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jake,
The amount of objective responses, so early, is indicative that "it's working", really well...
IMO, we'll know more- and soon, since the 15th patient dosed will be due for their first scan by the end of this month, which obviously means that patients 12, 13, 14 will be due for scans before the end of this month, and likely earlier patients will be due for their second scans as early as 'today', (since the first patient dosed was announced 16 weeks ago today).
I hope Peregrine will be aggressive with PR's from this important trial.
j
early data - 5 out of 11 objective,
the rest stable,
none progressive.
Those who understand how Bavi works, will understand that the results, as impressive as they were at the first scan, should improve with each scan.
This is/was not an 8 week trial.
This was a very early interim report (because the data was so impressive), on a trial that could go up to six months of combo therapy, and indefinite bavi monotherapy.
As I had said previously, the way this trial is set up, PPHM can issue interim reports whenever they feel it's warranted.
Yesterday's was warranted, and IMO, based on how Bavi works, any further incremental increases in efficacy also warrant PR's.
Based on the fact that they now have 5 objective responses, and I think they need 6 or 7 to expand the trial, (based on typical "Simon" trial parameters), I would assume the next interim update will likely announce that they've made it over that hurdle, probably without needing to wait to scan the 15th patient at the end of this month. Hopefully when they report on that milestone they'll include mention that the patients are even better off than they were in today's report, (via an increased percentage of objective responses).
I think part of what they need to talk about is how Bavi is different, and how it should KEEP working, it should KEEP improving the patient's condition, in those that it is working in.
Until?........
Looking at yesterday's info - no patient had progressive disease, and 5 out of 11 already had objective responses, at such an early point in the therapy, I'd say it could be working in all of them.
IMO,
* PPHM should issue additional PR's if there are additional objective responses.
* PPHM should issue additional PR's if there are further tumor responses in those patients already with objective responses. If second/third/fourth scans show additional tumor shrinkage, it should be reported, and IMO - the way Bavi works, if a patient has responded, there WILL be further tumor shrinkage, until....?....
* PPHM should issue additional/separate PR's if there are complete responses.
* PPHM should issue additional/separate PR's if the therapy reduces metastatic tumors.
* PPHM should drive home the fact that Bavi is battling cancer in a completely different way than any other therapy. Due to the newness of the concept that PS suppresses the immune system, and especially due to the fact that deliberately targeting PS defies traditional immunological common sense, PPHM still needs to aggressively explain the multiple MOA's of Bavi. They now have a great opportunity to do that - they can tell the story, backed up by this great data that is just starting to unfold in these breast cancer patients - data which I expect to be unprecedented data in terms of it's success.
AS the data unfolds, AS primary tumors shrink, AS metastatic tumors shrink, is a great time to explain why and how these things are happening, via, (as most here know already),
* the initial MOA of macrophages destroying bavi-tagged tumor vasculature,
* and then - as the macrophages, induced by Bavi, alter the local cytokine environment, secreting more TNF-alpha and less TGF-beta, facilitating dendritic cells to identify tumor-specific antigens and train other immune cells for a specific attack of the individual patient's cancer, kicking in the later 'adaptive' immune response.
PR's like the ones I propose, along with more education into Bavi's UNIQUE MOA's, could then make each "news" event into a "new rumor" as people understand that Bavi results should get better with each scan, over time. As good as each news event may be, in terms of great data, it should be seen also as a teaser/indicator for an even better news event down the road.
JMO
j
BKT - good and important question
The first patient was dosed on (or likely before) Feb 12th
http://pphm.client.shareholder.com/releasedetail.cfm?ReleaseID=293434
The 15th patient was dosed on (or likely before) April 29th
http://pphm.client.shareholder.com/releasedetail.cfm?ReleaseID=307128
According to what we've heard, they're scanned at roughly 8 week intervals. The first patient will then be due for her second scan roughly on (or likely before) June 3rd, which happens to be tomorrow.
The important thing is this - how quickly did they enroll the first several patients?
and - how proactive will the company be in announcing any further improvements in objective (or hopefully also complete) responses?
I expect a very nice improvement in the numbers after 16 weeks of therapy as compared to 8.
Will they have a number of patients through their second scans in a matter of a week or two?
I think they only need 6 or 7 objective responses to qualify for the trial moving into the largert group. They may not even have to wait to scan that 15th patient the first time.
(or the 14th patient, or 13th, or 12th)....
With how well it's been going, if they see another objective response in the next wek or two, that could put them over the hurdle much earlier than scanning the 15th patient. and that would be a very newsworth event - qualifying to expand the trial, earlier than I expected - and reporting even better details than we saw this morning.
j
Comparing the phase 1B results with phase 2 interim results -
(What a difference a better immune system makes)....
Phase 1B:
12 patients
12 weeks of therapy
50% had progressive disease
50% had objective response or stable disease
Phase 2:
11 patients (so far)
likely 8 weeks of therapy (so far, at scan time)
NO PATIENTS have progressive disease
100% have objective response or stable disease
Excitedly awaiting the next scans.......
j
jake,
If you can find any Avastin or Erbitux data after only about 8 weeks of therapy let me know. I doubt you'll find any.
This is such a short time of therapy to announce results, especially results like this, I'm stunned.
Also - I don't think Avastin or Erbitux see a 45% (5 out of 11) objective response rate.
This is early, but very promising news.
The most important thing to me is that, unlike Erbitix or Avastin, which only potentially work on a certain subset of people with certain tumor types, Bavi should work on ANY AND ALL of 'em, due to HOW it works.
AND IT SHOULD KEEP WORKING.
IMO, We'll be seeing that "OBJECTIVE RESPONSE" percentage CLIMBING in the coming weeks.
We'll be seeing even BETTER headlines, SHORTLY, IMO, on the patients' next scans, based on Bavituximab's MOA's.
j
PHASE II BAVI 100% OBJECTIVE OR STABLE -
5 out of 11 objective responses
other 6 out of 11 stable.
Judging by the trial dosing start, that's likely on their very first scan at 8 weeks. The 15th treated patient was announced to have been treated on April 29th. That would place that person's first scan time around the end of June, at the latest.
IMHO, if Bavi is working, Bavi KEEPS working.
These people are eligible for up to 6 months of chemo/Bavi, and eligible for indefinite continued Bavi if they don't progress.
Cancer does not, can not, mutate around bavi therapy.
Bavi attaches to the tumor's vasculature, which exposes PS due to hypoxic condition inside the tumor.
Five out of 11 objective responses.
What will their future scans show???????!!!!!!!!
VERY HAPPY,
j
Q: "WHY DOES JAZZ KEEP POSTING STUFF THAT PEOPLE THINK PPHM HAS ANYTHING TO DO WITH? "
A: because it does.
j
"Bavi seems to be on the back burner"
I respectfully disagree.
unless you mean - like when you're cooking - things on the back burner are the things that are about to be attended to.
j
"effects of inhibition of apoptotic events"
What is "CHAVI" talking about there ?........
full quote -
"The team is planning to investigate the effects of inhibition of apoptotic events on the timing of immune responses and control of viremia in SIV-infected macaques."
They're saying they're going to INHIBIT APOPTOTIC EVENTS in monkeys, and observe any effect it may have on altering immune response and/or controlling the virus.
HOW?
How do they plan to alter/affect the apoptotic process/events?...
:)
Does it have immediate implications for HIV therapeutics?
YES.
j
CHAVI UPDATE - bullet points - May 2008
http://www.chavi.org/modules/chavi_reports/index.php?id=24
"the team has shown evidence of immune cell apoptosis with elevations of microparticles beginning just before plasma viral load ramp-up.
now proven/published in the recent Haynes paper.
They showed that CD4 T cell microparticles can suppress memory B cell activation in vitro.
now proven/published in the recent Haynes paper.
These data raise the hypothesis that one reason for delayed protective antibody responses may be due to apoptotic microparticle-mediated immunosuppression of antibody responses."
"the effects of apoptotic microparticles on DC function are currently being addressed."
this is work currently being performed by Nina Bhardwaj, Seph Borrow, Andrew McMichael, at both NYU and Oxford U., respectively. They will find similar results to Joel Shilyansky, who was the first to show that PS exposing microparticles suppress DC's. (See my "REQUIRED READING #2.") - and yes - Bhardwaj, Borrow, and McMichael are aware of Shilyansky's work...
"The team is planning to investigate the effects of inhibition of apoptotic events on the timing of immune responses and control of viremia in SIV-infected macaques."
------------
j
Remember the HAYNES (CHAVI/Gates) HYPOTHESIS -
(which, now with his new paper, is looking to be right on).
HAYNES HYPOTHESIS -
..."Acutely expressed cytokines and apoptotic microparticles
hypothesized to play a key role in AHI pathogenesis....
April 9, 2007
http://72.14.209.104/search?q=cache:BCKueWKqWTIJ:www.chavi.org/wysiwyg/downloads/CHAVI_012_protocol_v1.pdf+%22CHAVI+012%22&hl=en&ct=clnk&cd=1&gl=us&client=firefox-a
-------------------------------
"Their [Haynes group] current hypothesis is that HIV induces a massive apoptosis
before and during viral ramp-up, and that plasma microparticles (fragments of apoptotic CD3
and T cells) have a suppressive effect on Ab generation, thereby amplifying the apoptotic
cascade. "
May 22, 2007
http://www3.niaid.nih.gov/research/topics/HIV/vaccines/advisory/avrs/PDF/AVRS_May07_Summary.pdf
--------------------------------
Conclusion: These results demonstrate that at the time of viral load ramp-up
in acute HIV-1 infection, there are elevations in plasma levels of TRAIL, FAS
Ligand, and TNFR2 that were associated with the presence of microparticles
from apoptotic T cells.
The presence of these apoptotic markers suggests
apoptosis occurring at the time of initial HIV-1 viral load ramp-up.
That PS+ apoptotic cells and microparticles have been reported to suppress antigen specific
immune responses suggests the hypothesis that immune cell
apoptosis in the very earliest stages of acute HIV-1 infection may delay the
onset of potentially protective anti-HIV-1 immune responses."
August 20, 2007
http://www.hivvaccineenterprise.org/_dwn/poster_sessions.pdf
-----------
j
Haynes most recent paper, & PS-mediated immune suppression
Induction of Plasma (TRAIL), TNFR-2, Fas Ligand and Plasma Microparticles After
HIV-1 Transmission: Implications for HIV-1 Vaccine Design
Nancy Gasper-Smith1, Deanna M. Crossman1, John F. Whitesides1, Nadia Mensali2, Janet
S. Ottinger2, Steven G. Plonk1, M. Anthony Moody1, Guido Ferrari2, Kent J. Weinhold2,
Sara E. Miller3, Charles F. Reich III4, Li Qin5,6, Stephen G. Self5,6
, George M. Shaw7,
Thomas N. Denny1, Laura E. Jones8, David S. Pisetsky4, Barton F. Haynes1
1Duke Human Vaccine Institute, Departments of Medicine, 2Surgery, Immunology and
3Pathology, Duke University School of Medicine, Durham, NC 27710, 4Durham VA
Hospital, Durham, NC 27710, 5Fred Hutchinson Cancer Research Center, Seattle, WA
98109, 6Statistical Center for HIV/AIDS Research and Prevention, (SCHARP), Seattle,
WA, 98109, 7University of Alabama-Birmingham, Birmingham, AL 35223, and
8Department of Ecology and Evolutional Biology, Cornell University,
Ithaca, NY 14853
Running Title: Cell Death During Early HIV-1 Infection
Abstract
Death of CD4+, CCR5+ T cells is a hallmark of human immunodeficiency
virus infection. We studied the plasma levels of cell death mediators and products –
tumor necrosis factor-related apoptosis inducing ligand (TRAIL), Fas ligand, tumor
necrosis factor receptor type 2 (TNFR2) and plasma microparticles during the earliest
stages of infection following HIV-1 transmission in plasma samples from US plasma
donors. Significant plasma TRAIL elevations occurred a mean of 7.2 days before the
peak of plasma viral load (VL), while TNFR2, Fas ligand and microparticle elevations
occurred coincident with maximum VL. Microparticles have been previously shown to
mediate immunosuppressive effects on T cells and macrophages. We found that T cell
apoptotic microparticles also potently suppressed in vitro IgG and IgA antibody
production by memory B cells. Thus, release of TRAIL during the eclipse phase of HIV-
1 transmission may initiate or amplify early HIV-1 induced cell death. The window of
opportunity for a HIV-1 vaccine is from HIV-1 transmission until establishment of the
latently infected CD4+ T cells. Release of products of cell death and subsequent
immunosuppression following HIV-1 transmission could potentially narrow the window
of opportunity in which a vaccine has to plausibly extinguish HIV-1, and place severe
constraints on the time the immune system has to respond to the transmitted virus.
--------------------------
snips -
("MP" = MicroParticles)
In this study, we raise the hypothesis that in addition to gut CD4 T cell loss, delay in
HIV-1 protective immune responses early on after HIV-1 transmission may involve the
production of elevated levels of immunosuppressive moieties such as TRAIL, TNFR2
and Fas ligand as well as plasma microparticles. Microparticles (MPs) are small
membrane-bound vesicles that are released from the surface of apoptotic cells by
exocytic or budding processes; as such, MPs bear cell surface markers and can bind
annexin V because of the expression of phosphatidylserine (32-44, 39). MPs, which
circulate in the blood in many clinical conditions, are part of a spectrum of subcellular
structures that are released from cells and can be distinguished from exosomes which are
released from multivesicular bodies during activation. Unlike MPs, exosomes express
endosomal markers. MPs have immunomodulatory activities and can promote immune
cell death; exosomes are also immunologically active, can suppress immune responses
(20,34,42,55), and have been reported elevated in chronic HIV-1 (4).
...................
The presence of TRAIL, TNFR2 and elevated MPs during this early period of acute
HIV-1 infection suggests a number of potential mechanisms of immunosuppression. First,
direct HIV-1 infection results in loss of a substantial proportion of CD4+ T cells, although
the numbers of infected cells does not account for all CD4+ T cell depletion (9,26,51).
Second, TRAIL induces bystander killing in uninfected CD4+ T cells that have previously
interacted with gp120 and have upregulated TRAIL receptors DR5 (28-30,47). Indeed,
Miura et al. showed that administration of an anti-TRAIL mAb in HIV-1 infected hu-PBLNOD-
SCID mice markedly reduces CD4+ T cell apoptosis (52).
Third, suppression of immune responses can be mediated by T cell MPs (32,34,35).
CXCR4+ and CCR5+ MPs can transfer co-receptors to co-receptor negative cells, making
them susceptible to infection by HIV-1 (48,57). Phagocytosis of MPs by macrophages
releases TGF-beta, prostaglandin E2 and IL-10 that can inhibit antigen-specific T and B cell
responses (20,35,42). In this regard, Estes et al. have shown dramatic increases in lymph
node TGF-beta and IL-10 on day 12 following SIV infection (22). Importantly, we have
demonstrated that PBMC and tonsillar cell MPs can directly inhibit memory B cell activation
(Figure 8).
Fourth, both Fas ligand and TRAIL are incorporated into MPs (37,53). Fas ligand
expressing MPs can directly induce apoptosis in nearby cells (20,37,53), and activated T cells
can be the target of Fas ligand-mediated proapoptotic microvesicles (53). Salvato et al.
have recently suggested that treatment of SIV-infected macaques with a mAb against Fas
ligand attenuates disease and may lead to elevated antibody responses to SIV (58). However,
in this regard, in interpreting mechanisms of cell death and immunosuppression by MPs, it
should be noted that preparations of MPs may contain exosomes, depending on the
conditions for isolation and centrifugation (Figure 7A). Although exosome release usually
results from activation rather than apoptosis, both vesicle types may be present together in
plasma, making it difficult to identify definitively the basis for immunological activity of a
vesicle preparation. With induction of apoptosis by staurosporine, however, MPs appear to
be the predominant vesicle type, and it is likely that MPs are responsible for the observed B
cell suppressive activity seen in vitro in Figure 8. In the setting of HIV-1 infection where
both activation and apoptosis occur, however, MPs and exosomes may act concomitantly,
with exosomes suppressing immune responses (2,7,15,61), and MPs contributing to both
immune suppression and cell death (20,32,34,35,39,42,55).
Finally, elevations of plasma TRAIL, TNFR2 and Fas ligand may be predictors of IL-10
mediated immune exhaustion that eventually occur with HIV-1 (6). In this regard, Norris et
al and Stacey and colleagues have demonstrated IL-10 production in plasma donors soon
after the peak in viral load (54,60).
-----------
j
moby,
The work discussed involves PS shutting down the immune response to HIV. The most important "NEW" info involves, specifically, PS and it's suppressive effects on B cells, (the cells responsible for making antibodies). Additionally, and very importantly! - these top docs are agreeing with the work of Fadok, Henson etc. who laid the (very recent) foundations of PS being immunosuppressive, and shutting down the immune response of macrophages, and T cells. That's something that I think most people just don't get the magnitude of! The fact that guys like bart Haynes are now saying PS is immunosuppressive!
(See my "REQUIRED READING #1, which, of course, is cited in this new Haynes paper.....)
Yes, I'll tell you that it's important. It's pretty darn important actually.
They are saying that PS is responsible for HIV establishing a foot-hold early in the infection.
"why did you not mention Bavi explicitly when you said you "expect it to be anti-PS from Peregrine Pharmaceuticals?"
Because as you should know, (and be excited about), Duke, (according to King's many quotes over the past two years), is experimenting with many Peregrine mabs besides Bavi.
I hope to learn about any of several additional possibilities - (all Peregrine's, and maybe not just mabs) -
- the betabodies
- humanized Bavi
- other anti-PS mabs
- other anti-PS mabs with different ways of binding PS
- anti-PS joined to IL-2
- anti-PS joined to IFN
- PS binding peptides
- Schroit's twin beta-2-glycoprotein-I molecules
I sure hope Duke is looking into all of them, or as many as possible! Can you imagine how valuable that research is? The work Duke is doing for Peregrine?
There are a LOT of possibilities....
The Anti-Phospholipid tech. is a platform, not a drug, that's what makes it all the more valuable.
Anti-PS is one part of the anti-Phospholipid platform.
Bavi is one anti-PS drug in the larger anti-phospholipid platform.
Bavi is their first drug in this platform, and obviously the most clinically advanced, and it looks to be doing very well. I sure hope there are many more coming along.
and - If you've been reading Peregrine PR's the past few years you should know about several other very promising drugs they've made, including the few I mentioned above (which are all based around anti-PS), but there are also other phospholipid targets, like phosphatidylethanolamine.... ("PE")..... That is also very promising... (and IMO will eventually make news as an anti-bacterial, as well as an anti mycobacterial drug...)
Who knows, maybe Duke is working with Peregrine's anti-PE too, besides the several Peregrine PS-targeting drugs referenced by King. We'll see.
We know that Duke is working with several PPHM mabs.
I hope to hear about more than just Bavi!
I certainly hope there is more to report on than Bavi alone after two+ years of Duke work along with 2+ years of additional development of the anti-PS platform.
Judging by King's comments on conf calls, I expect to read data on several PPHM mabs.
As one example- I've always thought that Bavi (or whatever other anti-PS, humanized, etc.,) joined with IL-2 should be very promising in HIV therapy.
- but - that's still IMO in addition to straight ahead naked Bavi (or other naked anti-PS), to "clean up the microparticles".
It's a very big and promising platform, which makes it all the more valuable to whomever may eventually partner or buy.
j
e,
I answered you here, on April 10th -
"Peregrine's anti-phospholipid platform is just that - a platform. The platform of naked mabs, mab conjugates, and also peptides, to various phospholipids will hopefully generate many many drugs over the next decade with different specificities for different phospholipids, with somewhat different MOA's etc. (and that's a good thing, which creates additional value for the platform itself and hence the company that eventually partners for it, or buys PPHM..). There are already quite a few other candidates from the platform that have shown promise in preclinical cancer and viral work, (including the PE-binding duramycin peptide conjugate mentioned today on the board), so yes, hopefully we'll hear news of other Peregrine mabs that Duke likes, as has been mentioned by King in the conference calls. "
j
at the peak virus point (which is a couple/few weeks after infection), there are over 400 times more microparticles than virions!
j
Similar mechanism involved in HIV & HCV viral load ramp up.....
It turns out that HCV uses the same method of disarming immune cells as HIV...
PS-exposing microparticles.
BTW - that Duke paper that came out yesterday is 44 pages...
I'm on my second read through this morning, and it's saying just what I thought it would. As I posted yesterday, CHAVI/Gates direction was basically laid out in the CHAVI Administrators meeting last January, (the two slides I post showing the illustrated detrimental immune response caused by exposed PS, and then the anti-PS binding the microparticles). Also, we got more detail in the Gates Vaccine conf abstract, where Haynes gave the plenary speech last August. Then the ideas were really hit upon again, repeatedly, in the most recent CHAVI update. That CHAVI update shows how much the work moved towards exposed PS being the culprit that facilitates the viral load ramp-up. The majority of the update centered around microparticle-mediated immune suppression in early HIV infection. That's what this paper focusses on - measurements into the timing of apoptosis and and it's effects on immune response in early HIV infection, with focus on PS-exposing microparticles as the culprit that lets the virus get out of hand.
facinating stuff!
It's a very important contribution to the HIV vaccine effort, and "raises the bar" in terms of how to tough it may be to design a successful vaccine.
Of course, the promising possibilities for successful therapeutics should now be more obvious :)
it's happening,
j
"It seems they are setting the stage...."
Absolutely.
Also nice to see both SCIENCE and NATURE - in the past few weeks! -
publish that PS IS IMMUNOSUPPRESSIVE and that PS HAS AN ESSENTIAL ROLE TO PLAY IN ENVELOPED VIRAL INFECTION OF CELLS, WITH OBVIOUS STRONG IMPLICATIONS IN HIV INFECTION.
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=29629391
it's happening,
j
as I had said about the recent CHAVI update -
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=29516189
"If I had to pick one paragraph from the most recent CHAVI update as the most important, it would be this-
from the B cell discovery team, (which happens to be the Bart Haynes team)-"
j
Using assays for plasma levels of soluble Fas ligand, TRAIL and TNFR2, the team has shown evidence of immune cell apoptosis with elevations of microparticles beginning just before plasma viral load ramp-up. They showed that CD4 T cell microparticles can suppress memory B cell activation in vitro. These data raise the hypothesis that one reason for delayed protective antibody responses may be due to apoptotic microparticle-mediated immunosuppression of antibody responses.
--------
The whole CHAVI update was incredible in how much the CHAVI work has now moved toward PS-mediated immunosuppression in early HIV infection. That paragraph above was very telling in where they are (and have been) headed with their work.
Now, sure enough, we see this paper from Haynes and other top CHAVI docs today-
Induction of Plasma (TRAIL), TNFR-2, Fas Ligand and Plasma Microparticles After HIV-1 Transmission: Implications for HIV-1 Vaccine Design
http://jvi.asm.org/cgi/content/abstract/JVI.00605-08v1
Abstract
Death of CD4+, CCR5+ T cells is a hallmark of human immunodeficiency virus infection. We studied the plasma levels of cell death mediators and products – tumor necrosis factor-related apoptosis inducing ligand (TRAIL), Fas ligand, tumor necrosis factor receptor type 2 (TNFR2) and plasma microparticles during the earliest stages of infection following HIV-1 transmission in plasma samples from US plasma donors. Significant plasma TRAIL elevations occurred a mean of 7.2 days before the peak of plasma viral load (VL), while TNFR2, Fas ligand and microparticle elevations occurred coincident with maximum VL. Microparticles have been previously shown to mediate immunosuppressive effects on T cells and macrophages. We found that T cell apoptotic microparticles also potently suppressed in vitro IgG and IgA antibody production by memory B cells. Thus, release of TRAIL during the eclipse phase of HIV-1 transmission may initiate or amplify early HIV-1 induced cell death. The window of opportunity for a HIV-1 vaccine is from HIV-1 transmission until establishment of the latently infected CD4+ T cells. Release of products of cell death and subsequent immunosuppression following HIV-1 transmission could potentially narrow the window of opportunity in which a vaccine has to plausibly extinguish HIV-1, and place severe constraints on the time the immune system has to respond to the transmitted virus.
----------
Gee! WHAT'S NEXT????!!!!!!!!!
LOL!!!!!!!!!!
:)
j
(hint)...........
Free - nice find! WOW!!
I've been waiting for that one!
That's likely got the details (and then some) of what Haynes was discussing when he was the Plenary speaker at the Gates vaccine conf. last August.
Remeber the abstract for that one -
Now we see this today -
J. Virol.
JVI Accepts, published online ahead of print on 28 May 2008
Induction of Plasma (TRAIL), TNFR-2, Fas Ligand and Plasma Microparticles After HIV-1 Transmission: Implications for HIV-1 Vaccine Design
Nancy Gasper-Smith, Deanna M. Crossman, John F. Whitesides, Nadia Mensali, Janet S. Ottinger, Steven G. Plonk, M. Anthony Moody, Guido Ferrari, Kent J. Weinhold, Sara E. Miller, Charles F. Reich III, Li Qin, Stephen G. Self, George M. Shaw, Thomas N. Denny, Laura E. Jones, David S. Pisetsky, and Barton F. Haynes*
Duke Human Vaccine Institute, Departments of Medicine, Surgery, Immunology,
and Pathology, Duke University School of Medicine, Durham, NC 27710;
Durham VA Hospital, Durham, NC 27710;
Fred Hutchinson Cancer Research Center, Seattle, WA 98109;
Statistical Center for HIV/AIDS Research and Prevention, (SCHARP), Seattle, WA, 98109;
University of Alabama-Birmingham, Birmingham, AL 35223; and
Department of Ecology and Evolutional Biology, Cornell University, Ithaca, NY 14853
Abstract
Death of CD4+, CCR5+ T cells is a hallmark of human immunodeficiency virus infection. We studied the plasma levels of cell death mediators and products – tumor necrosis factor-related apoptosis inducing ligand (TRAIL), Fas ligand, tumor necrosis factor receptor type 2 (TNFR2) and plasma microparticles during the earliest stages of infection following HIV-1 transmission in plasma samples from US plasma donors. Significant plasma TRAIL elevations occurred a mean of 7.2 days before the peak of plasma viral load (VL), while TNFR2, Fas ligand and microparticle elevations occurred coincident with maximum VL. Microparticles have been previously shown to mediate immunosuppressive effects on T cells and macrophages. We found that T cell apoptotic microparticles also potently suppressed in vitro IgG and IgA antibody production by memory B cells. Thus, release of TRAIL during the eclipse phase of HIV-1 transmission may initiate or amplify early HIV-1 induced cell death. The window of opportunity for a HIV-1 vaccine is from HIV-1 transmission until establishment of the latently infected CD4+ T cells. Release of products of cell death and subsequent immunosuppression following HIV-1 transmission could potentially narrow the window of opportunity in which a vaccine has to plausibly extinguish HIV-1, and place severe constraints on the time the immune system has to respond to the transmitted virus.
-------------------------
That's huge.
They are saying that PS-exposing microparticles shut off effective responses from T cells, and macrophages, and B cells
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
and it's from some big CHAVI names. (Haynes, Self, Shaw, etc.)
They're on board the PS immunosuppressive train.
CHAVI's vaccine work is paving the way to what I expect to be the most effective HIV therapeutic ever....
Actually- CHAVI's vaccine work is paving the way to what I expect to be the most effective broad-spectrum antiviral drug ever...
and yes, I expect it to be anti-PS from Peregrine Pharmaceuticals.
This is incredibly exciting.
They basically laid out their hypothesis last January in this illustration:
How did they plan to deal with the immunosuppressive PS, which also happens to be a target on the virus?
IMHO -
it's happening :)
j
PS - it's essential role in viral entry - recently from the top journals -
NATURE REVIEWS: Molecular Cell Biology -
Virus plays dead
http://www.nature.com/nrm/journal/v9/n6/full/nrm2420.html
..."Apoptotic bodies are macropinocytosed by phagocytes and other cell types, and uptake of apoptotic debris is triggered by the presence of exposed phosphatidylserine (PS) on the plasma membrane. The MV (Mature Virus) membrane is known to be enriched in PS, which is required for infection. The authors showed that PS is exposed on the MV surface and that virus particles extracted with a detergent that removes all lipids fail to induce blebbing and infection. Reconstitution of the extracted virus particles with PS restored infection. Because MVs are comparable in size to apoptotic bodies and the uptake mechanisms are the same, Mercer and Helenius concluded that viral PS might be analogous to cellular PS and triggers the uptake of virus particles by mimicking apoptotic bodies. Consistent with this, late-stage vaccinia-infected cells were shown to undergo apoptosis.
The clever use of apoptotic mimicry and subsequent entry by macropinocytosis allows large particles such as vaccinia virus to infect many different potential host cells. And by posing as apoptotic bodies, MVs may also avoid immune detection."
----------------
recently in Science -
A One-Sided Signal
Gregory D. Fairn and Sergio Grinstein
"The involvement of phosphatidylserine
may not be limited to infection
by vaccinia viruses."
"The role of phosphatidylserine
in the entry of HIV and other
viruses will surely be explored in greater
detail now."
Here's a great illustration -------------
It doesn't get much clearer than this.
This was in Science last month.
The thing is- This mechanism of viral infection and immune shut-off (via dependence on exposed PS), was JUST elucidated in Science last month!
It was "NEW" to most everyone who read it, (but not really new to folks familiar with the things I post). Never the less, this mechanism of viral infection was new in terms of appearing in one of the most respected scientific journals, and made a big splash, with even Nature subsequently commenting on the work, (the first reference at the top of this post).
- This NEW insight into how viruses get away with what they do just hit the top journals the past month.
Authors in the journal Science say,
"The role of phosphatidylserine
in the entry of HIV and other
viruses will surely be explored in greater
detail now."
- Duke University, through the Gates Foundation and the NIH (CHAVI), have not published yet, but have also been working in this area.
- But there's one difference.
- The recent work in Science and Nature outlined the way viruses utilize (and depend upon) exposed PS.
- The work at Duke is looking into ways to DEAL with it, to cover the PS that the virus relies on to enter cells and shut down the immune response.
- The work at Duke is using Peregrine's anti-PS.
--------
it's happening,
j
KT,
Whatever the reason, far fewer shares are actively betting against PPHM's stock moving up...
Also, IMHO, it means to me that nobody on the street believes in a PIPE coming soon.
If Peregrine were shopping, or even inquiring - even as discretely as possible (which is virtually impossible :) - for a private placement to raise money, wouldn't we be seeing a big jump in short shares? Especially with the PPS where it is - to raise enough money to avoid a "going concern" clause would take a lot of shares...
The current historically unprecedented low short percentage looks to me that there is no upcoming PIPE, and not even any rumor of a PIPE among the folks who should be hip to such things.
They're not betting on a PIPE.
Why?
j
hi kgoodrich,
the info is available after 4:00 pm on the 'Dissemination Date' at "nasdaqtrader.com"
j
Peregrine Pharmaceuticals Inc. Short Interest
05/15/2008 1,016,583
04/30/2008 1,004,668
04/15/2008 4,224,568
03/31/2008 3,977,830
03/14/2008 4,209,266
02/29/2008 4,061,311
02/15/2008 4,138,762
01/31/2008 4,185,323
01/15/2008 4,251,204
12/31/2007 4,309,024
12/14/2007 4,316,727
11/30/2007 4,287,426
11/15/2007 4,372,716
10/31/2007 4,361,370
10/15/2007 4,458,667
09/28/2007 4,429,317
09/14/2007 4,522,468
08/15/2007 4,818,099
07/13/2007 4,900,510
06/15/2007 16,999,394
-----------
j
short interest still way down, basically unchanged,
at 1 million.
Extremely low for PPHM, historically speaking....
I wonder why.
What's different this time?
j
OT: HIV movie -
http://www.rockefeller.edu/
Amazing,
j
e,
re: I guess they just didn't "feel" like it yet, huh?
Ha! :)
No, of course not.
You apparently don't understand how the phase I and phase II trials are set up.
The first possible meaningful ("warranted" as I said) update/report (in the current phase II trial which is what my post was about) will be (IMHO), to announce that the trial has passed the "Simon" or "two-stage" hurdle, meaning that the trial has achieved sufficient superiority, in the first 15 patients, over standard of care (which is chemo alone), to move into the second stage of the trial, (the additional 30-some patients).
I expect this to happen, and yes- hopefully soon!
I would love to hear as much detail as possible regarding specific numbers and classification of tumor response.
The least they have to say is that they "made it", and the trial will be treating the rest of the 31 patients.
(which would be great news in itself!).
More than that, it would be great to hear more details. We'll see. I'll be very happy to hear the simple fact that the Bavi/chemo combo has done well enough, (better than SOC!) to advance into the rest of the patient pool, but additionally, any further details would be icing on the cake as they say.
After that - is what I was referring to in my post (which you seem to have misunderstood due to ignorance of the trial protocols.)
To expalin:
Now that we are in this particular phase II trial,
This is NOT a dose escalation trial.
and NOT a "safety" trial.
You need to understand the trial parameters, and the defined endpoints of this phase II trial to understand why they now have the liberty to provide periodic updates as I was explaining previously.
It should be very exciting!
and I expect several updates with very nice info.
Personaly, I think we'll witness a major turning point in cancer therapy!
I also think that once the two Indian cancer trials get started, we'll be seeing, (due to having THREE similarly designed phase II cancer trials up and running), MANY "updates" - with very good info :)
j
mojo,
Sure, Bavituximab worked incredibly well against breast cancer, but it was just a couple women.
but, IMO, that's about to change.
Soon we'll hear about the first stage of the phase II breast cancer trial. These patients' immune systems are much better off than the few women who did so well in the Indian trial.
I would think that the women in this trial should respond better and faster to Bavituximab.
Also, the way Bavi works, it would seem to me that anyone that it works in, it should KEEP working in, unitl....?
Also, according to the way the trial is set up, anyone that it works in, is eligible for continued Bavi therapy for as long as it keeps working.... until?.....
Also, the way that the trial is set up, Peregrine can report on the progress of the patients basically anytime they feel it's warranted. With the way bavi works combined with the relative health of these peoples' immune systems, I would think periodic updates will be very good news.
j
terry,
There has yet to be a published paper on Thorpe's anti-PS viral work. I think we'll see it soon though. Also, as jessme suggested, it may be published concurrently with a separate paper out of Duke on their work with Peregrine's mabs. That would not surprise me. It would be great to see both papers in a top journal, and frankly, I expect it, due to the importance of the discoveries.
Thorpe's grant was up on Jan 31, 2008. You can read about the original info on the grant, with a brief outline of the proposed work, here at the CRISP database, if you go to the querry form, and put thorpe's name into the field,
http://crisp.cit.nih.gov/
Notice that the work culminates with experiments using the real lassa fever virus in a biosafety level 4 lab in Galveston TX.
"Key findings will then be validated for Lassa fever virus itself and with other arenaviruses that cause hemorrhagic fevers in a BSL-4 containment facility at UTMB Center for Biodefense, Galveston."
Well, the director of that lab, Clarence J. Peters, is now listed on Peregrine's SRB,
ANTI-VIRAL – HEMORRHAGIC FEVER
Daniel G. Bausch, M.D., M.P.H.
Associate Professor, Department of Tropical Medicine, Tulane School of Public Health and Tropical Medicine, New Orleans LA
Clarence J. Peters, M.D.
University of Texas Medical Branch, Galveston, TX
Director, Center for Biodefense
Professor of Pathology and of Microbiology and Immunology
Director of the Biosafety Level 4 Laboratory
WHO Collaborating Center for Tropical Diseases
Member
http://www.peregrineinc.com/index.php?option=com_content&task=view&id=15&Itemid=29
There is indeed an upcoming anti-PS viral paper out of UTSW, as mentioned in this slide at a recent Peregrine presentation in New York -
I expect the upcoming Thorpe viral paper to include the things we already know as well as new info. (Things we know like the in vitro work investigating anti-PS binding to all enveloped viruses, anti-PS preventing viral infection of cells, anti-PS binding to virus infected cells, the lassa fever guinea pig experiment, the CMV mice experiment, etc.)
I also expect the Thorpe viral paper to include many new things, obviously for instance the work from the BSL4 lab in Galveston, but also I'm expecting new influenza data and other data, namely - "other arenaviruses that cause hemorrhagic fevers" as mentioned in he grant...
It should be very exciting.
and again, I wouldn't be surprised to see it come out along with the paper out of Duke, both in a "top" journal, and get major press...
JMO,
j
NATURE : "Virus Plays Dead" -----------
NATURE REVIEWS: Molecular Cell Biology, June 2008
Virus plays dead
http://www.nature.com/nrm/journal/v9/n6/full/nrm2420.html
Research Highlight
Nature Reviews Molecular Cell Biology 9, 422 (June 2008) | doi:10.1038/nrm2420
Cellular microbiology: Virus plays dead
Arianne Heinrichs
Vaccinia virus is a large, complex, enveloped DNA virus that belongs to the Poxviridae family of viruses, which includes variola, the causative agent of smallpox. The infectious mature virus (MV) form of vaccinia has been shown to bind to actin-containing finger-like protrusions (filopodia) of the host cell and enter the cell in a pH-dependent manner. In a new study published in Science, Jason Mercer and Ari Helenius report that the MV form of vaccinia virus enters host cells using macropinocytosisand apoptotic mimicry.
The authors prepared fluorescent MV particles and used live imaging to follow the entry of individual particles into host cells that expressed fluorescent actin. Virus particles that bound to filopodia moved towards the cell body in an actin-dependent manner. Once they reached the cell body, membrane blebs formed at the site of contact with virus, followed by the formation of further blebs along the entire cell body. The blebs eventually retracted, which coincided with virus entry. Inhibiting membrane blebbing caused a large reduction in infection, which suggests that blebbing is required for infection.
The Ser/Thr kinase p21-activated kinase-1 (PAK1) is essential for MV infection; indeed, knockdown of PAK1 reduced infection significantly. MV infection is accompanied by phosphorylation of residue Thr423 of PAK1, which is known to be essential for macropinocytosis. PAK1 knockdown studies also showed that PAK1 is required for events that occur prior to viral fusion, including blebbing. Perturbance of several other factors, including the PAK1 activator GTPase Rac1 and Na+/H+ exchangers, inhibited both blebbing and infection. This further confirmed that these processes are linked. Sensitivity to Na+/H+ exchangers is also characteristic of macropinocytosis. Indeed, MVs co-internalized with fluid-phase cargo, but not clathrin-dependent cargo, in a macropinocytosis-type endocytic process.
Apoptotic bodies are macropinocytosed by phagocytes and other cell types, and uptake of apoptotic debris is triggered by the presence of exposed phosphatidylserine (PS) on the plasma membrane. The MV membrane is known to be enriched in PS, which is required for infection. The authors showed that PS is exposed on the MV surface and that virus particles extracted with a detergent that removes all lipids fail to induce blebbing and infection. Reconstitution of the extracted virus particles with PS restored infection. Because MVs are comparable in size to apoptotic bodies and the uptake mechanisms are the same, Mercer and Helenius concluded that viral PS might be analogous to cellular PS and triggers the uptake of virus particles by mimicking apoptotic bodies. Consistent with this, late-stage vaccinia-infected cells were shown to undergo apoptosis.
The clever use of apoptotic mimicry and subsequent entry by macropinocytosis allows large particles such as vaccinia virus to infect many different potential host cells. And by posing as apoptotic bodies, MVs may also avoid immune detection.
----------------
it's happening,
j
I notice that the HCV/HIV trial, according to a recent Peregrine update of the clinicaltrials.gov page, is now listed to be completed in Sept.
- so is the US Bavi monotherapy cancer trial :)
So, I'd assume that each trial is now, or already has been, dosing patients at the optimal dose (which I think is around 3mg/kg).
http://www.clinicaltrials.gov/ct2/results?term=bavituximab
j
If I had to pick one paragraph from the most recent CHAVI update as the most important, it would be this-
from the B cell discovery team, (which happens to be the Bart Haynes team)-
Using assays for plasma levels of soluble Fas ligand, TRAIL and TNFR2, the team has shown evidence of immune cell apoptosis with elevations of microparticles beginning just before plasma viral load ramp-up. They showed that CD4 T cell microparticles can suppress memory B cell activation in vitro. These data raise the hypothesis that one reason for delayed protective antibody responses may be due to apoptotic microparticle-mediated immunosuppression of antibody responses.
the team has shown evidence of immune cell apoptosis with elevations of microparticles beginning just before plasma viral load ramp-up.
CD4 T cell microparticles can suppress memory B cell activation
These data raise the hypothesis that one reason for delayed protective antibody responses may be due to apoptotic microparticle-mediated immunosuppression of antibody responses.
HOW CAN THAT BE DEALT WITH?
HOW CAN YOU INTERFERE WITH (PS-EXPOSING) "MICROPARTICLE-MEDIATED IMMUNOSUPPRESSION" ?
What is CHAVI planning on doing further?
They're planning to see/measure the timing and the extent to which apoptotic cells and the microparticles from those cells suppresses the immune response to HIV.
The team is planning to investigate the effects of inhibition of apoptotic events on the timing of immune responses and control of viremia in SIV-infected macaques.
HOW WILL THEY DO THAT?...............
--------
I'm looking forward to reading about results with Peregrine's mabs.
The work discussed above is in the context of seeing what a successful vaccine will need to do- and how quickly it will need to do what it needs to do, and any promising results using anti-PS in the CHAVI studies to gain insights into vaccine design, have immediate implications and applications for HIV therapeutics.
j
CHAVI progress reports, reporting progress --------
http://www.chavi.org/modules/chavi_reports/index.php?id=5
Year 01 Plans and Progress Report Nov 2005
"microparticle" mentions: NONE
"apoptosis/apoptotic" mentions: NONE
CHAVI Progress Report - Organizational Achievements Sept 2006
"microparticle" mentions: NONE
"apoptosis/apoptotic" mentions: NONE
CHAVI Y02 Fall Progress Report Dec 2006
"microparticle" mentions: NONE
"apoptosis/apoptotic" mentions: NONE
CHAVI Y03 First Quarter Progress Report Sept 2007
"microparticle" mentions: NONE
"apoptosis/apoptotic" mentions: 1
D. Recent CHAVI Manuscripts and Publications
Greenland, JR, Geiben, R, Ghosh, S, Pastor, WA, Letvin, NL Plasmid DNA vaccine –elicited cellular immune responses limit in vivo vaccine antigen expression through Fas-mediated apoptosis.
CHAVI Y03 Second Quarter Progress Report May 2008
"microparticle" mentions: 4
"apoptosis/apoptotic" mentions: 8
"microparticle" mentions in context:
The Innate Immunity Discovery Team has shown that although peripheral blood dendritic cell numbers are dramatically reduced in AHI, the remaining cells appear functional, undergoing phenotypic maturation and producing cytokines in response to TLR ligation. The interaction of these cells with T cells and the effects of apoptotic microparticles on DC function are currently being addressed.
Using assays for plasma levels of soluble Fas ligand, TRAIL and TNFR2, the team has shown evidence of immune cell apoptosis with elevations of microparticles beginning just before plasma viral load ramp-up. They showed that CD4 T cell microparticles can suppress memory B cell activation in vitro. These data raise the hypothesis that one reason for delayed protective antibody responses may be due to apoptotic microparticle-mediated immunosuppression of antibody responses.
"apoptosis/apoptotic" mentions in context:
The kinetics of cytokine responses in acute HIV infection have been correlated with those of apoptotic events and the onset of antibody production in the same subjects.
To give more insight into the relationship between early innate and subsequent adaptive responses, the team has recently begun a kinetic analysis of plasma cytokine levels and peripheral blood DC, NK and NKT cell frequency, subset composition, phenotype and functions in CHAVI 001 AHI subjects, data from which will be correlated with results from analysis of apoptotic events and T and B cell responses in the same subjects (being performed by other CHAVI groups).
The Innate Immunity Discovery Team has shown that although peripheral blood dendritic cell numbers are dramatically reduced in AHI, the remaining cells appear functional, undergoing phenotypic maturation and producing cytokines in response to TLR ligation. The interaction of these cells with T cells and the effects of apoptotic microparticles on DC function are currently being addressed.
Taken together, the data generated by the innate team indicate that innate responses are activated very rapidly in response to HIV infection. Some components of the early innate response may help to contain viral replication, acting either directly or via activation of other innate or adaptive responses. Ongoing relational studies will give more insight into protective aspects of the early innate response. However the early immune activation may also have detrimental consequences, driving viral replication and cellular apoptosis. The team is planning to investigate the effects of inhibition of apoptotic events on the timing of immune responses and control of viremia in SIV-infected macaques.
A key question is whether vaccine immunization can modify innate responses or modulate apoptotic events to respond faster/qualitatively better upon challenge with HIV-1 compared to the unimmunized state.
Using assays for plasma levels of soluble Fas ligand, TRAIL and TNFR2, the team has shown evidence of immune cell apoptosis with elevations of microparticles beginning just before plasma viral load ramp-up. They showed that CD4 T cell microparticles can suppress memory B cell activation in vitro. These data raise the hypothesis that one reason for delayed protective antibody responses may be due to apoptotic microparticle-mediated immunosuppression of antibody responses.
-----------
it's happening,
j
I'll tell you -
I've already outlined the general areas I expect to see covered in an upcoming paper ot of Duke, (which I've included below).
but basically,
1. I expect the research to point toward a new way of seeing how disease gets away with what it does. (and that is- via PS).
2. I expect the experimental results in the paper to point toward a new way of treating disease. (and that is - via anti-PS).
Those two things would be viewed as an extremely important, broad, and fundamental discovery - a turning point in how we understand and treat disease.
In my opinion, a good maxim for this new fundamental perspective would be:
"Evolution has favored pathogenesis that resembles apoptosis".
Here's what I'm expecting to see.
- and yes, I expect Peregrine's mabs to play the central role in this 'important, broad, and fundamental discovery', and I expect the link to Peregrine to be clear).
----------------------------------------------------------
* I expect them to show that targteting phospholipids alone with antibodies can neutralize HIV.
* I expect them to show that you don't need to target the virus itself.
* I expect them to show that by targeting phospholipids alone with abs you can prevent fusion of the virus with a cell.
* I expect them to show that targeting host-cell phospholipids stuck on the virus and on virally infected cells with abs is safe.
* I expect them to discuss or reference various details as to the binding specificities of anti-phospholipid abs which further delineate pathogenic and non-pathogenic abs. That's still quite a grey area among most scientists. Carl Alving, who heads the vaccine dept at Walter Reed has written on this topic in the past couple years and, along with Haynes' team, Pojen Chen and Schroit/Thorpe & PPHM, is on the forefront of the present understanding of what delineates pathogenic and non-pathogenic autoantibodies, and is a proponent of the concept of SAFE abs to phospholipids. This grey area (at best, presently), is the reason many scientists and others look funny when you tell them about Bavituximab's target.
* I expect this paper to sound the bell, most authoratively, which ushers in the concept of phospholipid-targeting as a new promising field of therapy, with implications for treating numerous diseases - viral, cancer, and others. I expect it to change how science views disease and disease therapy in a fundamental way.
* I expect them to discuss and lend authority to the fact that PS is immunosuppressive, and that covering PS blocks the immunosuppressive signals of PS.
* I expect to see specific cytokine data from experiments which detail this PS-induced cytokine change.
* I expect them to discuss the overwhelming apoptosis in early HIV infection, and that the shed apoptotic debris, in the form of microvesicles which contain cell membrane phospholipids, expose PS, and that this debris with exposed PS is responsible for blunting the immune system's response.
* Further, and importantly, I hope they put forth the idea that this way that HIV hides from the immune system, (via exposed PS), is quite possibly a general escape mechanism involved in other diseases. I hope to read discussion that, since PS is an early external apoptotic sign, and the cells of the immune system have evolved to recognize it as a fundamental signal for a friendly/homeostasis/pro-growth/repair response, that viruses and protozoan parasites logically seem to have independently evolved to take advantage of this signaling, since it provides the best advantage for their own survival and growth in the body.
we'll see, JMO,
j
PS triggers TGF-beta & immune suppression ------------------
other examples:
FEBS Lett. 2007 Jan
Involvement of phosphatidylinositol-3-kinase and ERK pathways in the production of TGF-beta1 by macrophages treated with liposomes composed of phosphatidylserine.
* Otsuka M,
* Negishi Y,
* Aramaki Y.
School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
We explored the involvement of phosphatidylinositol 3-kinase (PI3K) and ERK pathways in the production of TGF-beta1 by macrophages treated with liposomes composed of phosphatidylserine (PS-liposomes). PS-liposomes activated Akt, downstream of the PI3K signal cascade, and ERK which led to the expression of TGF-beta1. PI3K inhibitors, LY294002 and wortmannin, inhibited the activation of Akt and ERK following the treatment with PS-liposomes. These inhibitors also suppressed the production of TGF-beta1. Furthermore, PS-liposomes activated macrophages to induce TGF-beta1 expression through PS-specific receptors. These findings suggested that a PI3K-ERK signaling pathway via the PS-receptor is intimately involved in the production of TGF-beta1 which regulates macrophage functions.
------------------------------------------
Biol Pharm Bull. 2005 Sep
Phosphatidylserine-specific receptor contributes to TGF-beta production in macrophages through a MAP kinase, ERK.
* Otsuka M,
* Goto K,
* Tsuchiya S,
* Aramaki Y.
School of Pharmacy Tokyo University of Pharmacy and Life Science, Tokyo, Japan.
We explored the involvement of the phosphatidylserine (PS)-receptor in the production of TGF-beta by macrophages treated with PS-liposomes. The binding of anti-PS-receptor antibody to macrophages was specifically inhibited by PS-liposomes. The antibody led to an increase in the production of TGF-beta, and also activated ERK, a member of the MAP kinase. But no activations in p38 and JNK were observed. ERK inhibitor, U0126 completely prevented TGF-beta production. On the addition of a TGF-beta neutralizing antibody or U0126, the inhibitory effect of the anti-PS-receptor antibody on macrophage function, nitric oxide production, was restored. These findings suggested that TGF-beta is one of factors produced by PS-liposomes, and the ERK signaling pathway via the PS-receptor is intimately involved in the production of TGF-beta in macrophages.
------------------------------------------------------------
Cell Death Differ. 2005 Feb
Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release.
* Monks J,
* Rosner D,
* Geske FJ,
* Lehman L,
* Hanson L,
* Neville MC,
* Fadok VA.
Program in Cell Biology, D509, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206, USA.
Clearance of apoptotic cells is critical to tissue homeostasis and resolution of inflammatory lesions. Macrophages are known to remove dying cells and release anti-inflammatory mediators in response; however, many cells traditionally thought of as poor phagocytes can mediate this function as well. In the lactating mammary gland following weaning, alveolar epithelial cell death is massive, yet the gland involutes rapidly, attaining its prepregnancy state in a matter of days. We found histologic evidence of apoptotic cell phagocytosis by viable mammary epithelial cells (MEC) in the involuting mouse mammary gland. Cultured MEC were able to engulf apoptotic cells in vitro, utilizing many of the same receptors used by macrophages, including the phosphatidylserine receptor (PSR), CD36, the vitronectin receptor alpha(v)beta3, and CD91. In addition, MEC, like macrophages, produced TGFbeta in response to stimulation of the PSR by apoptotic cells or the anti-PSR ab 217G8E9, and downregulated endotoxin-stimulated proinflammatory cytokine production. These data support the hypothesis that amateur phagocytes play a significant role in apoptotic cell clearance and its regulation of inflammation.
----------------------------------------------------------
J Clin Invest. 2002 Jan
Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-beta1 secretion and the resolution of inflammation.
* Huynh ML,
* Fadok VA,
* Henson PM.
Department of Pulmonary and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, Colorado, USA.
Ingestion of apoptotic cells in vitro by macrophages induces TGF-beta1 secretion, resulting in an anti-inflammatory effect and suppression of proinflammatory mediators. Here, we show in vivo that direct instillation of apoptotic cells enhanced the resolution of acute inflammation. This enhancement appeared to require phosphatidylserine (PS) on the apoptotic cells and local induction of TGF-beta1. Working with thioglycollate-stimulated peritonea or LPS-stimulated lungs, we examined the effect of apoptotic cell uptake on TGF-beta1 induction. Viable or opsonized apoptotic human Jurkat T cells, or apoptotic PLB-985 cells, human monomyelocytes that do not express PS during apoptosis, failed to induce TGF-beta1. PS liposomes, or PS directly transferred onto the PLB-985 surface membranes, restored the TGF-beta1 induction. Apoptotic cell instillation into LPS-stimulated lungs reduced proinflammatory chemokine levels in the bronchoalveolar lavage fluid (BALF). Additionally, total inflammatory cell counts in the BALF were markedly reduced 1-5 days after apoptotic cell instillation, an effect that could be reversed by opsonization or coinstillation of TGF-beta1 neutralizing antibody. This reduction resulted from early decrease in neutrophils and later decreases in lymphocytes and macrophages. In conclusion, apoptotic cell recognition and clearance, via exposure of PS and ligation of its receptor, induce TGF-beta1 secretion, resulting in accelerated resolution of inflammation.
----------------------------------------------------------------
Cancer Biol Ther. 2005 Sep
Cancer cell immune escape and tumor progression by exploitation of anti-inflammatory and pro-inflammatory responses.
* Kim R,
* Emi M,
* Tanabe K.
International Radiation Information Center, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.
Apoptotic cells can be eliminated by phagocytosis, which is mediated by antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), through phosphatidylserine (PS) on apoptotic cells and phosphatidylserine receptor (PSR) on APCs. The phagocytosis of apoptotic cells by macrophages is strictly regulated by not only the inflammatory reaction, but also by an increase in anti-inflammatory factors such as IL-10, TGF-beta, and prostaglandin E2 (PGE2), leading to an anti-inflammatory situation, whereby apoptosis contributes to a noninflammatory response. However, because PS and PSR are expressed in cancer cells, shed soluble phosphatidylserine (sPS) can interact with the PS receptor on macrophages, which promotes an anti-inflammatory response to macrophages that may lead to immune escape. The sPS derived from cancer cells also reacts with the PSR on the cancer cells to produce IL-10, TGF-beta, and PGE2, which can cause suppression of anti-tumor immunity through the anti-inflammatory response to macrophages, which produces tumor-associated macrophages. Furthermore, sPS and TGF-beta inhibit the maturation of immature DCs, resulting in a functional inhibition of DCs. The potential roles of PS and PSR in cancer cells and macrophages in immune escape mediated by sPS and anti-inflammatory factors are discussed, which may explain their dual regulation of anti- and pro-inflammatory responses during tumor progression.
-------------------------------------------------------------
Immunobiology. 2003
Exposure of anionic phospholipids serves as anti-inflammatory and immunosuppressive signal--implications for antiphospholipid syndrome and systemic lupus erythematosus.
* Gaipl US,
* Beyer TD,
* Baumann I,
* Voll RE,
* Stach CM,
* Heyder P,
* Kalden JR,
* Manfredi A,
* Herrmann M.
Institute for Clinical Immunology and Rheumatology, Department of Internal Medicine III, Erlangen, Germany
In contrast to necrotic cells, the clearance of apoptotic ones usually is an anti-inflammatory process which elicits only a marginal immune response. During apoptosis phosphatidylserine (PS) is exposed on the outer leaflet of the cytoplasmic membrane and serves as target for the PS receptor of phagocytes. The latter is responsible for anti-inflammatory signalling and the induction of TGFbeta. We were interested whether the immunogenicity of apoptotic cells can be increased by masking PS. We observed that treatment of xenogeneic apoptotic cells with annexin V (AxV) significantly increased the humoral immune response against surface epitopes of these cells. Furthermore, AxV-coated irradiated tumour cells were able to elicit a long lasting tumour specific cytotoxic T lymphocyte response. AxV efficiently blocked the uptake of irradiated cells by macrophages but not by dendritic cells. Furthermore, AxV skewed the phagocytosis of irradiated cells towards inflammation. Investigation of patients with autoimmune diseases further supported the role of anionic surface phospholipids for anti-inflammatory clearance of apoptotic cells. Impaired clearance and opsonisation with anti-phospholipid-antibodies are discussed to be responsible for the development of systemic lupus erythematosus and anti-phospholipid-syndrome, respectively. Presentation of cryptic epitopes from late apoptotic cells in a proinflammatory context may challenge T cell tolerance. In addition, accumulation of uncleared apoptotic debris in the germinal centres of lymph nodes may result in the survival of autoreactive B cells.
----------------------------------------------------------------
Biochem Biophys Res Commun. 2001 Mar 2
Involvement of TGF-beta in inhibitory effects of negatively charged liposomes on nitric oxide production by macrophages stimulated with lps.
* Matsuno R,
* Aramaki Y,
* Tsuchiya S.
Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Tokyo, Hachioji, 192-0392, Japan.
We examined the role of TGF-beta in the inhibitory effects of negatively charged liposomes composed of phosphatidylserine (PS-liposomes) on nitric oxide (NO) production by macrophages stimulated with LPS. The expression of TGF-beta mRNA increased when mouse peritoneal macrophages were treated with PS-liposomes. The inhibitory effect of PS-liposomes on NO production was restored by treatment with anti-TGF-beta antibody. Furthermore, NO production, iNOS mRNA expression, and iNOS protein induction by LPS were inhibited by treatment of macrophages with TGF-beta as well as PS-liposomes. These results indicated that PS-liposomes down-regulate NO production by macrophages through the induction of TGF-beta and suggested that TGF-beta may suppress NO production upstream of the transcription of iNOS mRNA. Copyright 2001 Academic Press.
-------------------------------------------------------------
J Immunol. 2005 Feb 1
Interaction between phosphatidylserine and the phosphatidylserine receptor inhibits immune responses in vivo.
* Hoffmann PR,
* Kench JA,
* Vondracek A,
* Kruk E,
* Daleke DL,
* Jordan M,
* Marrack P,
* Henson PM,
* Fadok VA.
Department of Pediatrics, Program in Cell Biology, National Jewish Medical and Research Center, Denver, CO 80206, USA.
Phosphatidylserine (PS) on apoptotic cells promotes their uptake and induces anti-inflammatory responses in phagocytes, including TGF-beta release. Little is known regarding the effects of PS on adaptive immune responses. We therefore investigated the effects of PS-containing liposomes on immune responses in mice in vivo. PS liposomes specifically inhibited responses to Ags as determined by decreased draining lymph node tissue mass, with reduced numbers of total leukocytes and Ag-specific CD4(+) T cells. There was also a decrease in formation and size of germinal centers in spleen and lymph nodes, accompanied by decreased levels of Ag-specific IgG in blood. Many of these effects were mimicked by an agonistic Ab-specific for the PS receptor. TGF-beta appears to play a critical role in this inhibition, as the inhibitory effects of PS were reversed by in vivo administration of anti-TGF-beta Ab. PS-containing liposomes did not appear to directly inhibit dendritic cell maturation in vitro in response to a variety of stimuli, nor did it prevent their migration to regional lymph nodes in vivo, suggesting that the inhibitory effects may have resulted from complicated interactions between tissue cells and dendritic cells, subsequently inhibiting their ability to productively activate T lymphocytes.
---------------------------------------------------------------
Cancer Res. 2006 Jun 1
Tumor-driven evolution of immunosuppressive networks during malignant progression.
* Kim R,
* Emi M,
* Tanabe K,
* Arihiro K.
International Radiation Information Center, Research Institute for Radiation Biology and Medicine, Hiroshima University, Japan
Tumors evolve mechanisms to escape immune control by a process called immune editing, which provides a selective pressure in the tumor microenvironment that could lead to malignant progression. A variety of tumor-derived factors contribute to the emergence of complex local and regional immunosuppressive networks, including vascular endothelial growth factor, interleukin-10, transforming growth factor-beta, prostaglandin E(2), and soluble phosphatidylserine, soluble Fas, soluble Fas ligand, and soluble MHC class I-related chain A proteins. Although deposited at the primary tumor site, these secreted factors could extend immunosuppressive effects into the local lymph nodes and the spleen, promoting invasion and metastasis. Vascular endothelial growth factors play a key role in recruiting immature myeloid cells from the bone marrow to enrich the microenvironment as tumor-associated immature dendritic cells and tumor-associated macrophages. The understanding of the immunosuppressive networks that evolve is incomplete, but several features are emerging. Accumulation of tumor-associated immature dendritic cells may cause roving dendritic cells and T cells to become suppressed by the activation of indoleamine 2,3-dioxygenase and arginase I by tumor-derived growth factors. Soluble phosphatidylserines support tumor-associated macrophages by stimulating the release of anti-inflammatory mediators that block antitumor immune responses. Soluble Fas, soluble FasL, and soluble MHC class I-related chain A proteins may help tumor cells escape cytolysis by cytotoxic T cells and natural killer cells, possibly by counterattacking immune cells and causing their death. In summary, tumor-derived factors drive the evolution of an immunosuppressive network which ultimately extends immune evasion from the primary tumor site to peripheral sites in patients with cancer.
----------------------------------------------------------------
Biochem Biophys Res Commun. 2004 Nov 12
Toxoplasma gondii exposes phosphatidylserine inducing a TGF-beta1 autocrine effect orchestrating macrophage evasion.
* Seabra SH,
* de Souza W,
* Damatta RA.
Laboratorio de Biologia Celular e Tecidual, Centro de Biociencias e Biotecnologia, Universidade Estadual do Norte Fluminense, 28013-600 Campos dos Goytacazes, RJ, Brazil.
Toxoplasmosis is a worldwide disease caused by Toxoplasma gondii. Activated macrophages control T. gondii growth by nitric oxide (NO) production. However, T. gondii active invasion inhibits NO production, allowing parasite persistence. Here we show that the mechanism used by T. gondii to inhibit NO production persisting in activated macrophages depends on phosphatidylserine (PS) exposure. Masking PS with annexin-V on parasites or activated macrophages abolished NO production inhibition and parasite persistence. NO production inhibition depended on a transforming growth factor-beta1 (TGF-beta1) autocrine effect confirmed by the expression of Smad 2 and 3 in infected macrophages. TGF-beta1 led to inducible nitric oxide synthase (iNOS) degradation, actin filament (F-actin) depolymerization, and lack of nuclear factor-kappaB (NF-kappaB) in the nucleus. All these features were reverted by TGF-beta1 neutralizing antibody treatment. Thus, T. gondii mimics the evasion mechanism used by Leishmania amazonensis and also the anti-inflammatory response evoked by apoptotic cells.
-----------------------------------------------------------------
J Immunol. 2006 Feb 1
Mimicry of apoptotic cells by exposing phosphatidylserine participates in the establishment of amastigotes of Leishmania (L) amazonensis in mammalian hosts.
* Wanderley JL,
* Moreira ME,
* Benjamin A,
* Bonomo AC,
* Barcinski MA.
Divisao de Medicina Experimental, Coordenacao de Pesquisa, Instituto Nacional de Cancer, Rio de Janeiro, Brazil.
Signaling through exposed phosphatidylserine (PS) is fundamental for the TGFbeta1-dependent, noninflammatory phagocytosis of apoptotic cells. This same mechanism operates in the internalization of amastigotes of Leishmania (L) amazonensis (L(L)a) in a process quoted as apoptotic mimicry. Now we show that the host modulates PS exposure by the amastigotes and, as a consequence, BALB/c mice-derived amastigotes expose significantly more PS than those derived from C57BL/6 mice. Due to this difference in the density of surface PS molecules, the former are significantly more infective than the latter, both in vivo, in F1 (BALB/c x C57BL/6) mice, and in vitro, in thioglycollate-derived macrophages from this same mouse strain. PS exposure increases with progression of the lesion and reaches its maximum value in amastigotes obtained at the time point when the lesion in C57BL/6 mice begins to decrease in size and the lesions in BALB/c mice are still growing in size. Synthesis of active TGFbeta1, induction of IL-10 message, and inhibition of NO synthesis correlate with the amount of surface PS displayed by viable (propidium iodide-negative) infective amastigote. Furthermore, we also show that, similar to what happens with apoptotic cells, amastigotes of L(L)a are internalized by macropinocytosis. This mechanism of internalization is consistent with the large phagolysosomes characteristic of L(L)a infection. The intensity of macrophage macropinocytic activity is dependent on the amount of surface PS displayed by the infecting amastigote.
----------
j
Bavituximab & change in TGF-beta -----------
- from Rolf Brekken's September 2007 "Bavituximab preclinical studies" presentation, consisting of collaborative research into Bavituximab's immune-modulating MOA at Duke University & SRI.
One of the most important graphs I've ever seen, with implications for treating tumors, enveloped viruses, malaria, trypanosoma, toxoplasma, leishmania..
j
full "Bavituximab preclinical studies" presentation:
http://presentations.cancerconferences.com/7th_monoclonal/files/2007_0817/ws02_brekken.pdf
---------
j
Peter Henson's insightful Oct 2006 PS / TGF-beta paper --------
--------
Apoptotic cells, through transforming growth factor-beta, coordinately induce
anti-inflammatory and suppress pro-inflammatory eicosanoid and NO
synthesis in murine macrophages.
Celio G. Freire-de-Lima∗†‡2, Yi Qun Xiao†‡, Shyra J. Gardai†, Donna L. Bratton†, William P.
Schiemann† and Peter M. Henson†
*Instituto de Biofísica Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro,
RJ 21944-970, Brazil
†Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research
Center, 1400 Jackson Street, Denver, Colorado 80206
Running title: TGF-beta modulates inflammatory eicosanoids
link to full text:
http://www.jbc.org/cgi/reprint/M605146200v1
snips below-
j
--------------------
In the studies reported here, we showed that the
TGF-beta induction by apoptotic cells was
dependent on exposed PS
...............
These results strongly
suggest that the apoptotic cell inhibition of
pro-inflammatory mediator production is
pleiotropic and significantly dependent on the
stimulation of TGF-beta production.
...............
The
implication is that recognition of PS drives the
production of TGF-beta and the downstream antiinflammatory
responses reported herein.
..............
The induction of TGF-beta itself
could be attributed to exposed
phosphatidylserine on the apoptotic cells,
which therefore, appears to drive the
balanced inflammatory mediator responses.
..............
Apoptotic cells are rapidly engulfed by
adjacent tissue cells or macrophages before
they can release pro-inflammatory/proimmunogenic
intracellular contents. In
addition, recognition of the apoptotic cells is
actively anti-inflammatory and antiimmunogenic
with generation of antiinflammatory
mediators such as
transforming growth factor-beta (TGF-beta) and
anti-inflammatory eicosanoids. Here, we have
investigated the role played by the induction
of TGF-beta in the coordinate expression of antiinflammatory
eicosanoids or PPARγ and in
the suppression of pro-inflammatory lipid
mediators and nitric oxide (NO).
................
As a
cell becomes apoptotic, it is generally removed
in situ by near-neighbor cells or macrophages in
a quiet, almost invisible fashion; that is, the
process does not induce a local tissue reaction.
In fact, recognition and removal of apoptotic
cells is normally both anti-inflammatory and
anti-immunogenic (6-9).
................
The interaction and recognition are triggered by
surface changes on the apoptotic cells.
.................
there is considerable evidence to implicate PS as
the main stimulus for the anti-inflammatory or
anti-immunogenic effects (6-8,14-16).
...............
A major anti-inflammatory mediator induced in
response to apoptotic cells, mAb217 or PS
liposomes is TGF-beta (6,8,16). Blockade of TGF-
beta has been shown to reverse the suppressive
effects of apoptotic cells or PS in vivo on either
inflammation or adaptive immunity (7,8).
................
A key issue, therefore, is whether apoptotic cellinduced
TGF-beta, acting in an autocrine/paracrine
fashion, mediates the alterations in eicosanoid
generation. By use of a dominant negative TGF-
beta receptor construct we have been able to show
that apoptotic cells stimulate via their induction
of active TGF-beta, a co-ordinate production of
generally anti-inflammatory, and simultaneous
inhibition of generally pro-inflammatory,
eicosanoids.
................
Results
Apoptotic cells or antibody to PSRS on murine
macrophages stimulate production of TGF- beta
and concomitant blockade of LPS-induced
TNF alpha, NO and iNOS.
.................
Discussion
Apoptotic cells are known to induce an antiinflammatory
and anti-immunogenic response,
in part mediated by their induction of active
TGF-beta in responding cells. Here we show that
the effect of the apoptotic cells is to drive a
complex coordinated inhibition of potentially
inflammatory mediators along with induction of
potentially anti-inflammatory molecules in
macrophages that are orchestrated by the TGF-beta
production.
.................
The observations required the demonstration of
TGF-beta production in response to the apoptotic
cells – shown earlier by numerous investigators
and confirmed herein. A number of ligands have
been demonstrated on apoptotic cells that can
interact with a number of “receptors” on
responding cells, in this case macrophages.
Additionally there are a large group of “bridge”
molecules (see ref. 39) that can link the
apoptotic cell ligands to the receptors. We have
suggested that two important ligands are
phosphatidylserine (PS) and calreticulin. The
latter, as well as the collectin family of bridge
molecules (40) has been suggested to interact
with LRP as a receptor and, in isolation, seems
to induce a more pro-inflammatory response
(5,13). On the other hand, PS and its receptors
and possibly some or all of its bridge molecules
appear to induce the anti-inflammatory effects
and, in most cases, to act in a dominant fashion
in the normal response to apoptotic cells.
Necrotic cells are usually thought to be proinflammatory
(see for example 9,15) and may
have reversed this PS-driven dominance. Other
studies that have suggested that apoptotic cells
can in some circumstances act in a proinflammatory
fashion may also reflect variations
in balance between pro-inflammatory (e.g. LRP)
versus anti-inflammatory (e.g. PS driven)
responses.
................
The results indicate a complex effect of
apoptotic cells acting through release of TGF-beta
to upregulate generally anti-inflammatory
mediators and inhibit the production of proinflammatory
molecules.
________________________________________________
FULL TEXT BELOW -
__________________________________________________
Apoptotic cells, through transforming growth factor-β, coordinately induce
anti-inflammatory and suppress pro-inflammatory eicosanoid and NO
synthesis in murine macrophages.
Apoptotic cells are rapidly engulfed by
adjacent tissue cells or macrophages before
they can release pro-inflammatory/proimmunogenic
intracellular contents. In
addition, recognition of the apoptotic cells is
actively anti-inflammatory and antiimmunogenic
with generation of antiinflammatory
mediators such as
transforming growth factor-β (TGF-β) and
anti-inflammatory eicosanoids. Here, we have
investigated the role played by the induction
of TGF-β in the coordinate expression of antiinflammatory
eicosanoids or PPARγ and in
the suppression of pro-inflammatory lipid
mediators and nitric oxide (NO). By use of a
dominant negative TGFβII receptor, TGF-β
signaling was blocked and its participation in
the consequences of apoptotic cell stimulation
determined. The induction of TGF-β itself
could be attributed to exposed
phosphatidylserine on the apoptotic cells,
which therefore, appears to drive the
balanced inflammatory mediator responses.
Arachidonic acid release, COX-2 and
prostaglandin (PG) synthase expression were
shown to be significantly dependent on the
TGF-β production. On the other hand, a
requirement for TGF-β was also shown in the
inhibition of thromboxane synthase and
thromboxanes, of 5-lipoxygenase and
sulfidopeptide leukotrienes as well as iNOS
and NO. TGF-β-dependant induction of
arginase was also found and would further
limit the NO generation. Finally, apoptotic
cells stimulated production of 15-
lipoxygenase and 15-HETE, a potentially
anti-inflammatory pathway acting though
PPARγ, and lipoxin A4 production which
were also upregulated by a TGF-β-dependant
pathway in this system. These results strongly
suggest that the apoptotic cell inhibition of
pro-inflammatory mediator production is
pleiotropic and significantly dependent on the
stimulation of TGF-β production.
Recognition and clearance of apoptotic cells by
phagocytes play pivotal roles in development,
maintenance of tissue homeostasis, control of
the immune response, and resolution of
inflammation (1,2). Apoptotic cells are removed
by professional phagocytes, members of the
mononuclear phagocyte system (MPS) such as
macrophages and immature dendritic cells, or by
non-professional phagocytes such as fibroblasts,
endothelial, epithelial, smooth muscle or stromal
cells (3). Uptake of the apoptotic cell is by a
specialized and highly conserved form of
phagocytosis termed efferocytosis (4,5). As a
cell becomes apoptotic, it is generally removed
in situ by near-neighbor cells or macrophages in
a quiet, almost invisible fashion; that is, the
process does not induce a local tissue reaction.
In fact, recognition and removal of apoptotic
cells is normally both anti-inflammatory and
anti-immunogenic (6-9).
The interaction and recognition are triggered by
surface changes on the apoptotic cells. Two
widely distributed surface ligands on apoptotic
cells are phosphatidylserine, PS (10,11) and
calreticulin (5) which become associated in
patches together on the cell surface. Indirect
effects of the collectin family of molecules or
direct action of calreticulin leads to stimulation
of LRP (LDL receptor related protein) on the
phagocytosing cell (5,12). However, LRP
activation seems to induce production of proinflammatory
mediators (13). On the other hand,
there is considerable evidence to implicate PS as
the main stimulus for the anti-inflammatory or
anti-immunogenic effects (6-8,14-16). We
suspect that these two stimuli, acting through
different signaling pathways, are balanced, with
a normal bias towards the anti-inflammatory.
Unfortunately, the receptor(s) that recognizes PS
(PS recognition structures, PSRS) that is
responsible for this effect is unknown although it
does seem to distinguish between stereoisomeric
forms of the phosphoserine head group (10,14)
and does seem to react with an activating IgM
antibody mAb217 (17). whose binding is
blocked by PS. The antibody binds to, and
activates cells and mimics exactly the effects of
PS on apoptotic cells in contributing to uptake
and on the generation of anti-inflammatory
mediators (7,18) and has been used here along
with apoptotic cells to stimulate macrophages
for production or suppression of eicosanoids.
A major anti-inflammatory mediator induced in
response to apoptotic cells, mAb217 or PS
liposomes is TGF-β (6,8,16). Blockade of TGF-
β has been shown to reverse the suppressive
effects of apoptotic cells or PS in vivo on either
inflammation or adaptive immunity (7,8). On the
other hand, earlier studies also suggested
induction of other candidates such as IL-10 (19),
PGE2 (6) and even PAF (6) although the last
two can have both pro- or anti-inflammatory
effects. The ability of apoptotic cell recognition
to alter the production of eicosanoids had first
been noted for thromboxane (20,21) and
exemplified by roles for TGF-β and
prostaglandins in apoptotic cell enhancement of
Trypanosoma cruzi growth in macrophages (22).
A key issue, therefore, is whether apoptotic cellinduced
TGF-β, acting in an autocrine/paracrine
fashion, mediates the alterations in eicosanoid
generation. By use of a dominant negative TGF-
β receptor construct we have been able to show
that apoptotic cells stimulate via their induction
of active TGF-β, a co-ordinate production of
generally anti-inflammatory, and simultaneous
inhibition of generally pro-inflammatory,
eicosanoids. The effect is mediated by effects on
the synthases for these mediators. Additional
coordinate effects were seen on related proteins
including iNOS which was downregulated and
PPARγ or arginase, which were induced, i.e.
combining to reduce NO production and also
potentially in keeping with the antiinflammatory
balance.
Results
Apoptotic cells or antibody to PSRS on murine
macrophages stimulate production of TGF- β
and concomitant blockade of LPS-induced
TNF α, NO and iNOS.
Murine macrophages (peritoneal or RAW 264,
1.0 x106 cells/ml) were stimulated with LPS
(100 ng/ml) as positive control or mAb217 (50
µg/ml) or apoptotic Jurkat T cells (3x106
cells/ml) for 18 h. These stimuli each induced
TGF-β production in both types of macrophages
(Figure 1A). Isotype control IgM or viable
Jurkat T cells were inactive. These data are in
accordance with previous reports (6,16). The
stimulation of TGF-β production by apoptotic
cells in this system was blocked by
preincubation of the targets with the PS-binding
protein Factor Va (Figure 1B) as had been
shown earlier with annexin V (29). PS
liposomes also stimulated the production of
TGF-β but less efficiently. This is probably
because the presentation to the PSRS from PS
exposed on the apoptotic cell is from a quite
different environment compared with a
liposome. However, PS liposomes increased the
production of TGF-β in the presence of LPS or
Cyclosporine A (Figure 1C). These findings
suggest that PS liposomes themselves may upregulate
TGF-β translation when TGF-β
message has been induced by other stimuli
(17,30). Classically activated macrophages (LPS
and IFNγ stimulation) exhibit release of TNFα
and NO as well as upregulation of iNOS. As
shown in figures 1D-E, these three responses to
stimulation with LPS and IFNγ were inhibited
by exposure of the macrophages to apoptotic
cells or mAb217. Previous studies implicated the
TGF-β produced, in the suppression of TNFα
induction and might be expected to serve the
same role for suppression of iNOS and NO.
To demonstrate this presumed requirement for
TGF-β in the suppression, a dominant negative
form of the TGFβ RII was employed.
Transfection of RAW 264 cells with this
construct was shown to block the ability of
TGF-β to signal for 3 x PT-luc reporter (which
contains three consecutive TPA response
elements (TREs) and a portion of the
plasminogen activator inhibitor (PAI-1)
promoter region) gene activation (Figure 2A).
Since TGF-β can induce its own synthesis, the
effect of the dominant negative receptor was
also examined on the production of TGF-β itself
after stimulation with apoptotic cells, mAb217
or LPS. As shown in Figure 2B, this treatment
blocked 60-70% of the TGF-β produced by each
of the stimuli i.e. supporting an additional
autocrine/paracrine effect of TGF-β on its own
induction in these systems.
Transfection of the truncated TGFβRII was
found to completely reverse the suppression of
TNFα and NO production caused by apoptotic
cells or PSRS stimulation and also restored the
upregulation of iNOS protein (Figures 2C-E). In
keeping with the suppression of NO production
by blocking upregulation of iNOS, exposure of
macrophages to apoptotic cells or PSRS stimuli
also led to increases in intracellular levels of
arginase 1 (Figure 2F) which could further
reduce the production of nitric oxide.
Macrophages with truncated TGF βRII are
defective in prostaglandin production and
prostaglandin synthase expression in response
to stimulation with apoptotic cells or LPS.
In the original studies of anti-inflammatory
effects of apoptotic cells, PGE2 was also shown
to be generated and it too seemed to play a role
in suppression of inflammatory mediators (27).
Accordingly we next examined the effect of
apoptotic cells and stimulation with mAb217 on
induction of potentially anti-inflammatory
prostaglandins as well as the role of TGF-β in
their regulation. The original studies did not
address the probable induction of PGI2 (detected
as PGF1α) along with PGE2 and, accordingly,
this was included in the analysis. Supernatants
from the cell culture were collected 18 h after
stimulation and analyzed for PGE2 and PGF1α.
The cell lysates were collected and the levels of
synthases for PGE2 (PGES1), PGD2 and PGI2
determined by Western blotting. As expected,
the two stimuli induced production of PGE2 and
PGF1α starting at 2 h or earlier and extending
out to 18 h incubation (Figure 3A-C).
Importantly, they also increased the intracellular
levels of the prostaglandin synthases (Figure
3D).
It has previously been reported that TGF-β can
induce prostaglandin production (22,31,32) and
in data not shown, direct addition of active TGF-
β to the macrophage cultures did stimulate
production of PGE2 and PGF1α. When the
macrophage response to TGF-β was blocked
with the dominant negative receptor, induction
of PGE2 and PGF1α by either apoptotic cells or
mAb217 was prevented (Figure 3A and B).
Interestingly, LPS-induced PGE2 and PGF1α
were also reduced by about 80%. We suspect
that the lack of complete blockade with this
stimulus reflects the possible use of alternative
pathways not involving TGF-β. In keeping with
the data on the prostaglandins themselves, the
truncated receptor also reduced upregulation of
the synthases (Figure 3D) although not to as
great an extent as seen for the secreted
prostaglandins.
These prostaglandins are lipid mediators that
like TGF-β have been reported to have pro- or
anti-inflammatory properties in different
circumstances. For example, we earlier showed
PGE2 to decrease TNF-α production from
macrophages (6). A possible contributory effect
of prostaglandins themselves to T G F-β
production is depicted in Figure 3E wherein
indomethacin was shown able to reduce the
amount of TGF-β produced in response to
apoptotic cells and mAb217 (see also ref 6).
This, along with the autostimulation of TGF-β
by TGF-β (see above), further shows the
extensive feedback responses inherent in these
systems.
TGF- β-dependent suppression of thromboxane
synthesis.
Early studies of responses to apoptotic cells
showed that thromboxane production was
decreased (20,21) and also see ref. (6). This
suggests co-ordinate up-regulation of potentially
anti-inflammatory prostanoids along with downregulation
of pro-inflammatory thromboxane
and raises the question of whether TGF-β is
responsible for both effects. Since macrophages
do not produce TXA2 spontaneously, and did
not do so at any time after incubation with
apoptotic cells or mAb217 alone (data not
shown), they were activated with LPS in order to
demonstrate the suppressive effect of costimulation
with either apoptotic cells or
mAb217 (Figure 4A). Almost complete
inhibition of TXA2 (measured as TXB2) was
seen, and transfection of the macrophages with
the dominant negative TGF-β receptor reversed
this inhibition. Since LPS also induces TGF-β
production, enhancement of the LPS effect on
thromboxane production might have been seen if
this TGF-β was blocked. However, this was not
observed, probably because the generation of
active TGF-β following LPS occurred after the
majority of the thromboxane had already been
produced. Once again, the effect of the TGF-β
appeared to be at the level of the synthase.
Thromboxane synthase levels in the
macrophages were suppressed by apoptotic cells
and mAb217, but not after transfection of the
truncated TGF-β receptor (Figure 4B).
Stimulation of macrophages with mAb217 or
apoptotic cells induced COX-2 expression and
arachidonic acid release through TGF- β
dependant signaling
The demonstrated reciprocal effect of TGF-β on
the prostanoid synthases raised the possibility
that the induction of PGE2 and PGI2 reflected a
diversion of the precursor PGH2 from utilization
by thromboxane synthase. However, since the
prostaglandins were increased directly in
macrophages that did not express the
thromboxane synthase, it seemed likely that the
apoptotic cells also led to increased levels of one
or other PGH synthases (COX enzymes) and
thereby, increased production of PGH2. No
evidence was found for altered amounts of
COX-1 (data not shown) but both apoptotic cells
and mAb217, as well as LPS as expected, did
increase the intracellular amounts of COX-2
(Figure 5A). In keeping with the theme of this
study, macrophages expressing the truncated
TGF-β receptor did not show upregulation of
COX-2 in response to the apoptotic cells and
markedly reduced that stimulated by LPS.
In order to initiate eicosanoid production, a
source of free arachidonate must be available
and, presumably, must be initiated by the
apoptotic cell or mAb217 stimulus. This is
shown in Figure 5B where free 3H arachidonate
(including its metabolites) was measured in the
supernatant after previous incorporation into
macrophage phospholipids before stimulation. In
this case, blocking the TGF-β effects with the
dominant negative receptor reduced the amounts
of arachidonate released by about 50% in the
case of all three stimuli. This presumably
reflects either a timing issue as noted for the
LPS-induced thromboxane or the availability of
preformed phospholipases that were stimulated
by either the LPS or PSRS engagement in
addition to an effect from TGF-β. TGF-β has
been reported to induce prostaglandin
production (22,31,32), which would necessitate
its ability to initiate released arachidonic acid, an
effect which was shown here in Figure 5B.
Acting through TGF- β, apoptotic cells or
mAb217 decreased LPS-enhanced 5-
lipoxygenase and leukotrienes but increased 15-
LO, 15-HETE and LXA4 as well as PPAR γ.
The pattern of TGF-β induction of potentially
anti-inflammatory, but suppression of proinflammatory,
eicosanoids was further explored
by examining the effects on lipoxygenases and
leukotrienes. Our earlier study (6) had shown
suppression of leukotriene release from
macrophages by apoptotic cells. Stimulation of
macrophages with apoptotic cells or mAb217 by
themselves did not induce cysteinyl leukotrienes
at 18 h and suppressed that induced by priming
with LPS (Figure 6A). However, a time course
study (Figure 6B) did show an early production
at 2 h but none subsequently. Similarly, LTB4
was seen only 2 h after stimulation, after this
time the production was downregulated (data not
shown). Examination of the key upstream
enzyme, 5-lipoxygenase revealed, as expected,
that its levels were increased after priming with
LPS but that this did not occur in the presence of
the apoptotic cells or mAb217 (Figure 6C). In
the presence of the truncated TGF-β receptor,
the LPS-primed increase in levels of 5-LO was
no longer prevented (Figure 6C). We attribute
the early induction of leukotrienes to an effect of
endogenous 5-LO acting on early released
arachidonate before the TGF-β effect has time to
kick in.
By contrast, when 15-LO was examined, the
apoptotic cell stimulus directly increased
intracellular levels of the enzyme and production
of 15-HETE (Figure 6 D-F). The 15-HETE
production was seen 4-8 h after stimulation and
peaked at 18 h (Figure 6F). Once again, the
induction by apoptotic cells or mAb217 was
blocked in the presence of the truncated TGF-β
receptor. On the other hand, TGF-β did not
appear to play a significant role in the induction
of 15-LO or 15-HETE by LPS. Products of 15-
LO have been suggested to participate in lipoxin
A4 (LXA4) production and activation of PPARγ
(33,34), which may also have anti-inflammatory
effects in macrophages (35-37). Accordingly, we
also examined the effect of apoptotic cells and
mAb217 to induce LXA4 and alter the levels of
PPARγ. As shown in Figure 7A-B, both stimuli
initiated LXA4 production and increased the
amounts of PPARγ protein in the cells, as did
direct addition of TGF-β. This supports a
possible additional anti-inflammatory effect of
apoptotic cells via LXA4 and/or activation of
PPARγ
Discussion
Apoptotic cells are known to induce an antiinflammatory
and anti-immunogenic response,
in part mediated by their induction of active
TGF-β in responding cells. Here we show that
the effect of the apoptotic cells is to drive a
complex coordinated inhibition of potentially
inflammatory mediators along with induction of
potentially anti-inflammatory molecules in
macrophages that are orchestrated by the TGF-β
production. By study of the responses induced
by apoptotic cells in macrophages that are
unresponsive to TGF-β by virtue of their
transfection with a dominant negative TGF-β
receptor, the role of this important mediator on a
wide variety of eicosanoids, nitric oxide and
related molecules was delineated. Thus, the
earlier demonstration that apoptotic cells
induced the production of PGE2 but suppressed
thromboxane (6,20) were shown both to be due
to the effects of TGF-β and these observations
extended to other potentially pro- and antiinflammatory
arachidonate metabolites. It
should be noted that the prostanoids PGE2 PGI2
and PGD2 are known to exhibit both pro- and
anti-inflammatory actions, in part for PGE2,
depending upon the receptors that are engaged
(eg (38)). Similar pleomorphic effects should be
noted for NO. In our earlier study (6), we
showed that PGE2 suppressed LPS-induced
inflammatory mediators from macrophages (6)
and in the data reported herein, prostanoid
production also showed a feedback enhancement
of TGFβ production. Thus, in this limited
context we see these prostanoids as potentially
anti-inflammatory and their induction as
contributing to the overall anti-inflammatory
consequences of apoptotic cell recognition.
The observations required the demonstration of
TGF-β production in response to the apoptotic
cells – shown earlier by numerous investigators
and confirmed herein. A number of ligands have
been demonstrated on apoptotic cells that can
interact with a number of “receptors” on
responding cells, in this case macrophages.
Additionally there are a large group of “bridge”
molecules (see ref. 39) that can link the
apoptotic cell ligands to the receptors. We have
suggested that two important ligands are
phosphatidylserine (PS) and calreticulin. The
latter, as well as the collectin family of bridge
molecules (40) has been suggested to interact
with LRP as a receptor and, in isolation, seems
to induce a more pro-inflammatory response
(5,13). On the other hand, PS and its receptors
and possibly some or all of its bridge molecules
appear to induce the anti-inflammatory effects
and, in most cases, to act in a dominant fashion
in the normal response to apoptotic cells.
Necrotic cells are usually thought to be proinflammatory
(see for example 9,15) and may
have reversed this PS-driven dominance. Other
studies that have suggested that apoptotic cells
can in some circumstances act in a proinflammatory
fashion may also reflect variations
in balance between pro-inflammatory (e.g. LRP)
versus anti-inflammatory (e.g. PS driven)
responses.
In the studies reported here, we showed that the
TGF-β induction by apoptotic cells was
dependent on exposed PS by blockade with the
PS-binding protein Factor Va, which has the
advantage over the more usually employed
Annexin V by binding in physiologic
concentrations of calcium. Although direct
stimulation of TGF-β by PS-containing
liposomes was weak, these agents readily
enhanced TGF-β production to other stimuli.
The amounts of TGF-β measured by ELISA
only represents that in the supernatant and is
therefore relatively insensitive for a molecule
that binds to surfaces and cell membranes. Early
in vivo demonstration of PS liposome effects
were also relatively weak in comparison with
apoptotic cells (8) and, we suspect, may reflect
issues of presentation and avidity.
The other approach employed here was to
examine the effect of mAb217, an IgM
activating antibody that is suspected of binding
to an as yet unidentified receptor for PS on
responding cells. Its ability to bind all cells that
respond to PS-exposing apoptotic cells, to be
blocked itself by pretreatment of the cells with
L- but not D-phosphatidylserine, and its close
mimicking of the activation induced by PSexposing
apoptotic cells supports this
contention. In all cases in this study, mAb217
and apoptotic cells behaved identically. The
implication is that recognition of PS drives the
production of TGF-β and the downstream antiinflammatory
responses reported herein.
The eicosanoids are derived from metabolism of
arachidonate through a number of pathways. In
this study we focused on prostanoid synthesis
through PGH synthases (COX) and downstream
prostaglandin and thromboxane synthases as
well as through lipoxygenases and their
downstream products of leukotrienes and
HETES. Apoptotic cell and TGF-β induction of
PGE2, and PGI2 were shown. The effect
appeared to be on upregulation of the respective
synthases PGES and PGIS whose protein levels
were increased in response to the apoptotic cells
and blocked in the presence of the dn-TGF-β
receptor. Likewise, PGD synthase protein levels
were upregulated by the apoptotic cells and
mAb217 but not in the absence of TGF-β
effects. By contrast the pro-inflammatory
eicosanoid thromboxane was suppressed. In this
case resting macrophages or those stimulated
with apoptotic cells alone did not produce
thromboxane or exhibit significant levels of its
synthase. When stimulated to induce
thromboxane synthase by LPS or LPS and IFNγ
however, the apoptotic cells or mAb217
suppressed the upregulation and the mediator
production, again in a TGF-β-dependent fashion.
These effects on prostanoid balance implied an
upregulation of both COX and of sources of
substrate, namely arachidonic acid and both
increases in COX2 (but not COX1) and of
released arachidonate were demonstrated in
response to apoptotic cells. The effect on COX2
was also shown to be dependent on TGF-β (see
41,42) and at least part of the arachidonate
release. TGF-β itself also induced liberation of
arachidonate. While not explored directly herein,
it seems reasonable to assume that the apoptotic
cells and/or TGF-β induce activation of existing
phospholipases 2 and that the TGF-β may also
initiate some upregulation of these that
contributes to greater and/or more prolonged
release of the arachidonate.
Our earlier study had suggested that apoptotic
cells also suppressed the production of
potentially pro-inflammatory sulfidopeptide
leukotrienes (6). Here we show that levels of the
upstream enzyme 5-lipoxygenase, as well as of
the leukotrienes themselves, were also
suppressed by the apoptotic cells and that this
too was reversed in the absence of TGF-β
signaling. The decrease in 5-lipoxygenase levels
noted here are at odds with studies showing
enhanced production in response to TGF-β
during macrophage maturation under the
influence of Vitamin D3 (43). We suspect,
therefore, a discordant effect of TGF-β on
maturing versus mature macrophages.
Intriguingly, and in keeping with the coordinated
anti-inflammatory effects, 15-lipoxygenase was
upregulated and its stable product 15-HETE
increased by the apoptotic cells or mAb217,
again due to TGF-β. Products of 15-HPETE are
generally thought to be anti-inflammatory (44-
47) and some may possibly achieve this effect
via activation of PPARγ (34). Accordingly we
also addressed the effect of the apoptotic cells
and TGF-β pathways on LXA4 production and
the upregulation of PPARγ and showed that
those too were increased.
The other pathway and mediator addressed in
this study was the potentially immunoregulatory
and broad spectrum signaling molecule nitric
oxide (NO). Apoptotic cells and mAb217
suppressed the generation of NO and the
upregulation of iNOS. TGF-β is well known to
inhibit production of iNOS (48-50) and,
therefore, the TGF-β dependence of this was
expected and shown by the dominant receptor
approach. Intriguingly, arginase, which reduces
the substrate for NOS enzymes, was reciprocally
upregulated by the apoptotic cells – again via
TGF-β. This would provide an additional brake
on the NO generation by macrophages
responding to apoptotic cells.
The results indicate a complex effect of
apoptotic cells acting through release of TGF-β
to upregulate generally anti-inflammatory
mediators and inhibit the production of proinflammatory
molecules. The systems employed
here support an effect on synthesis of the various
enzymes involved in generating these mediators,
i.e. acting on transcription, translation or both.
However, we cannot exclude additional effects
on metabolism or secretion of the mediators
acting in the shorter term. In response to
apoptotic cells, macrophages appear to release
preformed TGF-β almost immediately (30-60
minutes, (8,16)) followed by new synthesis and
more prolonged generation of the active
molecule. Combined, this would achieve a fairly
rapid and then prolonged effect on the
inflammatory state of the cell. However, it
should be noted that the immediate generation of
free arachidonate in response to apoptotic cells
might lead to generation of potentially proinflammatory
eicosanoids by enzymatic
pathways constitutively present in the cell (e.g.
5-LO and leukotriene synthases) before the
slower effects of TGF-β had a chance to kick in
(see for example refs. 51-54). Given the
pluripotentiality and plasticity of this cell type,
we are generally skeptical of the concept of
stable macrophage “phenotypes” (3).
Nevertheless, it should also be noted that some
of the effects of apoptotic cell exposure mimic
those seen in the so called alternatively activated
macrophage (55,56) raising intriguing questions
for the future of how long these effects persist
following the interaction with apoptotic cells
and/or the consequences of prolonged exposure
as might be experienced in a resolving
inflammatory response in vivo.
---------
j
j
TGF-beta / NIH / Sharon Wahl ----
I assume people are aware of the now well-proven PS : TGF-beta connection.
BTW - Sharon Wahl was also the lead author on the NIH paper that showed how HIV enters macrophages via exposed PS.
j
----------------------------------------
Curr Opin Immunol. 2008 May 15
TGF-beta and tumors-an ill-fated alliance.
Moutsopoulos NM, Wen J, Wahl SM.
Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4352, United States.
Mechanisms of host defense can form an unwitting alliance with tumor cells to promote tumor progression, invasion, and dissemination to distant sites. By secreting TGF-beta, an immunoregulatory molecule designated for both promoting inflammation and dampening immune responses, the tumor tricks the host into supporting its expansion and survival. TGF-beta not only recruits leukocytes to secrete chemokines, growth factors, cytokines, and proteases in support of a tumor-friendly niche but also in a context-specific manner, incapacitates the emergent immune response. As a profound immunosuppressant, TGF-beta, both directly and through the generation of regulatory T cells, blunts immune surveillance, favoring tumor escape. Collectively, the ability of the tumor to hijack these host defense pathways can tip the balance in favor of the tumor.
-----------
It's happening,
j
CHAVI UPDATE & HAYNES HYPOTHESIS ---------
CHAVI UPDATE - May 19, 2008
http://www.chavi.org/modules/chavi_reports/index.php?id=24
"the team has shown evidence of immune cell apoptosis with elevations of microparticles beginning just before plasma viral load ramp-up.
They showed that CD4 T cell microparticles can suppress memory B cell activation in vitro.
These data raise the hypothesis that one reason for delayed protective antibody responses may be due to apoptotic microparticle-mediated immunosuppression of antibody responses."
"the effects of apoptotic microparticles on DC function are currently being addressed."
"The team is planning to investigate the effects of inhibition of apoptotic events on the timing of immune responses and control of viremia in SIV-infected macaques."
-----------------------------------------
HAYNES HYPOTHESIS -
..."Acutely expressed cytokines and apoptotic microparticles
hypothesized to play a key role in AHI pathogenesis....
April 9, 2007
http://72.14.209.104/search?q=cache:BCKueWKqWTIJ:www.chavi.org/wysiwyg/downloads/CHAVI_012_protocol_v1.pdf+%22CHAVI+012%22&hl=en&ct=clnk&cd=1&gl=us&client=firefox-a
-------------------------------
"Their [Haynes group] current hypothesis is that HIV induces a massive apoptosis
before and during viral ramp-up, and that plasma microparticles (fragments of apoptotic CD3
and T cells) have a suppressive effect on Ab generation, thereby amplifying the apoptotic
cascade. "
May 22, 2007
http://www3.niaid.nih.gov/research/topics/HIV/vaccines/advisory/avrs/PDF/AVRS_May07_Summary.pdf
--------------------------------
Conclusion: These results demonstrate that at the time of viral load ramp-up
in acute HIV-1 infection, there are elevations in plasma levels of TRAIL, FAS
Ligand, and TNFR2 that were associated with the presence of microparticles
from apoptotic T cells.
The presence of these apoptotic markers suggests
apoptosis occurring at the time of initial HIV-1 viral load ramp-up.
That PS+ apoptotic cells and microparticles have been reported to suppress antigen specific
immune responses suggests the hypothesis that immune cell
apoptosis in the very earliest stages of acute HIV-1 infection may delay the
onset of potentially protective anti-HIV-1 immune responses."
August 20, 2007
http://www.hivvaccineenterprise.org/_dwn/poster_sessions.pdf
-------------------------------
The one specific agent yet mentioned in CHAVI updates -
January 2007 -
---------
j
jess,
I would think that most 3rd party readers of our back and forth discussion today would understand what I said pretty clearly.
I can only suggest that maybe if you read my posts again it may become more clear to you.
j
djohn,
It's been discussed, here -
http://www-lmmb.ncifcrf.gov/~dimitrov/bibliogr/pdf/Zhang2007CurrPharmDesign.pdf
then also at CROI-
http://www.retroconference.org/2008/Abstracts/32719.htm
Also at the Gates August 07 vaccine conf-
http://www.hivvaccineenterprise.org/_dwn/Late_Breaker_Abstracts.pdf
j