysis using restriction-fragment-length polymorphisms.
N Engl J Med 1992, 326:231-5. [PubMed Abstract]

Return to citation in text: [1]

10. Deneke SM, Fanburg BL: Regulation of cellular glutathione.
Am J Physiol 1989, 257(4 Pt 1):L163-73. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1] [2]

11. de Quay B, Malinverni R, Lauterburg BH: Glutathione depletion in HIV-infected patients: role of cysteine deficiency and effect of oral N-acetylcysteine.
AIDS 1992, 6(8):815-9. [PubMed Abstract]

Return to citation in text: [1] [2] [3]

12. Dorman SE, Holland S: Interferon-gamma and interleukin-12 pathway defects and human disease.
Cytokine Growth Factor Rev 2000, 11(4):321-33. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1]

13. Droge W, Holm E: Role of cysteine and glutathione in HIV infection and other diseases associated with muscle wasting and immunological dysfunction.
FASEB J 1997, 11(13):1077-89. [PubMed Abstract] [Publisher Full Text]

Review
Return to citation in text: [1]

14. Eck HP, Gmunder H, Hartmann M, Petzoldt D, Daniel V, Droge W: Low concentrations of acid-soluble thiol (cysteine) in the blood plasma of HIV-1-infected patients.
Biol Chem Hoppe Seyler 1989, 370(2):101-8. [PubMed Abstract]

Return to citation in text: [1] [2]

15. el-Ahmady O, Mansour M, Zoeir H, Mansour O: Elevated concentrations of interleukins and leukotriene in response to Mycobacterium tuberculosis infection.
Ann Clin Biochem 1997, 34(Pt 2):160-4. [PubMed Abstract]

Return to citation in text: [1]

16. Ellner JJ: Tuberculosis in the time of AIDS. The facts and the message.
Chest 1990, 98:1051-2. [PubMed Abstract]

17. Ellner JJ: Regulation of the human immune response during tuberculosis.
J Lab Clin Med 1997, 130:469-75. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1] [2]

18. Flynn JL, Chan J, Triebold KJ, Dalton DK, Stewart TA, Bloom BR: An essential role for interferon-γ in resistance to Mycobacterium tuberculosis infection.
J Exp Med 1993, 178:2249-2252. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1] [2]

19. Fraternale A, Casabianca A, Tonelli A, Chiarantini L, Brandi G, Magnani M: New drug combinations for the treatment of murine AIDS and macrophage protection.
Eur J Clin Invest 2001, 31(3):190-2. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1] [2] [3]

20. Godfried MH, van der Poll T, Weverling GJ, et al.: Soluble receptors for tumor necrosis factor as predictors of progression to AIDS in asymptomatic human immunodeficiency virus type 1 infection.
J Infect Dis 1994, 169:739-45. [PubMed Abstract]

Return to citation in text: [1]

21. Havlir DV, Torriani FJ, Schrier RD, et al.: Serum interleukin-6 (IL-6), IL-10, tumor necrosis factor (TNF) alpha, soluble type II TNF receptor, and transforming growth factor beta levels in human immunodeficiency virus type 1-infected individuals with Mycobacterium avium complex disease.
J Clin Microbiol 2001, 39:298-303. [PubMed Abstract] [Publisher Full Text] [PubMed Central Full Text]

Return to citation in text: [1] [2] [3]

22. Haug CJ, Aukrust P, Lien E, Muller F, Espevik T, Froland SS: Disseminated Mycobacterium avium complex infection in AIDS: immunopathogenic significance of an activated tumor necrosis factor system and depressed serum levels of 1,25 dihydroxyvitamin D.
J Infect Dis 1996, 173:259-62. [PubMed Abstract]

Return to citation in text: [1]

23. Helbling B, von Overbeck J, Lauterburg BH: Decreased release of glutathione into the systemic circulation of patients with HIV infection.
Eur J Clin Invest 1996, 26:38-44. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1] [2]

24. Herzenberg LA, De Rosa SC, Dubs JG, Roederer M, Anderson MT, Ela SW, Deresinski SC, Herzenberg LA: Glutathione deficiency is associated with impaired survival in HIV disease.
Proc Natl Acad Sci USA 1997, 94:1967-72. [PubMed Abstract] [Publisher Full Text] [PubMed Central Full Text]

Return to citation in text: [1] [2] [3]

25. Moreno C, Mehlert A, Lamb J: The inhibitory effects of mycobacterial lipoarabinomannan and polysaccharides upon polyclonal and monoclonal human T cell proliferation.
Clin Exp Immunol 1988, 74:206-10. [PubMed Abstract]

Return to citation in text: [1]

26. Nagabhushanam V, Solache A, Ting LM, Escaron CJ, Zhang JY, Ernst JD: Innate inhibition of adaptive immunity: Mycobacterium tuberculosis-induced IL-6 inhibits macrophage responses to IFN-gamma.
J Immunol 2003, 171:4750-7. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1] [2]

27. Nathan C, Xie QW: Regulation of biosynthesis of nitric oxide.
J Biol Chem 1994, 269:13725-8. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1]

28. Newman RM, Fleshner PR, Lajam FE, Kim U: Esophageal tuberculosis: a rare presentation with hematemesis.
Am J Gastroenterol 1991, 86(6):751-5. [PubMed Abstract]

Return to citation in text: [1]

29. Rook GA: Progress in the immunology of the mycobacterioses.
Clin Exp Immunol 1987, 69:1-9. [PubMed Abstract]

Return to citation in text: [1]

30. Rothstein JL, Lint TF, Schreiber H: Tumor necrosis factor/cachectin. Induction of hemorrhagic necrosis in normal tissue requires the fifth component of complement (C5).
J Exp Med 1988, 168:2007-21. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1]

31. Salvaggio A, Balotta C, Galli M, Clerici M: CD4 count in HIV infection is positively correlated to interferon-gamma and negatively correlated to interleukin-10 in vitro production.
AIDS 1996, 10:449-51. [PubMed Abstract]

Return to citation in text: [1]

32. Selwyn PA, Sckell BM, Alcabes P, Friedland GH, Klein RS, Schoenbaum EE: High risk of active tuberculosis in HIV-infected drug users with cutaneous anergy.
Jama 1992, 268:504-9. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1]

33. Silver RF, Li Q, Boom WH, Ellner JJ: Lymphocyte-dependent inhibition of growth of virulent Mycobacterium tuberculosis H37Rv within human monocytes: requirement for CD4+ T cells in purified protein derivative-positive, but not in purified protein derivative-negative subjects.
J Immunol 1998, 160(5):2408-17. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1] [2]

34. Stamler JS, Simon DI, Jaraki O, Osborne JA, Francis S, Mullins M, Singel D, Loscalzo J: S-nitrosylation of tissue-type plasminogen activator confers vasodilatory and antiplatelet properties on the enzyme.
Proc Natl Acad Sci USA 1992, 89:8087-91. [PubMed Abstract] [Publisher Full Text] [PubMed Central Full Text]

Return to citation in text: [1]

35. Stamler JS: Redox signaling: nitrosylation and related target interactions of nitric oxide.
Cell 1994, 78:931-6. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1]

36. Stenger S, Hanson DA, Teitelbaum R, Dewan P, Niazi KR, Froelich CJ, Ganz T, Thoma-Uszynski S, Melian A, Bogdan C, Porcelli SA, Bloom BR, Krensky AM, Modlin RL: An antimicrobial activity of cytolytic T cells mediated by granulysin.
Science 1998, 282(5386):121-5. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1] [2]

37. Strieter RM, Kunkel SL, Bone RC: Role of tumor necrosis factor-alpha in disease states and inflammation.
Crit Care Med 1993, 21:S447-63. [PubMed Abstract]

Return to citation in text: [1]

38. Vanham G, Toossi Z, Hirsch CS, Wallis RS, Schwander SK, Rich EA, Ellner JJ: Examining a paradox in the pathogenesis of human pulmonary tuberculosis: immune activation and suppression/anergy.
Tuber Lung Dis 1997, 78:145-58. [PubMed Abstract] [Publisher Full Text]

39. VanHeyningen TK, Collins HL, Russell DG: IL-6 produced by macrophages infected with Mycobacterium species suppresses T cell responses.
J Immunol 1997, 158:330-7. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1]

40. Venketaraman V, Dayaram YK, Amin AG, Ngo R, Green RM, Talaue MT, Mann J, Connell ND: Role of glutathione in macrophage control of mycobacteria.
Infect Immunity 2003, 71(4):1864-71. [Publisher Full Text]

Return to citation in text: [1] [2]

41. Venketaraman V, Dayaram YK, Talaue MT, Connell ND: Glutathione and nitrosoglutathione in macrophage defense against M. tuberculosis.
Infect Immunity 2005, 73(3):1886-9. [Publisher Full Text]

Return to citation in text: [1] [2] [3]

42. Wallis RS, Amir-Tahmasseb M, Ellner JJ: Induction of interleukin 1 and tumor necrosis factor by mycobacterial proteins: the monocyte western blot.
Proc Natl Acad Sci U S A 1990, 87:3348-52. [PubMed Abstract] [Publisher Full Text] [PubMed Central Full Text]

Return to citation in text: [1]

43. Wallis RS, Ellner JJ: Cytokines and tuberculosis.
J Leukoc Biol 1994, 55:676-81. [PubMed Abstract]

Return to citation in text: [1]

44. Wallis RS, Fujiwara H, Ellner JJ: Direct stimulation of monocyte release of interleukin 1 by mycobacterial protein antigens.
J Immunol 1986, 136:193-6. [PubMed Abstract] [Publisher Full Text]

Return to citation in text: [1]

Richard Jefferys,

It has not been all that long ago since you came out of the woodwork, in the snakepit. I remember it well. And now you are trying to blow first horn, trying to tell us about CMV being an "opportunistic infection" that was very rare before HIV was invented, but is prevalent among newborn babies now, all of a sudden.

As so many viruses, CMV is quite common and omnipresent. It always was, and it doesn't usually cause any problems. The reason we didn't hear much about CMV before the eighties is that nobody was looking for it. Only after the HIV hullabaloo started, and it became clear even the HIV-hustlers that HIV could not be the sole cause of AIDS, other viruses such as CMV and Herpes were dragged into the fray.
A newborn baby doesn't need HIV to have weak immunity. Apart from some antibodies inherited from the mother, the whole system still has to be built up. That also holds for the cellular immunity system. And if a baby is already sick after being born prematurely, and in need of blood transfusions (!) inverted T-cell subset ratios need not surprise us.
Recurrent otitis media, hepatomegaly? They are certainly not AIDS-defining diseases. The first paper is from Nov. 1984, shortly after the Gallo-craze broke loose. Everybody was eager to see the new fashionable virus reflected in their own research. Just like the medical student, who goes on hospital rounds, and diagnoses each patient he sees with the disease he has just learned about that morning in class.

Wow! Tell 'em, James! Good to see you here. This thread could use some real substance.

Wilhelm

Richard wrote:

"As regards Liam Scheff (truly one of the vilest creatures I have ever encountered on the internet), it is notable that a standard tactic of propaganda is to try and provoke an emotional response that precludes rational evaluation of whether the propaganda is based on fact...."

Ummm.... isn't your ad hominem attack on Liam Scheff there, actually trying to provoke an unsubstantiated emotional response?

Exactly what you are supposedly decrying.

As to the science of AIDS... do the words "endogenous retrovirus" strike a cord of plausability with you.

After all, it would rather explain Padian, eh?

Fintan, If HIV were not sexually transmissible then it would be found in sexual partners of HIV seropositive individuals at about the same frequency as it is found in the general population. In fact, HIV is found at much higher frequencies in sexual partners (but not other family members) of HIV positive individuals. So no, HIV does not behave as an "endogenous retrovirus".

REPOSTING WITHOUT MISSED WORDS OR TYPOS:

After 25 years of keeping track of AIDS cases, all the while assuming that AIDS cases have something to do with a person's sexual activities, I am surprised by the shockingly tiny number of alleged "heterosexual" AIDS cases found among women in San Francisco.

There have been over 26,000 cases of AIDS recorded in San Francisco since 1981....... but only 263, roughly around 10 per year, are attributed to a woman's heterosexual activities.

How is this to be explained, especially since my friend and I live near SF and many of us can assure you that heterosexual SF women engage lustily in all sorts of sexual behaviors, usually without condoms since they take charge of their own birth control devices.

Also, I was amazed to discover how few actual cases of AIDS or even HIV-positive test results have ever been found at the sexually-active campuses of universities in California.

How is that to be explained among a cohort of sexually-active 20-26 year olds when there are large numbers of cases of genital warts, herpes simplex and chlamydia?

References to key scientific studies and logical, evidence-based explanations would be much appreciated.

Thanks.

Charles,

Two words: Padian Paper

"We found no seroconversions..." (Padian, pg 354.)

Hank

James, have you tried writing or emailing the authors of the studies you cite?

If you want to assist in getting studies set up this would be a logical move.

I find it very encouraging that you are a) making a positive proposal and b) providing evidence to support your case.

However, I don't agree with your conclusions about PCP and KS having nothing to do with HIV and immune suppression and I strongly doubt that any of the authors of the studies you cite would either.

My advice is to contact the scientists that are currently researching possible treatments such as NAC and vitamin supplements and see what you can do to support their research.

I note that the NCCAM in the US sponsors various studies researching alternative treatments for HIV/AIDS

I also note that Leonore Herzenberg's work on NAC which you cite is supported by an NIH grant.

Wafaie Fawzi's work on vitamin supplements is supported by a National Institute of Child Health and Human Development grant.

The Bill and Melinda Gates Foundation is also a logical granting body to approach.

I think that a positive approach like this has a good probability in achieving many of your goals.

I also think that there are many people that would also support such an iniative.

Best wishes.

To Charles:

361 women died of AIDS in the UK in 2000. 559 died falling down the stairs. The authority is a little weak, coming from a youth magazine, but consider this an invitation to disprove it.

Yours
RS

Interesting that it's OK to be skeptical of anyone but the "rethinkers," and then even saying that I'll take something they say with a grain of salt results in charges of "libel." Funny, that.

I suppose it's too much to ask to keep the vile attacks down to a single thread. Closing this.