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The release of microparticles by apoptotic cells and their effects on macrophages ---------
from David Pisetsky of Duke U. who is now working closely with Bart Haynes on HIV -----
http://www.springerlink.com/content/m1v03873757tl2q0/
The release of microparticles by apoptotic cells and their effects on macrophages
J. H. W. Distler3, L. C. Huber1, A. J. Hueber2, C. F. Reich III3, S. Gay1, O. Distler1 and D. S. Pisetsky3
(1) Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, CH-8091, Switzerland
(2) Department of Rheumatology and Internal Medicine, University of Erlangen-Nuremberg, Erlangen, D-91054, Germany
(3) Division of Rheumatology, Durham VA Hospital and Duke University Medical Center, Durham, NC, USA
Microparticles are small membrane vesicles released from the cell membrane by exogenous budding. To elucidate the interactions of microparticles with macrophages, the effect of microparticles released from Jurkat T cells on RAW 264.7 cells was determined. Microparticles were isolated by differential centrifugation, using FACS analysis with annexin V and cell surface markers for identification. Various inducers of apoptosis increased the release of microparticles from Jurkat cells up to 5-fold. The released microparticles were then cultured with RAW 264.7 cells. As shown by confocal microscopy and FACS analysis, RAW 264.7 macrophages cleared microparticles by phagocytosis. In addition, microparticles induced apoptosis in RAW 264.7 cells in a dose-dependent manner with up to a 5-fold increase of annexin V positive cells and 9-fold increase in caspase 3 activity. Cell proliferation as determined by the MTT test was also reduced. Furthermore, microparticles stimulated the release of microparticles from macrophages. These effects were specific for macrophages, since no apoptosis was observed in NIH 3T3 and L929 cells. These findings indicate that microparticles can induce macrophages to undergo apoptosis, in turn resulting in a further increase of microparticles. The release of microparticles from apoptotic cells may therefore represent a novel amplification loop of cell death.
--------
j
new immunosuppressive microparticle paper ----------
a must-read -
Erythrocyte-derived ectosomes have immunosuppressive
properties
Salima Sadallah, Ceylan Eken, and Ju¨ rg A. Schifferli1
Department of Biomedicine, University Hospital Basel, Basel, Switzerland
Abstract: Several clinical studies have suggested
that blood transfusions are immunosuppressive.
Whereas there have been reports describing immunosuppression
induced by leukocytes or fragments
thereof, the possibility that microparticles, released
by erythrocytes during storage, are also involved
was not investigated. We present evidence
here that such microparticles have all the properties
of ectosomes including size, the presence of a
lipid membrane, and the specific sorting of proteins.
These erythrocyte-derived ectosomes (Eecto)
fixed C1q, which was followed by activation
of the classical pathway of complement with binding
of C3 fragments. Similarly to ectosomes released
by PMN, they express phosphatidylserine on
their surface membrane, suggesting that they may
react with and down-regulate cells of the immune
system. In vitro, they were taken up by macrophages,
and they significantly inhibited the activation
of these macrophages by zymosan A and LPS,
as shown by a significant drop in TNF- and IL-8
release (respectively, 80% and 76% inhibitions).
In addition, the effect of E-ecto was not transient
but lasted for at least 24 h. In sum, E-ecto may
interfere with the innate immune system/inflammatory
reaction. Therefore, E-ecto transfused with
erythrocytes may account for some of the immunosuppressive
properties attributed to blood transfusions.
J. Leukoc. Biol. 84: 000–000; 2008.
----------------------
snips -
The exposure of APCs to PS-expressing particles induces
transient tolerance for danger signals with ensuing down-regulation
of the immune response, as suggested by the literature
about cells undergoing apoptosis [20], PMN-derived ectosomes
[21], and PS-expressing liposomes [22]. Many clinical studies
suggest that transfusions might be immunosuppressive, although
these observations are not generally accepted [23–25].
However, a recent clinical study indicated that transfusions of
erythrocytes might be responsible for a diminished survival in
cancer patients [26].
In the present study, we analyzed the pool of microparticles
released by stored leukocyte-depleted erythrocytes; after having
demonstrated that they had all of the properties of ectosomes,
we tested their capacity to interfere with the activation
of macrophages by zymosan A and LPS. The main finding was
that they down-regulated the capacity of these Toll-like ligands
to activate macrophages.
.....................
HMDM bind and ingest ectosomes
The expression of PS and possibly other undefined changes in
the membrane structure is likely to induce macrophages to
recognize, bind, and phagocytose E-ecto. This was evident
when studied by confocal microscopy. Fluorescently labeled
E-ecto incubated for 30 min with HMDM were phagocytosed,
as reflected by the intense fluorescent staining of the cells (Fig.
5A). In contrast, when HMDM were preincubated with cytochalasin
D, a potent inhibitor of phagocytosis, they showed
almost no fluorescence and hence, no uptake of E-ecto (Fig.
5B).
E-ecto have an immunosuppressive effect on
macrophages
Apoptotic cells as well as PMN-derived ectosomes have antiinflammatory
and immunosuppressive properties [20, 21, 32].
This may be related to the expression of PS [22]. Accordingly,
it was of interest to test whether E-ecto that express PS as well
could affect HMDM when these cells were activated. Thus, we
coincubated E-ecto with HMDM in the presence or absence of
zymosan A or LPS for 15 h. SN were analyzed for TNF-,
IL-10, IL-8, and TGF-1. First and as a control, we did not
observe any modification in cytokine release by resting HMDM
when exposed to E-ecto (not shown), but E-ecto inhibited the
activation of HMDM by zymosan A. Whereas zymosan A
induced release of TNF-, IL-10, and IL-8 by HMDM, the
simultaneous addition of E-ecto was responsible for a significant
decrease in the release of all three cytokines (Fig. 6,
A–C). The inhibition was dose-dependant (Fig. 6, A–C). We
next investigated the time-dependent effect of E-ecto on cytokine
secretion by zymosan-activated HMDM. E-ecto induced a
rapid and sustained, inhibitory effect on the release of TNF-,
IL-10, and IL-8 (Fig. 6, A–C).
In this experiment, we also
tested as control the effect of erythrocytes alone and in the
presence of E-ecto. Erythrocytes in excess by themselves had
no effects on the release of TNF- by zymosan A-activated
macrophages (Fig. 6D) nor did their addition to E-ecto; i.e., the
E-ecto effect was not modified by the presence of erythrocytes.
These effects were similar to those observed previously with
PMN-ectosomes, with a major difference; i.e., E-ecto did not
induce the release of TGF-1 (Fig. 6E). Indeed, PMN-ectosomes
induce on their own the release of TGF-1 by HMDM,
a release that is not modified by activating agents such as
zymosan A or LPS [21]. The induction of TGF-1 release was
thought to explain the down-regulation of HMDM so that they
react only weakly or not at all when exposed to zymosan A. As
we could find no TGB-1 release induced by E-ecto, we
repeated the experiments in parallel with PMN-ectosomes prepared
as described previously (Fig. 6F). The results confirmed
the differences between the two types of ectosomes. To see
whether these differences were intrinsically a result of differences
in the cell origin or possibly to level of PS expression, we
repeated the experiments with E-ecto, formed by in vitro aging
of erythrocytes or (ATP depletion, not shown) by incubating
erythrocytes with Ca ionophores (Fig. 6F). Again, PMNderived
ectosomes induced TGF-1 release, whereas none of
the E-ecto could do so (Fig. 6F). By contrast, all three types of
ectosomes had inhibitory effects on the release of TNF-,
IL-10, and IL-8 (Fig. 6F).
LPS induced the release of TNF-, IL-8, and IL-10 but had
no effect on TGF-1 release. When HMDM were stimulated
with LPS, E-ecto produced an inhibition of TNF-, IL-8 (Fig.
6G), and IL-10 (not shown) release similar to that induced on
the zymosan A-activated HMDM. In a control experiment,
LPS-stimulated HMDM released no TGF-1 when exposed to
E-ecto (not shown).
The inhibition of macrophages by E-ecto is longlasting
Macrophages were preincubated with E-ecto for 60 min and
next washed with prewarmed medium to remove the remaining
ectosomes. Macrophages were then left to recover (1 h, 24 h)
before overnight stimulation with zymosan A. The release of
TNF-, IL-8, and IL-10 by zymosan A-activated macrophages
was inhibited up to 24 h after the contact between ectosomes
and macrophages (Fig. 7, A–C), indicating a lasting alteration
of the macrophage signaling machinery. The release of TGF-1
was not modified (not shown).
.....................
DISCUSSION
In the present study, we could demonstrate that ectosomes
derived from erythrocytes during storage have inhibitory potential
on activated macrophages, indicating that they are
active players in the regulation of inflammation.
-------------
j
It's not a matter of cell-neighbors doing friendly clean-up as opposed to immune cells kicking arse. Macrophages react differently depending on what the sense/feel. PS signals a "self"-cleanup type response. It is looking like Pathogens have learned (evolved) to take advantage of that, to trick cells into the wrong response, by wearing PS, the universally agreed-upon sign for friendly metazoan clean-up). Or think of it this way - the pathogens that have proven to be most successful (survive) are the ones that happen to have an exposed phospholipid that triggers the "self"-cleanup response. It makes perfect sense, and it implies a new way of seeing pathogenesis in general that explains things very clearly.
To quote Dr. Barton Haynes of Duke U., in his patent application where he proposes using anti-PS mabs as therapy for patients infected with HIV, discussing work which has yet to appear in a published paper...
[0051] It will be appreciated from a reading of the foregoing that if HIV has evolved to escape the host immune response by making the immune system blind to it, other infectious agents may have evolved similarly. That is, this may represent a general mechanism of escape. That being the case, approaches comparable to those described herein can be expected to be useful in the treatment of such other agents as well.
http://appft1.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=3&p=1&f=G&l=50&d=PG01&S1=%28%22haynes+barton%22.IN.%29&OS=in/%22haynes+barton%22&RS=IN/%22haynes+barton%22
---------
"How do healthy cells benefit from ingesting leftovers?"
The organism stays alive, that's how. Multicellular organisms that don't 'lick the plate / ingest leftovers' as you say, ( delayed clearance of apoptotic cells) wind up with auto-immune disease, or worse - dead. A healthy cell with a dying neighbor cell better clean it up or call a professional, or that healthy cell won't stay healthy too long. There's a window of opportunity, as a cell dies, when it needs to be dealt with, or else nasty things escape, and harm their neighbors, and so on...
The apoptotic program developed out of necessity in parallel (or preceded) metazoans.
j
microbeman,
Barton Haynes Recently Published Papers and Patent Applications As They Relate to the Immunosuppressive Signaling of Exposed PS
On July 24, 2008, another Haynes patent application went public on the World Intellectual Property Organization database.
Pertinent excerpts follow:
"The time of appearance of antibodies in the development of acute HIV infection has been recently mapped and it has been shown that most
of the antibodies arise after a delay in the peak response to HIV envelope epitopes of approximately two to three weeks. Indeed, the most
protective antibodies, those that neutralize autologous virus, can be delayed for up to a year."
"To begin to understand the "delay" in induction of antibodies at the time of HIV transmission, the first question to be addressed was whether
there are immunosuppressive events, such as massive apoptosis, with release of phosphatidylserine microparticles at the time of viral load
ramp up during acute HIV infection."
"Apoptotic microparticles are the products of either activated or apoptotic cells, that are increased in the plasma of a number of diseases,
including autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, Crohn's disease, coronary artery disease
and other forms of heart disease, and chronic HIV-I infection."
"Apoptotic microparticles can bind to non-apoptotic cells and induce apoptosis, are procoagulant, proinflammatory, and can be
immunosuppressive for T and B cell responses to specific antigen."
"Thus, the massive apoptosis that occurs with acute HIV infection with resulting release of TRAIL, mediation of apoptosis via FAS-FASL
interactions, and release of PS containing viral and other particles, all conspire to initially immuno suppress the host, preventing rapid
protective B cell responses."
"Thus, the production of high levels of biologically active plasma mediators and byproducts of cell death during the first two to three weeks of
HIV- 1 transmission raises the notion that the window of opportunity for a preventive vaccine to work may be shorter than previously thought,
ie within the first 14-17 days of transmission, placing considerable constraints on the time available for development of robust anti-HIV-1
immunity following transmission."
"Inhibition of cell death and immunosuppressive MP mediated effects by a vaccine for HIV or other infectious agents may be important as
well. This could be accomplished, for example, by an HIV vaccine component inducing anti-lipid antibodies or antibodies against other
components of microparticles to facilitate clearance of microparticles and/or to block microparticle toxic effects."
"Another use of the data herein is as a rationale for the treatment of HIV-I. For example, antibodies against TNFR or TNF-alpha;,
antiphosphatidylserine antibodies or other inhibitors of cell death (Fas-Fc as an inhibitor of FAS-FAS ligand interactions and DR5-Fc as an
inhibitor of TRAIL DR5 interactions) can be used to inhibit cell death in HIV as a therapy."
http://www.wipo.int/pctdb/en/wads.jsp?IA=US2008000412&LANGUAGE=EN&ID=id00000006622149&VOL=89&DOC=00fca1&WO=08/088747&WEEK=30/2008&TYPE=A2&DOC_TYPE=PAMPH&PAGE=1
Barton Haynes Recently Published Papers and Patent Applications As They Relate to the Immunosuppressive Signaling of Exposed PS continued
Haynes recent paper in the August issue of the journal Virology has generated much discussion in the HIV research community, as well as several popular news articles.
Examples of follow-on popular news stories:
HIV Conquers Immune System Faster Than Previously Realized.
HIV Overpowers Immune System Quicker than Previously Thought
These popular articles do a nice job of summarizing the basic insights found by the Haynes team which shed light on what is responsible
for the rapid immune cell death found in early HIV infection. What the popular articles fail to clearly discuss is the fundamental culprit
responsible for the quick immunosuppression which paves the way for the ramp-up of virions (and the lack of effectiveness of all previous experimental HIV immunogens). The popular articles go no further than
mentioning "microparticles" as players in the overwhelming immunosuppression. Haynes actual paper as well as his several patent
applications that have recently appeared in the various US and international patent databases provide the all-important details. Haynes work
discusses exposed phosphatidylserine (PS) on microparticles overwhelming immune cells, facilitating the rise in viral counts.
The following document details this fascinating area of research, and points to this PS-induced immune suppression pathway as being an
important mechanism involved in the pathogenesis of several diverse diseases.
First, some background.........
Recent insights into enveloped virus infection, from the journal Science
In the April 2008 journal Science, authors Mercer and Helenius discuss data showing how vaccinia viruses (the pox family of viruses), utilize
phosphatidylserine (PS) exposed on its surface to facilitate entry into cells. Besides PS aiding viral entry, the authors also discuss recent
research showing that exposed PS also prevents an immune response to the virus. They use the term "apoptotic mimicry", (masquerading
as a dying native cell), a term showing up more often in recent research involving how various unrelated pathogens use similar means to
evade the cells of the immune system, thereby surviving and thriving in the body.
Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells
The Mercer and Helenius paper spawned several well-written layman's articles on the new insight into viral infection. For instance:
Trojan Horse Of Viruses Revealed
excerpts:
ScienceDaily (Apr. 25, 2008) — Viruses use various tricks and disguises to invade cells. Researchers have now discovered yet another
strategy used by viruses: the vaccinia virus disguises itself as cell waste, triggers the formation of evaginations in cells and is suspected to
enter the cell interior before the immune defense even notices.
The invasion strategy
In order to infiltrate a cell, the vaccinia virus exploits the cellular waste disposal mechanism. When a cell dies, other cells in the vicinity ingest
the remains, without needing waste disposal experts such as macrophages. The cells recognize the waste via a special molecule,
phosphatidylserine, which sits on the inner surface of the double membrane of cells.
This special molecule is pushed out as soon as the cell dies and is broken into parts. The vaccinia virus itself also carries this official waste
tag on its surface.
"The substance accumulates on the shell of vaccinia viruses", Jason Mercer explained. The pathogen disguises itself as waste material and
tricks cells into digesting it, just as they normally would with the remains of dead cells. As the immune response is simultaneously
suppressed, the virus can be ingested as waste without being noticed.
Dirty Rotten Poxviruses
excerpts:
"poxviruses have engineered a way to sneak into cells through the garbage chute."
"the virus disguises itself as junk"
"the team wondered if the virus was playing dead. They found that the virus's surface was studded with phosphatidylserine, a lipid that also
flags dead cells as garbage. Removing lipids from the virus's surface stopped infection, and recoating the virus with phosphatidylserine
restarted it. The results suggest that the virus is "more clever than originally thought" because it exploits a garbage-collection process found
in almost all cells, says Mercer.
Where does Barton Haynes fit in?
Over the past couple years, in his work looking into the pathogenesis of HIV, Barton Haynes has been following the hypothesis that the
reason HIV manages to proliferate so quickly after infection, and the reason no vaccines have yet been effective, have both been due to a
massive amount of "apoptotic debris" (microparticles from dying cells) which occurs early in HIV infection. This debris contains exposed
phosphatidylserine (PS), and is responsible for the depletion of CD4+ T cells, as well as an overall blunting of immune response to the virus.
Haynes puts forth the idea (documented below) that the fundamental culprit is phosphatidylserine (PS). He cites recent research (Henson,
Fadok, etc.) showing how exposed PS alters the function of macrophages, dendritic cells, and T cells. In the August Journal of Virology he
also provides new data showing how microparticles with exposed PS alter the fuctions of antibody producing B cells.
here are earlier excerpts illustrating Haynes overall working hypothesis:
Haynes / CHAVI / Gates hypothesis example 1:
"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
Haynes / CHAVI / Gates hypothesis example 2:
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
Haynes most recent publication
The following are excerpts from Bart Haynes' new paper in the August 2008 Journal of Virology, which discuss detailed data showing how
exposed PS on microparticles is responsible for the weak immune response to HIV.
Induction of plasma (TRAIL), TNFR-2, Fas ligand, and plasma microparticles after human immunodeficiency virus type 1 (HIV-1)
transmission: implications for HIV-1 vaccine design.
Excerpts:
("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).”
“suppression of immune responses can be mediated by T cell MPs (32,34,35). CXCR4+ and CCR5+ MPs can transfer co-receptors to coreceptor
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).”
“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”
“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).”
August 2008, Journal of Virology
http://jvi.asm.org/cgi/content/abstract/82/15/7700
Where Does Haynes Go From Here? Patents Point The Way...
We can see where Haynes is going in his work by looking at two recent patent applications.
In one recent patent application, in the World Intellectual Property Organization database, Haynes discusses what a future successful
HIV vaccine must do. He specifically states that any future successful vaccine must stop exposed PS from blunting immune response. He
states that an essential goal of a future vaccine is to induce anti-PS antibodies, which would bind/block the PS from downregulating the
immune response.
In Haynes second recent patent application, in the US database, he discusses how monoclonal anti-PS antibodies can be safe therapy in
people already infected with HIV.
excerpts and links to the two recent patent applications:
The “Vaccine” Patent -
- Thus, HTV virions and HIV envelope can directly induce T cell death in AHI, soluble TRAIL can bind to uninfected cells and induce death in
AHI, and with both HTV infection of cells and with massive apoptosis, high levels of phosphatidylserine containing cells and particles likely
abound in AHI. It has recently been shown that PD-I (programmed death molecule- 1) is present on the surface of human B cells in chronic
HIV infection. This suggests that human B cells are primed for apoptosis in HIV infection (Figure 6). HIV specific CD8+ T cell PD-I expression
correlates with a CD8+ T cell response to poorly controlled chronic HIV infection (Petrovas et al, JEM 203: 2281 (2006)). Phosphatidylserine
(PS) on the surface of HIV infected cells and virions has been found (Figure 7) and Callahan et al have found PS is a cofactor for HIV
infection of monocytes (Callahan, J. Immunol 170:4840 (2003)). PS-dependent ingestion of apoptotic cells promotes TGF-βl secretion
(Huynh et al, J. Clin. Invest. 109:41 (2002)) and interaction between PS and PS receptor inhibits antibody responses in vivo (Hoffman et al,
J. Immunol. 174:1393 (2005)). INF-α, an anti-viral cytokine, sensitizes lymphocytes to apoptosis (Carrero et al, JEM 200:535 (2004)). There
are increases in PS+ shed membrane particles in chronic HIV infection (Aupeix et al, J. Clin. Invest. 99:1546 (1997)), and apoptotic
microparticles modulate macrophage immune responses (Distler et al, Apoptosis 10:731 (2005)).
The present invention relates to a multicomponent vaccine that addresses problems resulting from the diversity of HIV by the use consensus
and/or mosaic HIV genes coupled with strategies designed to break immune tolerance to allow for induction of the desired specificity of
neutralzing antibodies at mucosal sites (e.g., through the use of T regulatory cell inhibition and/or TLR-9 agonist adjuvants), and strategies
designed to overcome HIV-I induced apoptosis (e.g., induction of anti- phosphatidylserine (PS) antibodies, anti-CD36 antibodies, and/or antitat
antibodies).
WHAT IS CLAIMED IS:
1. A method of inducing the production of an immune response against HIV-I in a mammal comprising administering to said mammal: i) a
centralized HIV-I gene sequence, ii) an agent that breaks mammalian immune tolerance, and iii) an agent that inhibits HIV-I -induced
apoptosis or an immunosuppressive effect of HIV-I -induced apoptosis, wherein (i), (ii) and (iii) are administered in amounts sufficient to
effect said production.
22. The method according to claim 1 wherein said agent that inhibits HIV-1-induced apoptosis induces anti-phosphatidylserine (PS)
antibodies, anti- CD36 antibodies, or anti-HIV tat antibodies.
http://www.wipo.int/pctdb/en/wads.jsp?IA=US2007024122&LANGUAGE=EN&ID=id00000006427540&VOL=87&DOC=0084d3&WO=08/063586&WEEK=22/2008&TYPE=A2&DOC_TYPE=PAMPH&PAGE=1
The “therapeutic” patent
Here Haynes discusses the use of anti-phospholipid monoclonal antibodies as HIV therapy. Haynes has moved from seeing the lipid binding
characteristics of the well-known broadly-neutralizing HIV abs (2F5, 4E10) as unfortunate, and now sees the lipid-binding characteristics as
“the key” to broad and safe HIV neutralization. Haynes goes so far as to say that there is no need for an HIV neutralizing antibody to bind a
viral epitope. The idea is presented that binding a host-cell 'tag-along' phospholipid results in successful HIV neutralization.
WHAT IS CLAIMED IS:
1. A method of treating HIV comprising administering to a patient in need thereof an antibody derivable from a normal subject or from an
autoimmune disease subject that binds to a lipid on the surface of HIV or on the surface of HIV-infected cells and thereby neutralizes HIV
1, wherein said antibody is administered in an amount sufficient to effect said treatment.
2. The method according to claim 1 wherein said antibody is derivable from an anti-phospholipid syndrome subject.
3. The method according to claim 1 wherein said antibody is non-pathogenic.
4. The method according to claim 1 wherein said antibody is IS1, IS4 or IS6, or binding fragment thereof.
5. The method according to claim 1 wherein said antibody is IS1, or binding fragment thereof.
[0105] ....That IS1 neutralized HIV evidences the facts that: a) humans can make non-pathogenic anti-lipid antibodies that neutralize HIV,
and b) IS1 is an antibody that can be safely used as a therapeutic Mab for treatment of HIV infected subjects or in the setting of postexposure
prophylaxis of subjects following needle, sexual or other exposure to HIV or HIV infected materials.
[0108] Alving and colleagues have made a mouse mab against phosphatidyl inositol phosphate and have shown that it neutralizes HIV in a
PBMC assay. What the present studies show is that humans can spontaneously make anti-lipid antibodies and that these antibodies can
broadly neutralize HIV in an unprecedented manner.
[0109] Summarizing, autoimmune disease patients can make antibodies that bind to virus-infected cells and, presumably, to budding HIV
virions by virtue of their reactivity to HIV membranes and host membranes. Certain anti-lipid antibodies from autoimmune disease patients
can also react with the Envelope trimer (such as IS6) but not all of the antibodies react also with the trimer (i.e., IS1 and IS4 do not react).
Therefore, reactivity with the HIV envelope is not a prerequisite for neutralization in these antibodies.
http://appft1.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearchadv.html&r=2&p=1&f=G&l=50&d=PG01&S1=%28%22haynes+barton%22.IN.%29&OS=in/%22haynes+barton%22&RS=IN/%22haynes+barton%22
The Big Picture
In the Haynes "therapeutic patent" above, one additional quote stands out:
[0051] It will be appreciated from a reading of the foregoing that if HIV has evolved to escape the host immune response by making the
immune system blind to it, other infectious agents may have evolved similarly. That is, this may represent a general mechanism of escape.
That being the case, approaches comparable to those described herein can be expected to be useful in the treatment of such other agents
well.
In their recent paper in Science, (the beginning of this post) Mercer and Helenius also speculate on this broad-based pathogenic immune evasion mechanism:
Also, in the same issue of Science, Fairn and Grinstein discuss the broader implications of Mercer and Helenius's findings:
A One-Sided Signal
Gregory D. Fairn and Sergio Grinstein
"Because the infected cells undergo apoptosis, and thus experience scrambling of plasma membrane lipids, the budding virus also acquires
an envelope that exposes phosphatidylserine on its external surface"
"the presence of exofacial phosphatidylserine is required for viral entry. "
"The involvement of phosphatidylserine may not be limited to infection by vaccinia viruses."
"It is therefore conceivable that HIV similarly requires phosphatidylserine for infection"
"The role of phosphatidylserine in the entry of HIV and other viruses will surely be explored in greater detail now”
----------
It's happening :)
j
FYI: froms Haynes most recent WIPO app --------------
To begin to understand the "delay" in induction of antibodies at the time of HIV transmission, the first question to be addressed was whether there are immunosuppressive events, such as massive apoptosis, with release of phosphatidylserine microparticles at the time of viral load ramp up during acute HIV infection (Mattapallil et al, Nature 434:1093 (2005), Veazey et al, Science 280:427 (1998), Guadalupe et al, J. Virol. 77:11708 (2003), Benchley et al, J. Exp. Med. 200:749 (2004), Mehandru et al, J. Exp. Med. 200:761 (2004), Esser et al, J. Virol. 75: 6173-6182 (2001), Aupelx et al, J. Clin Invest. 99:1546-1554 (1997), Callahan et al, J. Immunol. 170:4840-4845 (2003)). Apoptotic microparticles are the products of either activated or apoptotic cells, that are increased in the plasma of a number of diseases, including autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (Distler et al, Arth. Rheum. 52:33337-3348 (2005), Tesse et al, Arterioscler. Thromb. Vase. Biol. 25:2522-2527 (2005), Cerri et al, J. Immunol. 177:1975-1980 (2006)), Crohn's disease (Chamouard et al, Dig. Dis. Sci. 50:574-580 (2005)), coronary artery disease and other forms of heart disease (Boulanger et al, Cardiovas. Res. 67:1-3 (2005)), and chronic HIV-I infection (Esser et al, J. Virol. 75:6173-6182 (2001), Aupelx et al, J. Clin Invest. 99:1546-1554 (1997)). Apoptotic microparticles can bind to non-apoptotic cells and induce apoptosis (Distler et al, Apoptosis 10:731- 741 (2005)), are procoagulant (Distiller et al, Apoptosis 10:731-741 (2005)), proinflammatory (Tesse et al, Arterioscler. Thromb. Vase. Biol. 25:2522-2527 (2005), Cerri et al, J. Immunol. 177:1975-1980 (2006)), and can be immunosuppressive for T and B cell responses to specific antigen (Esser et al, J. Virol. 75:6173-6182 (2001), Fadok et al. J. Immunol. 174:1393 (2005)). Microparticle levels correlate with the levels of IL-6 in healthy adults (Chirinos et al, Amer. J. Card. 95:1258-1260 (2005)), are increased in acute coronary syndromes and correlate with severity of angiographic coronary lesions (reviewed in Mezentsev, Am. J. Physiol. Heart Circ. Physiol. 289:H1106-H11 14 (2005)). CD31/annexin V+ apoptotoc microparticles correlate with coronary endothelial function in patients with coronary artery disease (Werner et al, Arterioscler. Throm. Vase. Biol. 26:112-116 (2006), Epub Oct. 2005).
http://www.wipo.int/pctdb/en/fetch.jsp?SEARCH_IA=US2008000412&DBSELECT=PCT&C=10&TOTAL=37&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41248521-KEY&QUERY=%28IN%2F%22haynes+barton%22%29+&RESULT=1&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=1444025&IA=US2008000412&LANG=ENG&DISPLAY=DESC
------------
:)
j
atherothrombosis -
Apoptosis, a major determinant of atherothrombosis.
http://www.ncbi.nlm.nih.gov/pubmed/12868350?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum
Procoagulant microparticles: 'criminal partners' in atherothrombosis and deleterious cellular exchanges.
http://www.ncbi.nlm.nih.gov/pubmed/16855341?dopt=Abstract
The Role of Microparticles in Inflammation and Thrombosis
http://www3.interscience.wiley.com/journal/118494948/abstract?CRETRY=1&SRETRY=0
Circulating CD31+/Annexin V+ Apoptotic Microparticles Correlate With Coronary Endothelial Function in Patients With Coronary Artery Disease
http://atvb.ahajournals.org/cgi/content/full/26/1/112
------------
Evolution has favored pathogenesis that resembles apoptosis.
j
thanks mouse,
That's fascinating. Crohn's disease is associated with people making antiphospholipid antibodies...
Taking her off the Crohn's medication, her anti-phospholipid abs would return....
j
Bart Haynes quote from Duke PR about his recent paper/discoveries
outlined in the August Journal of Virology-------------
Bart Haynes: (re: HIV vaccine design)
"It is becoming clearer why we have failed in our efforts to date, and what we need to confront to succeed in the future."
http://www.dukemednews.com/news/article.php?id=10364
HAYNES MOST RECENT PATENT APPLICATION - JULY 24 ------------
July 24, 2008 Haynes WIPO patent application ----------
"The time of appearance of antibodies in the development of acute HIV infection has been recently mapped and it has been shown that most
of the antibodies arise after a delay in the peak response to HIV envelope epitopes of approximately two to three weeks. Indeed, the most
protective antibodies, those that neutralize autologous virus, can be delayed for up to a year."
"To begin to understand the "delay" in induction of antibodies at the time of HIV transmission, the first question to be addressed was whether
there are immunosuppressive events, such as massive apoptosis, with release of phosphatidylserine microparticles at the time of viral load
ramp up during acute HIV infection."
"Apoptotic microparticles are the products of either activated or apoptotic cells, that are increased in the plasma of a number of diseases,
including autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, Crohn's disease, coronary artery disease
and other forms of heart disease, and chronic HIV-I infection."
"Apoptotic microparticles can bind to non-apoptotic cells and induce apoptosis, are procoagulant, proinflammatory, and can be
immunosuppressive for T and B cell responses to specific antigen."
"Thus, the massive apoptosis that occurs with acute HIV infection with resulting release of TRAIL, mediation of apoptosis via FAS-FASL
interactions, and release of PS containing viral and other particles, all conspire to initially immuno suppress the host, preventing rapid
protective B cell responses."
"Thus, the production of high levels of biologically active plasma mediators and byproducts of cell death during the first two to three weeks of
HIV- 1 transmission raises the notion that the window of opportunity for a preventive vaccine to work may be shorter than previously thought,
ie within the first 14-17 days of transmission, placing considerable constraints on the time available for development of robust anti-HIV-1
immunity following transmission."
"Inhibition of cell death and immunosuppressive MP mediated effects by a vaccine for HIV or other infectious agents may be important as
well. This could be accomplished, for example, by an HIV vaccine component inducing anti-lipid antibodies or antibodies against other
components of microparticles to facilitate clearance of microparticles and/or to block microparticle toxic effects."
"Another use of the data herein is as a rationale for the treatment of HIV-I. For example, antibodies against TNFR or TNF-alpha;,
antiphosphatidylserine antibodies or other inhibitors of cell death (Fas-Fc as an inhibitor of FAS-FAS ligand interactions and DR5-Fc as an
inhibitor of TRAIL DR5 interactions) can be used to inhibit cell death in HIV as a therapy."
http://www.wipo.int/pctdb/en/wads.jsp?IA=US2008000412&LANGUAGE=EN&ID=id00000006622149&VOL=89&DOC=00fca1&WO=08/088747&WEEK=30/2008&TYPE=A2&DOC_TYPE=PAMPH&PAGE=1
-----------------
Haynes is saying that there is a reason that all HIV vaccines have failed, and that reason is the massive PS-exposing apoptotic debris. Haynes is saying that these PS-exposing microparticles must be dealt with for any hope of a successful vaccine.
Is there a more likely reason why they just recently ditched the big vaccine trial they were planning?
I don't think so.....
I think they realized it would fail, because they were not doing anything to address exposed PS.
I believe that the reason they just scrapped the big planned vaccine trial was specifically because of immunosuppressive exposed PS.
The most recent CHAVI progress report made it pretty obvious how the CHAVI / Gates work has now focussed on PS-exposing microparticles as what needs to be dealt with.
It's happening,
j
BAVITUXIMAB :
definition, from back to front........
MAB = monoclonal antibody
XI = chimeric
TU = tumor applications
VI = virus applications
NOW.........
the FIRST syllable is supposed to be some "differentiator". It's supposed to be something that simply adds an individual characteristic to the word, which, due to the later syllables that I've already explained, will help differentiate it from the rest of the mabs....
So...........
Why "BA"?............
I've felt all along that it was no random pick, no coincidence.
IMHO, "BA" stands for "BACTERIA"
:)
BA - VI - TU - XI -MAB
anti-BActerial, anti-VIral, anti-TUmor, chimeric, Monoclonal AntiBody.
hey, ya heard it here first,
j
RCJ, re: antiviral -
Here are the past four months of all DTRA awards over one hundred thousand dollars.............
Actually, from my research, in the PAST YEAR, Peregrine's ANTI-VIRAL award is in the top five of ALL DTRA awards, and the four awards larger than Peregrine went to the following four companies-
Raytheon,
(revenues approx. $20 billion)
Bechtel National,
(revenues of $27 billion in 2007)
Northrop Grumman,
(revenues $30 billion)
and Science Applications International
(revenues of $8.9 billion last year)
I would think those guys have lobbying budgets bigger than PPHM's mkt cap...
here's the DTRA's record for the past four months again -
----------
j
moby,
Nw you understand - they enrolled the first stage, and the trial expanded to treat the rest of the 46 patients.
(Earlier you had said "the Georgia trial only took 3 months to complete enrollment." which I hope you now realize was in error.)
next -
re:"What I said was "have issued what sort of objective response is seen as part of the trial."
Yes, WHEN IT'S DONE. :)
As I've been telling you.
You will have to wait, AS IN EVERY OTHER PREVIOUS BAVI PHASE 1 TRIAL, until it's done enrolling. Thank you for agreeing with me that no previous Bavituximab phase 1 trial has ever issued any info until it's done enrolling.
re: IS1- I consider Chen's joining the CORE TECHNOLOGIES of PPHM's SAB, combined with PPHM licensing anti-PS mabs from his university, to be pretty obviously not a unrelated coincidence.
j
IMPORTANT NEW HAYNES / LETVIN PAPER -
The "Immunogen Paper".........
http://hwmaint.pnas.org/cgi/reprint/0803352105v1
This new paper in PNAS is discussing the work that I've been posting about in the important WIPO patent application -
the Multicomponent Vaccine patent app.
http://www.wipo.int/pctdb/en/fetch.jsp?SEARCH_IA=US2007024122&DBSELECT=PCT&C=10&TOTAL=37&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249635-KEY&QUERY=%28IN%2F%22haynes+barton%22%29+&RESULT=3&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=1415994&IA=US2007024122&LANG=ENG&DISPLAY=DESC
(The one I recently told y'all to read it's entire 97 pages :)...
In short, for those who choose not to read these things for themselves, they've found a really good immunogen.
Now, the other "component" of any successful vaccine, according to Haynes, is that they need to prevent the massive amount of exposed PS.
j
moby,
The Georgia phase 2 breast cancer trial is not completely enrolled.
The trial is for 46 patients.
There was never a press release saying it's completely enrolled.
You may be misunderstanding the Simon two-stage design of the trial.
please post any press release quotes you're referring to.
-------------------------
There has never been interim updates in ANY Bavituximab phase 1 trial.
Neither cancer, nor HCV Bavituximab phase 1 trials have ever issued interim data.
The 1st Bavituximab HCV trial did not.
The 2nd Bavituximab HCV trial did not.
The US cancer monotherapy trial has not.
The Indian cancer trial did not.
You will hear info when the trial is done, as has happened in every other phase 1 Bavituximab trial.
please post any interim data from these previous trials you are aware of.
------------------------
Pojen Chen is listed on PPHM's SAB.
He's not listed under "HIV", or HCV",
He's listed under "CORE TECHNOLOGIES".
Peregrine would not be paying the competition.
IS1 is Pojen Chen's.
I had figured Chen's mabs were a part of Peregrine Intellectual Property when I saw his name listed under Core Technologies, and Chen's mabs were being focussed on by Bart Haynes group, as reported several times in CHAVI/Gates literature.
Then I saw in the recent SEC report that Peregrine had licensed anti-PS mabs from Chen's university.
If you're looking for even more of a smoking gun, I think you'll have to wait for whatever paper comes out which details all the work done with Pojen Chen's (Peregrine's) IS1 mab.
and yes, of course there will be a paper all about it.
Haynes says in his recent patent application,
A method of treating HIV comprising administering to a patient in need thereof an antibody derivable from a normal subject or from an autoimmune disease subject that binds to a lipid on the surface of HIV or on the surface of HIV-infected cells and thereby neutralizes HIV-1, wherein said antibody is administered in an amount sufficient to effect said treatment.
The method according to claim 1 wherein said antibody is non-pathogenic.
The method according to claim 1 wherein said antibody is IS1, IS4 or IS6, or binding fragment thereof.
The method according to claim 1 wherein said antibody is IS1, or binding fragment thereof.
* IS1 is an antibody that can be safely used as a therapeutic Mab for treatment of HIV infected subjects
* these antibodies can broadly neutralize HIV in an unprecedented manner.
* reactivity with the HIV envelope is not a prerequisite for neutralization in these antibodies.
AND MY FAVORITE "broad implication" point mentioned by Haynes -
It will be appreciated from a reading of the foregoing that if HIV has evolved to escape the host immune response by making the immune system blind to it, other infectious agents may have evolved similarly. That is, this may represent a general mechanism of escape. That being the case, approaches comparable to those described herein can be expected to be useful in the treatment of such other agents well.
http://appft1.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2&p=1&f=G&l=50&d=PG01&S1=%28%22haynes+Barton%22.IN.%29&OS=in/%22haynes+Barton%22&RS=IN/%22haynes+Barton%22
----------
Obviously this is very important work, and no paper has yet been published on the work. But one will.
I think it'll turn some heads, and yes, I tihnk it will be reporting good things about PEREGRINE'S mabs as therapy for HIV.
King recently said the work has expanded, to 9 institutions and 50 scientists, which to me implies 'good' things.
As for clinical trials, if the data warrants it, (and Haynes patent application excerpts above sure seem like it!), I'd expect to see them happen at, and be run by Duke, likely sponsored by NIAID or the Gates.
j
BTW - When I said that Haynes is now "pinning his hopes" on anti-PS, IMO, I could just as easily and accurately say that The Gates Foundation is now pinning their hopes on anti-PS, and that CHAVI is now pinning their hopes on anti-PS.
j
moby,
re: "Fact remains that the results from the first HPC trial weren't earth-shattering. The party line then was that these people had hopelessly shot immune systems. "
HUH?
In the trial you refer to, a single dose of Bavituximab did better than a regimen of interferon. A single dose of Bavituximab also had a lasting effect, as measured 16 weeks later, which proved the all-important immuno-modulatory MOA of the mab. (it actually trains the immune system to see the pathogen for what it is, and stay on guard against it into the future. BTW - This is an MOA I expect to "kick in" around now in the Georgia breast cancer patients :) The earliest treated patients should be going off the docetaxel now, or very soon, and will then be getting only Bavituximab on a weekly basis. IMHO, I think we'll see the "adaptive" immune response kick in big, in an "autologous vaccination" type of way! :) ....
BTW, also - Folks that have been through chemo have much more wrecked immune systems than HCV patients. Actually, the HCV patients have provided valuable "safety data" in a more 'normal patient population', as opposed to cancer patients, whose immune systems are in pretty bad shape.
re:"It's a bit odd, to me anyway, that management would PR encouraging early data out of both the Cotara and Georgia trials and not do the same for the HPC/HIV trial if similar results had been seen early on."
HUH?
The Cotara trial is ONE DOSE and they're DONE. Easy to report about in a reasonable time.
The Georgia breast cancer trial is a trial in which all patients are getting a therapeutic dose. (Not a dose-escalation trial). More importantly, the Georgia trial is an EFFICACY trial, whereas the HCV/HIV trial is a SAFETY trial. When the results for the HCV/HIV trial are in, those results will be reported with a focus on pharmacokinetic data, not efficacy data. IF they see "promising signs of eficacy", that's about all they'll say- that they saw "promising signs of efficacy". It's a PHASE 1 trial. They are not measuring many efficacy parameters. That's what PHASE 2 is about. I suppose they could say, regarding the HCV/HIV trial, some sort of interim update if they felt like it, something like, "We've treated "X" number of patients so far, and it's looking safe!".
(Because after all it's a safety trial, not an efficacy trial). Do you remember that they (obviously, because it was a phase I safety trial) never issued interim reports on either of the previous two HCV trials? Also, OBVIOUSLY, because it was a SAFETY trial, they did not mention any interim reports in the Indian PHASE 1 SAFETY CANCER TRIAL. and OBVIOUSLY, they have not issued any interim info from the PHASE 1 Bavi SAFETY TRIAL with bavi as monotherapy in cancer in the US. The reason they have not issued an interim update on the Bavi US cancer trial, as compared to the Bavi Georgia cancer trial, is because the Bavi US cancer trial IS A SAFETY TRIAL, PHASE 1, while the Bavi Georgia trial IS AN EFFICACY TRIAL, PHASE 2.
re: "But, when you factor in what the Haynes team has reported regarding what HIV does to the immune system early in the infection cycle it tells me that there's simply been nothing to report..."
HUH?
Again, Haynes is saying that he thinks ANTI-PS MABS WILL BE GOOD THERAPY FOR TREATING HIV.
HE's NOT saying that they'll only work early before the immune system is wrecked, he's actually specifically saying that anti-PS
"can be safely used as a therapeutic Mab for treatment of HIV infected subjects"
and that anti-PS can
"broadly neutralize HIV in an unprecedented manner".
Again, you seem to be disagreeing with the man.
j
moby,
re: "the Georgia trial only took 3 months to complete enrollment."
HUH?
They've stated that they've treated 14 out of 46 patients so far.
not sure what you're talking about,
j
djohn,
re:"Any thoughts on what he means "multiple arms"?"
Yes, T cells, - and B cells.
Haynes recent contribution, (in the August Journal of Virology), shows how PS-exposing microparticles shut down B cells from producing neutralizing antibodies to the virus.
Previously, it has been shown how PS shuts down macrophages, (Hoffman, etc....)
and how PS shuts down dendritic cells, (Shilyanky, etc.)..
and how PS shuts down T cells, (Henson, etc...)
but Haynes has also just shown that PS shuts down B cells.
In sum, exposed PS is quite immuno-suppressive, and must be dealt with for any hope of beating the virus.
(That's another general insight thanks to the recent work of Haynes & team...)
- and that's why he's now on record as pinning his hopes on anti-PS.....
fascinating times.
it's happening :)
j
Peregrine's anti-PS DTRA award - IN PERSPECTIVE --------
http://www.dtra.mil/be/business_opp/awards.cfm
Here are the past four months of all DTRA awards over one hundred thousand dollars.............
( I like the look of this :)
----------
Incredible,
:)
j
flglf4,
Potentially, benefits in effectiveness, (like potentially a cure IMHO), as well as benefits in safety.
The only way anybody is really going to beat cancer or serious chronic viral diseases completely is by getting their immune system to recognize the disease and deal with it specifically, and stay on the watch for any reappearance of it into the future, and be equipped to fight it off again.
It's my opinion that the field of immunotherapy has seen such mediocre success in it's history because no immunotherapies have yet dealt with the elephant in the middle of the room, which is looking to be exposed PS.
see my 5 "required reading",
(which I should actually update to include the recent Mercer/Helenius vaccinia paper, as well as the Gasper-Smith/Haynes paper... but here's my old list :)
----------------------------
exposed PS - 5 must read papers
to understand the potential of therapeutic anti-PS
j
---------------------------------------------------------------------
REQUIRED READING #1 of 5 (abridged) ------
Interaction between Phosphatidylserine and the Phosphatidylserine Receptor Inhibits Immune Responses In Vivo
http://www.jimmunol.org/cgi/content/full/174/3/1393
Peter R. Hoffmann2,*, Jennifer A. Kench*, Andrea Vondracek{dagger}, Ellen Kruk*, David L. Daleke{ddagger}, Michael Jordan{dagger}, Philippa Marrack{dagger}, Peter M. Henson* and Valerie A. Fadok*,{dagger}
The Journal of Immunology, 2005
.
Interaction between Phosphatidylserine and the Phosphatidylserine Receptor Inhibits Immune Responses In Vivo
Introduction
The rapid engulfment of apoptotic cells by professional and nonprofessional phagocytes prevents the release of potentially toxic or immunogenic intracellular contents from the dying cells (1, 2, 3, 4). Much attention has recently focused on the effects of apoptotic cells on phagocytes engulfing them. Interactions between macrophages and apoptotic cells result in the secretion of anti-inflammatory mediators such as TGF-{beta}, IL-10, and PGE2, as well as inhibition of the production of proinflammatory mediators (5, 6, 7, 8, 9, 10, 11). Similar results have been found with nonleukocytic phagocytes including fibroblasts and epithelial cells (Ref.12 ; J. Monks and V. A. Fadok, unpublished observations). In addition, apoptotic cells have been shown in some cases to inhibit dendritic cell maturation and Ag presentation (13, 14, 15).
Apoptotic cells have also been shown to affect inflammation and adaptive immune responses in vivo, although the results are conflicting. For example, apoptotic cells have been shown to be poor adjuvants compared with their necrotic counterparts in the generation of delayed-type hypersensitivity (15). Other evidence suggests that the uptake of apoptotic cells is not an immunologically null event, but is capable of modulating immune responses to self-Ags through induction of T cell-tolerance (16). The tolerizing effects of apoptotic cells have been further demonstrated in studies in which injection of apoptotic cells inhibited rejection of bone marrow allografts (17). By contrast, it has been suggested that apoptotic tumor cells can stimulate antitumor responses in vitro and possibly in vivo (18, 19, 20, 21).
Surprisingly, little is known regarding the mechanisms by which apoptotic cells inhibit inflammation and adaptive immune responses. Exposure of the anionic phospholipid phosphatidylserine (PS)3 in the outer leaflet of the plasma membrane is one of the most striking and consistent changes on the surface of apoptotic cells (22, 23, 24, 25, 26, 27, 28, 29). It has been known for some time that PS can inhibit macrophage production of proinflammatory cytokines and NO, and that it can block macrophage killing of intracellular parasites (30, 31, 32, 33, 34, 35, 36). Data derived from experiments in vitro and in vivo suggest that PS exposure is crucial for the release of TGF-{beta} by phagocytes that accompanies apoptotic cell recognition and uptake (11, 37, 38). Furthermore, PS-containing liposomes can mimic the response to apoptotic cells by generating TGF-{beta} release (11, 38). Blocking PS on apoptotic cells with annexin V (a Ca2+-dependent, PS-binding protein) has been shown to eliminate the inhibitory effects of the apoptotic cells on the humoral responses (39).
We hypothesized that PS exposed on apoptotic cells was a key factor in inhibiting the inflammatory response required for survival of Ag-specific T lymphocytes and subsequent activation of the adaptive immune response (40, 41). If this is true, PS should inhibit immune responses in vivo. Using three different immunogens and several different assays to measure immune responses, we determined that PS stereo-specifically inhibited responses of Ag-specific CD4+ T and B cells in vivo, and that these effects could be mimicked using an activating Ab directed against the PS receptor. Furthermore, these inhibitory effects could be reversed, at least in part, with anti-TGF-{beta} Ab, implicating TGF-{beta} as an important mediator of the inhibitory response to PS. PS-containing liposomes did not appear to block maturation of bone marrow-derived dendritic cells in response to bacterial endotoxin, TNF-{alpha}, or other stimuli as determined by alternation in expression of surface markers or by ability to present Ag to CD4+ lymphocytes. PS-containing liposomes also failed to block the Ag-induced migration of dendritic cells into regional lymph nodes in vivo. Taken together, these findings suggest that PS inhibits the immune response by inhibiting inflammation in tissue.
-------------------------------------------------------------------------
REQUIRED READING #2 of 5 (abridged) ------
Phosphatidylserine Regulates the Maturation of Human Dendritic Cells
http://www.jimmunol.org/cgi/content/full/173/5/2985
The Journal of Immunology, 2004
Xiao Chen*, Kara Doffek*, Sonia L. Sugg{dagger} and Joel Shilyansky2,*
* Division of Pediatric Surgery and {dagger} Division of Endocrine Surgery, Department of Surgery, Medical College of Wisconsin, Milwaukee, WI 53226
Phosphatidylserine Regulates the Maturation of Human Dendritic Cells
Phosphatidylserine (PS), which is exposed on the surface of apoptotic cells, has been implicated in immune regulation. However, the effects of PS on the maturation and function of dendritic cells (DCs), which play a central role in both immune activation and regulation, have not been described. Large unilamellar liposomes containing PS or phosphatidylcholine were used to model the plasma membrane phospholipid composition of apoptotic and live cells, respectively. PS liposomes inhibited the up-regulation of HLA-ABC, HLA-DR, CD80, CD86, CD40, and CD83, as well as the production of IL-12p70 by human DCs in response to LPS. PS did not affect DC viability directly but predisposed DCs to apoptosis in response to LPS. DCs exposed to PS had diminished capacity to stimulate allogeneic T cell proliferation and to activate IFN-{gamma}-producing CD4+ T cells. Exogenous IL-12 restored IFN-{gamma} production by CD4+ T cells. Furthermore, activated CTLs proliferated poorly to cognate Ag presented by DCs exposed to PS. Our findings suggest that PS exposure provides a sufficient signal to inhibit DC maturation and to modulate adaptive immune responses.
INTRODUCTION
Dendritic cells (DCs),3 which are the most potent professional APCs of the immune system, play an important role in the initiation of adaptive immunity (1). DCs acquire, process, and present Ags derived from pathogenic organisms to activate naive T cells, which mount a specific anti-pathogen immune response. DCs also continuously acquire self-Ags from cells that undergo apoptosis as part of normal cell turnover. However, pathological autoimmune conditions occur rarely, suggesting that the ability of DCs to stimulate self-reactive T cells is tightly controlled. The mechanisms permitting DCs to activate vigorous immune responses to most pathogens and to maintain tolerance to tissue-derived "self" Ags are not fully understood (1, 2). There has been a growing body of evidence suggesting that phagocytosis of apoptotic cells under steady state conditions leads to impaired DC maturation and may induce tolerance (3, 4).
snip
.......Recent studies demonstrate that PS-dependent recognition and ingestion of apoptotic cells by macrophages triggers the release of anti-inflammatory cytokines and inhibits the production of proinflammatory cytokines (20, 21, 22). These studies offer a link between the recognition of apoptotic cells and the physiological consequences of their phagocytosis. Whereas the role of PS in the interaction of apoptotic cells with macrophages and the immunological consequence of PS ligation have been broadly delineated, the role of PS in regulating the phagocytosis of apoptotic cells by DCs and the immunological implication of such interactions are not known. Because of the close lineage relationship between macrophages and myeloid DCs, we hypothesized that PS might play a role in the inhibition of myeloid DC maturation. To test this hypothesis, we prepared large unilamellar liposomes containing PS as a simplified model of cell membrane alterations associated with apoptosis. We examined the effect of PS containing liposomes on DC maturation and immunostimulatory capacity. Our results suggest that PS inhibits the ability of DCs to undergo maturation, secrete IL-12, activate T cells, and stimulate IFN-{gamma}-producing CD4+ T cells.....
snip
DISCUSSION
An emerging paradigm suggests that under steady state conditions, apoptotic cells modulate DC function, inducing immune tolerance to self-Ags and preventing autoimmunity (3, 4, 9). Early apoptosis is characterized by loss of membrane asymmetry and exposure of PS on the cell surface (13). PS recently has been shown to play a central role in the recognition of apoptotic cells by macrophages and may be a signal to secrete anti-inflammatory cytokines (17, 20). However, the effects of PS on the maturation and function of human monocyte-derived DCs, whose lineage is closely related to macrophages, have not been well described. We found that PS, which is recognized specifically by DCs, triggers phagocytosis and plays a key role in modulating DC maturation and function....
....Our findings, for the first time, demonstrate that PS is specifically recognized via PSR expressed on the surface of DCs and triggers phagocytosis....
...Previous reports have suggested that iDCs can induce immune unresponsiveness in vitro and in vivo, possibly due to reduced Ag presentation and T cell costimulation (38, 39, 40, 41). Inhibition of DC maturation by exposure to PS is a potential mechanism for inducing immune unresponsiveness or tolerance. We found that DCs exposed to PS had impaired ability to activate allogeneic T cells (Fig. 5, A and B) and to stimulate IFN-{gamma} production (Fig. 6, A and C). Increasing the ratio of DCs to responders restored the proliferation of allogeneic T cells; however, IFN-{gamma} production by allogeneic CD4+ T cells was still impaired. Increased susceptibility to apoptosis by DCs exposed to PS after LPS treatment could not explain the inability to stimulate IFN-{gamma}-producing T cells in vitro, because IL-4-producing T cells were still induced. Furthermore, exogenous IL-12, which does not affect DC viability, restored IFN-{gamma} production by T cells. The findings suggest that PS did not simply inhibit the ability to activate T cells, but selectively modulated the ability to induce IFN-{gamma}-producing T cells.
....Taken together, these findings suggest that DCs exposed to PS would fail to initiate and may extinguish cellular immune responses.....
...Our findings suggest that PS exposure directly inhibits DC maturation and modulates their ability to activate T cell responses. PS is specifically recognized by DCs and triggers phagocytosis via the PSR. Our findings suggest that PS modulates the adaptive immune response at three different levels: 1) PS inhibits survival, Ag presentation, and costimulation by DCs, leading to ineffective T cell proliferation; 2) PS inhibits IL-12p70 secretion by DCs, diminishing the ability to stimulate IFN-{gamma}-producing T cells characteristic of Th1 responses; and 3) DCs exposed to PS have reduced ability to stimulate the proliferation of activated CD8+ T cells, even in the presence of abundant Ags. PS may synergize with additional signals, such as CD200, IL-10, and TGF-{beta}, expressed by apoptotic, inflammatory, or stromal cells (20, 22, 50, 51, 56, 65, 66). Further understanding of the mechanisms by which PS modulates DC function may lead to the development of effective immunotherapies for autoimmune diseases and for cancer.
----------------------------------------------------------------
REQUIRED READING #3 of 5 (abridged) ------
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
http://www.jbc.org/cgi/reprint/M605146200v1
--------------
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.
--------------------------------------------------------------
REQUIRED READING #4 of 5 (abridged) ------
The influence on the immunomodulatory effects of dying and
dead cells of Annexin V
The Journal of Leukocyte Biology, Jan, 2007
http://www.jleukbio.org/cgi/rapidpdf/jlb.0306166v1
-----------
Abstract: Apoptotic and necrotic cells expose
phosphatidylserine (PS). This membrane modifi-
cation ensures a swift recognition and uptake by
phagocytes of the dying and dead cells. Annexin
V (AxV) preferentially binds to anionic phospholipids
and thereby, modulates the clearance process.
First, we analyzed the influence of AxV on
the immunogenicity of apoptotic cells. The addition
to apoptotic cells of AxV prior to their injection
into mice increased their immunogenicity
significantly. Next, we studied the influence of
endogenous AxV on the allogeneic reaction
against apoptotic and necrotic cells. To preserve
heat-labile, short-lived “danger signals,” we induced
necrosis by mechanical stress. Wild-type
mice showed a strong, allogeneic delayed-type
hypersensitivity (DTH) reaction. In contrast,
AxV-deficient animals showed almost no allogeneic
DTH reaction, indicating that endogenous
AxV increases the immune response against dead
cells. Furthermore, AxV-deficient macrophages
had a higher immunosuppressive potential in
vitro. Next, we analyzed the influence of AxV on
chronic macrophage infection with HIV-1,
known to expose PS on its surface. The infectivity
in human macrophages of HIV-1 was reduced
significantly in the presence of AxV. Finally, we
show that AxV also blocked the in vitro uptake by
macrophages of primary necrotic cells. Similar
to apoptotic cells, necrotic cells generated by
heat treatment displayed an anti-inflammatory
activity. In contrast, mechanical stress-induced
necrotic cells led to a decreased secretion of
IL-10, indicating a more inflammatory potential.
From the experiments presented above, we
conclude that AxV influences the clearance of
several PS-exposing particles such as viruses,
dying, and dead cells. J. Leukoc. Biol. 81:
000–000; 2007.
In healthy situations, cells dying by apoptosis maintain their
membrane integrity until they get cleared. Cell shrinkage leads
to formation of apoptotic bodies containing organelles. Nevertheless,
the dead corpse still has an important message: “No
inflammation, please!”
We were asking the question of whether an interference with
the anti-inflammatory clearance by macrophages of apoptotic
cells, mainly mediated by PS recognition, consecutively restores
the immunogenicity of apoptotic cells in vivo. For this
purpose, mice were immunized with apoptotic cells in the
presence or absence of the PS-binding protein AxV. Using
AxV-deficient mice [21], we further analyzed the physiological
function of AxV in the induction of an immune response
against apoptotic and necrotic cells. PS is also expressed on
monocytes as part of their differentiation program [22]. Many
viruses including HIV cause extensive apoptosis, and infected
monocytes/macrophages therefore express elevated levels of
PS, which consecutively, can also be found in the outer membrane
of the enveloped retrovirus [23]. Using an in vitro culture
system, we examined whether, like the swift uptake of apoptotic
cells, the AxV can also block the silent entry of HIV-1 into
human macrophages. To better understand the mechanism how
PS exposing AxV-binding particles modulates the immune
response, we examined the cytokine secretion of activated
human macrophages and peritoneal macrophages from AxVdeficient
mice after contact with dying and dead cells.
DISCUSSION
Apoptosis is defined as programmed cell death or cellular
suicide, whereas necrosis arises as a result of a violent external
stimulus. A hallmark of apoptotic cells, in contrast to necrotic
ones, is that they maintain their membrane integrity over time.
Thus, the release of intracellular components, which could
damage the surrounding tissue, induce inflammation, or elicit
immune responses, is avoided [26]. To ensure immediate recognition
and uptake by phagocytes, apoptotic cells undergo
early membrane modifications. One such event is the exposure
of PS in the outer leaflet of the plasma membrane associated
with a loss of phospholipid asymmetry. Phagocytes interact
with PS on apoptotic cells mainly through secreted bridging
adaptor proteins, also called opsonins. The latter are important
for a high-capacity clearance of apoptotic cells [27].
The recognition of exposed PS triggers the release of immunosuppressive
cytokines [14, 15, 28], which quench inflammation
and prevent the maturation of antigen-presenting dendritic
cells (DC). Apoptotic cells per se do influence the production
of soluble pattern recognition receptors such as pertussis toxin
3 by maturing DC [29]. Furthermore, the opsonization of apoptotic
cells by soluble factors influences their antigen presentation
by DC and thereby, modulates their immunogenicity
[30]. Recently, it was discussed that alterations of the plasma
membrane phospholipid distribution have important influences,
not only for cell clearance but also for the execution of
apoptosis [31].
AxV is a natural-occurring, specific ligand for PS and may
consequently interfere in vivo with the immunosuppressive
effects of apoptotic cells. We showed that the immunogenicity
of apoptotic cells could be restored by the addition of AxV.
During apoptotic as well as necrotic cell death, autoantigens
are cleaved or otherwise modified, and these modifications may
render cryptic epitopes immune-dominant (reviewed in ref.
[32]). When interfering with the clearance of dying cells, DC
may acquire modified autoantigens such as apoptotic nuclei
and chromatin, and consequently, autoreactive T cells can be
activated. This may also lead to chronic autoimmunity, as is the
case in SLE [24]. It was also shown that an impaired clearance
of dying tumor cells can lead to tumor rejection. AxV decreased
apoptotic cell uptake by peritoneal macrophages and
concomitantly increased their uptake by CD8/11c DC [33].
When AxV binds to apoptotic cells, it “crystallizes” as an
extended two-dimensional network. It has an autocrine function
and activates away from cell entry. This results in the
internalization of the PS-expressing membrane patches from
the surfaces of apoptotic macrophages [35]. We have shown
previously that AxV shows positive cooperativity for PS binding
on membranes of apoptotic and necrotic but not of viable
cells. We suggested that phagocytes can differentiate between
dying and viable cells by means of PS clustering and consecutively,
by the lateral mobility of the cellular membranes [36].
PS-mediated phagocytosis of apoptotic cells suppresses inflammatory
signals such as TNF-, IFN- , and NO and also triggers
the production of TGF-, an anti-inflammatory cytokine
[15]. We have previously shown that necrotic cells, like apoptotic
ones, can also engage CD36 and a surface receptor
recognized by mAb 217G8E9. They thereby mediate antiinflammatory
signals [12]. Here, we show that necrotic cells
induced by heat treatment promote the release of anti-inflammatory
cytokines by LPS-activated macrophages, as is the case
for apoptotic cells. Heat-induced necrosis destroys heat-labile
and short-lived danger signals. The uptake of the necrotic cells
then becomes anti-inflammatory. In contrast, mechanical
stress-induced necrotic cells led to a decreased secretion of the
anti-inflammatory cytokine IL-10 and consecutively, displayed
a more immunostimulatory phenotype. We conclude that the
proinflammatory properties of necrotic cells depend on the
inductor of necrosis. Furthermore, we added AxV to this experimental
setting and found a significant, further up-regulation
of IL-10 secretion in the case of heat-necrotized cells but
not in the case of stress-induced necrotic cells (not shown),
further confirming the higher proinflammatory potency of mechanical
stress-induced necrotic cells when compared with
heat-induced necrosis.
The primary force of the immune system is the need to detect
and protect against danger [37]. One mode of action of danger
signals is to stimulate the maturation and activation of DC
necessary for the initiation of primary and secondary immune
responses. Endogenous danger signals, released by tissues
undergoing stress, damage, or abnormal death, and also exogenous
danger signals, elaborated by pathogens, can contribute
to this stimulation. Some endogenous danger signals, which
have been discovered recently are heat-shock proteins, nucleotides,
reactive oxygen intermediates, extracellular matrix
breakdown products, neuromediators, cytokines such as the
IFNs [38], as well as uric acid [39], ATP [40, 41], and HMGB1
[16, 42], which as well as its best-characterized receptor for
advanced glycation end products, is important for the maturation
of human plasmacytoid DC [43] and also controls T cell
activation [44]. Furthermore, the release of intracellular factors
from necrotic tumor cells can promote reactive angiogenesis,
stromal proliferation, and local immune suppression [45]. Future
work has to be focused strongly on all such “alarmins”
[46], which signal the body: “Attention, tissue damage has
occurred!”
Many viral infections (such as HIV) lead to the release of
several proinflammatory cytokines and to extensive apoptosis.
The latter may contribute to the impaired immune response
accompanying such infections [50, 51]. Recently, it has been
shown that PS can be detected at the surface of HIV-1 and that
AxV can be used to enrich these virus particles. Furthermore,
the infection by HIV-1 strains of monocytes can be compromised
upon the addition of AxV during the infection process
[23]. Here, we demonstrated that clinically relevant, chronic
infections of HMDM with HIV-1 isolates can be inhibited by a
single application of AxV. In addition, Ma et al. [52] showed
that Annexin II is necessary for HIV-1 uptake into human
macrophages. This HIV-1 PS interaction with Annexin II could
be disrupted by a secretory leukocyte protease inhibitor [52].
Taken together, AxV efficiently blocks the silent entry of
HIV into macrophages as well as the swift clearance of apoptotic
and necrotic cells. The disturbed, PS-dependent clearance
by macrophages of apoptotic cells leads to the accumulation
of the latter and to the occurrence of late apoptotic cells,
which have lost their membrane integrities. The proinflammatory
cytokine profile of the late clearance and endogenous
danger signals released from cells, which have lost their membrane
integrity, build a proinflammatory microenvironment. DC
may then pick up antigens derived from the dying cells in a
proinflammatory milieu and present the cell-derived antigens
together with costimulation (Fig. 5). We showed that AxV
increased the immunogenicity of apoptotic cells significantly.
In addition, immunization with mechanical stress-induced necrotic
cells was used to mimic the above-mentioned scenario.
Only in the presence of endogenous AxV (WT animals) was a
specific immune response against the dead cells to be observed.
Furthermore, endogenous AxV led to a decreased
secretion of IL-10 in peritoneal macrophages in response to
mechanical stress-induced necrotic cells. Examinations of the
in vitro mechanisms underlying the observed effects revealed
that mechanical stress-induced necrotic cells led to an inflammatory
modulation of macrophages in contrast to apoptotic
cells and heat-induced necrotic ones. In conclusion, apoptotic
and necrotic cells strongly influence the immune response.
AxV is an important modulator of this interaction. It inhibits
certain viral infections and increases the immune response
against tumor cells and more general, that against dying and
dead cells.
-----------------------------------------------------------------
REQUIRED READING #5 of 5 (abridged) ------
Cancer Cell Immune Escape and Tumor Progression by Exploitation
of Anti-Inflammatory and Pro-Inflammatory Responses
Cancer Biology & Therapy 4:9, 924-933, September 2005
http://www.landesbioscience.com/journals/cbt/article/kim4-9.pdf
-----------
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-b, 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-b, 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-b 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.
-----------------
the PS/PSR-mediated
anti-inflammatory response can be induced by soluble phosphatidylserine (sPS) derived
from cancer cells. The sPS can interact with either APCs or PSR on cancer cells, resulting
in the production of IL-10, TGF-b and PGE2, whereby the function of APCs is inhibited.16,17
Thus, the production of anti-inflammatory cytokines mediated by sPS/PSR on APCs and
cancer cells may promote immunosuppression, which impairs macrophage cytotoxicity
and DC function. The impairment
prevents an immune response in the
tumor microenvironment and facilitates
tumor progression and metastasis.18,19
-------------
The shift
to an anti-inflammatory response to immune cells mediated by
TAM in response to sPS from cancer cells may result in the inhibition
of maturation of DCs and their migration to a primary lymphoid organ
for activation of T-cells.
----------------
The
production of soluble factors such as IL-10 and TGF-b helps cancer
cells to avoid immune recognition through inhibition of development
and activity of immune cells.
-----------------
Given that the immunosuppressive mediators such as IL-10,
TGF-b, and PGE2 play an important role in promoting immune
escape in the microenvironment of cancer cells, the release of these
cytokines take place from TAMs as well as from cancer cells.42
-----------------
vascular endothelial cells in tumors
externalize PS to their luminal surface, whereas PS is absent from the
external surface of vascular endothelial cells in normal tissues.19 (Thorpe/Ran, j.)
-------------
the sequential immune response by TAM
and DCs can be abrogated by sPS derived from cancer cells. The
enforced anti-inflammatory response by sPS-not only by interaction
with the PSR on TAM but also with the PSR on immature DCs to
inhibit their maturation-may cause immune escape and tumor
proliferation. Furthermore, the sPS derived from cancer cells binds
to these cells’ own PSR, resulting in the production of IL-10,
TGF-b, and PGE2 in the tumor microenvironment and facilitation
of tumor proliferation.
---------------
During the anti-inflammatory response, the TGF-b derived from
TAM prevents the maturation of antigen-presenting DCs, thereby
inhibiting the activation of natural killer cells and CTLs.86 In addition,
the TGF-b promotes the proliferation of stromal cells such as fibroblasts
and macrophages and induces them to secrete angiogenic and
cell survival factors that stimulate tumor proliferation through
NF-kB, which is activated by Akt.52 These stromal cells also produce
Th2 cytokines, such as IL-4 and IL-10, as well as PGE2 as a
result of TGF-b secretion.
-----------------
Thus, the production of
IL-10, TGF-b, and PGE2 from TAM mediated by sPS or hydroxide
plays a critical role in immune escape.
-----------
j
j
flglf,
Understand the difference between a vaccine and a therapeutic.
Duke / Gates is using Peregrine's anti-PS, THERAPEUTICALLY!, to see if it is worth it to try to create a VACCINE that gets YOU to make your OWN anti-PS, to have "at the ready" to expand and quickly fend off HIV if it were to enter your body.
If Peregrine's anti-PS "works", (immediately, as THERAPY!), then Duke has accomplished "step 1" of the long, multi-step, perhaps impossible goal of a safe effective HIV vaccine.
The hard part would be to design a vaccine to make it happen, in you, safely.... (it defies the current immunological paradigm ,to deliberately try to induce "auto-antibodies"!)
If you were to read Haynes recent "Multicomponent Vaccine" patent application, you will see where the work is heading. Basically, the "multicomponent vaccine" plan is to find a good HIV immunogen, and combine that with something that induces anti-PS, so the immunized individual could prevent the massive PS-exposing debris from weakening his immune system, while also specifically fighting the virus.
But!-----------------
While the world's top brains work on a vaccine,
the IMMEDIATE take-away for Peregrine is a new type of promising HIV THERAPEUTIC! (Validated by the world's top brains), and IMHO published in one of the world's top journals.
- a therapy that works in a WHOLE NEW WAY.
you know-
("broadly neutralize HIV in an unprecedented manner.")
(as well as of course the implications that Haynes is now on record saying, - like the mention of all the other diseases that follow a similar pathogenesis (via PS-exposing immunosuppressive microparticles shed from apoptotic cells). As of yesterday's patent application, he was mentioning auto-immune disease such as lupus etc, as well as rheumatoid arthritis, atherosclerosis, etc....
(Read the past couple year's work of Haynes' Duke colleague, and now close partner in this work, David Pisetsky for insights into all that....)
He also speaks about the incredible breadth of therapeutic possibilities, in one of the other very recent patent applications, (the one in which he specifically says these things) -
*IS1 is an antibody that can be safely used as a therapeutic Mab for treatment of HIV infected subjects
*these antibodies can
broadly neutralize HIV in an unprecedented manner.
*reactivity with the HIV envelope is not a prerequisite for neutralization in these antibodies.
In that recent patent application, where he says anti-PS can be used as safe HIV therapy- (Oh BY THE WAY THAT'S SPECIFICALLY POJEN CHEN'S ANTI-PS, WHO IS ONE OF PEREGRINE'S "CORE TECHNOLOGY" CONTRIBUTORS...), Haynes says this, as to the potential breadth -
[0051] It will be appreciated from a reading of the foregoing that if HIV has evolved to escape the host immune response by making the
immune system blind to it, other infectious agents may have evolved similarly. That is, this may represent a general mechanism of escape.
That being the case, approaches comparable to those described herein can be expected to be useful in the treatment of such other agents
well.
-----------
j
moby,
re:"For me, the data out of Georgia and the recent announcements about how early after infection that HIV can compromise the immune system points why Bavi didn't perform as well in the HCV arena as was originally hoped."
Then you disagree with Haynes.
Haynes is proposing using anti-PS as HIV therapy.
He said it, not me.
He's basically saying it should work.
Why's he saying that?....
Haynes is saying any future successful vaccine needs to prevent immunosuppressive PS signaling.
Why's he saying that?....
Further, Haynes suggests that a good way to do it would be for the vaccine to induce anti-PS.
Why's he saying that?....
(That's rhetorical. You'd know why he's saying it if you were to read his papers and patent applications in their entirety).
Also, with regard to your statement above, it's funny you mention HCV in relation to this work... If you were to actually read the Haynes paper in the August Journal of Virology, you would see the differences- as discussed (and graphed!) by Haynes- illustrating the differences between the immunosuppressive PS-exposing microparticle aspect of the pathogenesis of HIV as compared to HCV and HBV.
j
The Official Duke Press Release ----------
about the Gasper-Smith / Haynes paper in the August issue of the Journal of Virology ----------
http://www.dukemednews.com/news/article.php?id=10364
Most pertinent bits-
Through a series of in vitro laboratory experiments with peripheral blood cells, scientists found that microparticles suppressed levels of IgG and IgA, two classes of antibodies that normally would protect a person against infection. "This is important because many scientists believe that a fast-acting memory B cell response as well as a T cell response will be necessary to fight HIV-1" said Nancy Gasper-Smith, PhD, the lead author of the study.
"These and other studies that recently revealed more about the singular nature of HIV-1 have given us valuable information that is helping us move closer to establishing a basic science foundation that can lead to novel technologies for vaccine design, Haynes said. Haynes. "It is becoming clearer why we have failed in our efforts to date, and what we need to confront to succeed in the future."
-----------------------------------------------
Entire Press Release:
DURHAM, N.C. – New research into the earliest events occurring immediately upon infection with HIV-I shows that the virus deals a stunning blow to the immune system earlier than was previously understood. According to scientists at Duke University Medical Center, this suggests the window of opportunity for successful intervention may be only a matter of days – not weeks – after transmission, as researchers had previously believed.
Appearing in the August issue of the Journal of Virology, the finding may make the challenge of designing an effective HIV/AIDS vaccine appear daunting. But researchers say the study has also yielded a blueprint for what a successful vaccine should look like, and moreover, when such a vaccine would need to work.
Until now, scientists believed that the window of opportunity to intervene in the process of HIV-1 infection lay in the three to four weeks between transmission and the development of an established pool of infected CD4 T cells. HIV-1 cripples the immune system by invading and killing CD4 T cells, key infection-fighters in the body.
"But this new study shows that HIV-I does a lot of damage to the immune system very early in that time frame, and now we feel that the opportunity to intervene most effectively may range from about five to seven days after infection," said Barton Haynes, M.D., the senior author of the study and director of the Center for HIV/AIDS Vaccine Immunology (CHAVI) at Duke University Medical Center.
Haynes said the findings suggest that an optimal vaccine strategy would have to pack a double punch: First, establishing as much immunity as possible before infection, much as classic vaccines do, and then following a few days later with a mechanism to provoke a strong, secondary, broad-based antibody response. "Vaccine candidates to date have pretty much followed a single strategy. Now we know that we need to activate multiple arms of the immune system and we have a better idea of when to do it."
The conclusion comes from the study of 30 people who were newly-infected with HIV-1. Plasma from these individuals was sampled every three days for several months – before, during, and after the "ramp-up" phase of infection, when HIV-1 is multiplying rapidly and heading toward its peak viral load. In measuring the levels of four products of CD4 T cell death during this period in these samples, they were able to track and establish a timetable of the virus's destructive path.
The four byproducts of CD4 T cell death include TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), Fas ligand, TNF receptor type 2 and plasma microparticles, tiny bits of cell membrane that are broken up and left floating around in the plasma when the cell dies and breaks apart.
The researchers found that TRAIL levels increased significantly a full week (7.2. days) before peak viral load, which is approximately 17 days after HIV-1 transmission, suggesting that during the earliest period of infection, called the eclipse phase, TRAIL may actually initiate or hasten HIV-1's destruction of CD4 T cells. In contrast, they found that the levels of the other three cell death products were most significantly elevated during peak viral load.
"What this demonstrates is that significant T cell death is occurring much earlier during this period than we previously believed, and that TRAIL itself may be a co-conspirator in enhancing cell death," Haynes said. "This leads us to believe that the time frame for successful intervention has to move even close to the point of infection."
Researchers also examined the effects of cell death products upon B cells, another arm of the immune system responsible for the creation of antibodies. Previous studies have shown that the antibody response to HIV-1 is "too little, too late" – appearing after the virus has peaked and after the reservoir of infected T cells has already been established.
Through a series of in vitro laboratory experiments with peripheral blood cells, scientists found that microparticles suppressed levels of IgG and IgA, two classes of antibodies that normally would protect a person against infection. "This is important because many scientists believe that a fast-acting memory B cell response as well as a T cell response will be necessary to fight HIV-1" said Nancy Gasper-Smith, PhD, the lead author of the study.
Daniel Douek, M.D., PhD, chief of the Human Immunology Section of the National Institutes of Health, said the study sheds new light on key events in the earliest phase of infection. "The cohort is a gem. It is clear from the raised levels of TRAIL that the body senses the virus before plasma viral loads have peaked. This suggests that the virus begins to cause damage in ways that may be unrelated to the well-described massive depletion of gut CD4 T cells that becomes apparent around peak viral load. For clinical practice, this means the window of opportunity in which antiviral therapies and vaccines must act is becoming ever narrower."
"These and other studies that recently revealed more about the singular nature of HIV-1 have given us valuable information that is helping us move closer to establishing a basic science foundation that can lead to novel technologies for vaccine design, Haynes said. Haynes. "It is becoming clearer why we have failed in our efforts to date, and what we need to confront to succeed in the future."
The study was supported by grants from the National Institutes of Health
Colleagues from Duke who contributed to the research include Deanna Crossman, John Whitesides, Nadia Mensali, Janet Ottinger, Steven Plonk, M. Anthony Moody, Guido Ferrari, Kent Weinhold, Sara Miller and Thomas Denny. Additional co-authors are David Pisetsky and Charles Reich, from the Durham Veterans Administration Hospital; Li Qin and Stephen Self, from Fred Hutchinson Cancer Research Center and the Statistical Center for HIV-AIDS Research and Prevention; George Shaw from the University of Alabama: and Laura Jones, from Cornell.
link to PR:
http://www.dukemednews.com/news/article.php?id=10364
--------------
j
The Barton Haynes patent application, which went up on the World Intellectual Property Organization database on July 24, 2008 -
Pertinent excerpts follow:
"The time of appearance of antibodies in the development of acute HIV infection has been recently mapped and it has been shown that most
of the antibodies arise after a delay in the peak response to HIV envelope epitopes of approximately two to three weeks. Indeed, the most
protective antibodies, those that neutralize autologous virus, can be delayed for up to a year."
"To begin to understand the "delay" in induction of antibodies at the time of HIV transmission, the first question to be addressed was whether
there are immunosuppressive events, such as massive apoptosis, with release of phosphatidylserine microparticles at the time of viral load
ramp up during acute HIV infection."
"Apoptotic microparticles are the products of either activated or apoptotic cells, that are increased in the plasma of a number of diseases,
including autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, Crohn's disease, coronary artery disease
and other forms of heart disease, and chronic HIV-I infection."
"Apoptotic microparticles can bind to non-apoptotic cells and induce apoptosis, are procoagulant, proinflammatory, and can be
immunosuppressive for T and B cell responses to specific antigen."
"Thus, the massive apoptosis that occurs with acute HIV infection with resulting release of TRAIL, mediation of apoptosis via FAS-FASL
interactions, and release of PS containing viral and other particles, all conspire to initially immuno suppress the host, preventing rapid
protective B cell responses."
"Thus, the production of high levels of biologically active plasma mediators and byproducts of cell death during the first two to three weeks of
HIV- 1 transmission raises the notion that the window of opportunity for a preventive vaccine to work may be shorter than previously thought,
ie within the first 14-17 days of transmission, placing considerable constraints on the time available for development of robust anti-HIV-1
immunity following transmission."
"Inhibition of cell death and immunosuppressive MP mediated effects by a vaccine for HIV or other infectious agents may be important as
well. This could be accomplished, for example, by an HIV vaccine component inducing anti-lipid antibodies or antibodies against other
components of microparticles to facilitate clearance of microparticles and/or to block microparticle toxic effects."
"Another use of the data herein is as a rationale for the treatment of HIV-I. For example, antibodies against TNFR or TNF-alpha;,
antiphosphatidylserine antibodies or other inhibitors of cell death (Fas-Fc as an inhibitor of FAS-FAS ligand interactions and DR5-Fc as an
inhibitor of TRAIL DR5 interactions) can be used to inhibit cell death in HIV as a therapy."
http://www.wipo.int/pctdb/en/wads.jsp?IA=US2008000412&LANGUAGE=EN&ID=id00000006622149&VOL=89&DOC=00fca1&WO=08/088747&WEEK=30/2008&TYPE=A2&DOC_TYPE=PAMPH&PAGE=1
repost: here's my 'old' "predictions" for what I expect to read in the upcoming PPHM-pertinent "Haynes paper"....
--------------------------------
What I expect from "Haynes paper" -
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=29353576
I expect the paper to be authored by several top-tier names not only from Duke but other places.
Some Duke names I expect:
Bart Haynes (who's name will be last), Munir Alam, David Pisetsky, Tony Moody, David Montefiori,
Some other names you might see:
Phil Thorpe, Melina Soares, Pojen Chen (also a PPHM SAB member, who has isolated and developed some very interesting mabs to phospholipids that most people probably haven't YET read about... ), also possibly - George Shaw, Beatrice Hahn, Andrew McMichael, Norm Letvin, Gary Nabel, Persephone Borrow, Nina Bhardwaj,
In short, I wouldn't be surprised to see names of top researchers at Duke University, UCLA, UTSW, Univ. of Alabama at Birmingham, NYU, Albert Einstein, Harvard University, Oxford University, NIAID/NIH, to also be listed.
Based on the the importance of the discoveries, I expect it to be published in a major journal, and I agree with microbeman, - Nature, Cell, or Science.
Haynes' previous big shocker in 2005 that got all the money thrown his way (CHAVI & Gates), which made him the #`1 top govt. funded doc in the country, was in Science. That's where he showed that the few rare broadly neutralizing abs found in people bound to not only viral bits, but also phospholipids. He reasoned that those rare BNAbs were not typically made in most people for reasons I've already discussed.
It's taken two short years to move from (most folks) seeing the lipid-binding characteristics of those now-famous rare mabs as a hindrance, to seing them as the KEY....
Or- to re-phrase that - Haynes' original paper sent a shock-wave through the research community, with his data that the extremely rare BNAbs (Broadly Neutralizing Abs) were polyspecific for viral epitopes AND PHOSPHOLIPIDS. The phospholipid binding was seen negatively, surely, as a likely reason for the abs rarity in people, and also with potential safety problems.
I expect the upcoming paper to change the view of that 180 degrees.
I expect the mabs used in work at Duke to be Peregrine's.
Here are a few of the things I expect to see in an upcoming Haynes paper.
---------------------------------------------
* I expect them to say/prove that targteting phospholipids alone with antibodies can neutralize HIV.
(That's a shocker with implications for many viruses and other diseases).
* I expect them to say/prove that you don't need to target the virus itself.
* I expect them to say/prove that by targeting phospholipids alone with abs you can prevent fusion of the virus with a cell.
* I expect them to say/prove that targeting certain host-cell phospholipids stuck on the virus and on virally infected cells with abs is safe.
(Anyone who understands the long-standing foundations of the immunological paradigm will realize how big a change this is).
* I expect them to discuss 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, (a little more on that later).
* 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. 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 resonse, that "successful" cancers, 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. I expect them to show with impressive data that anti-PS mabs safely deal with this fundamental loop-hole/compromise in our immune response that has been with us since the dawn of metazoans, (which has been proven to be, as you'd expect, also the dawn of the apoptotic process).
we'll see, JMO,
j
jess, again,
re: post # 26473,
what "DUKE PAPER" are you referring to?
j
what "DUKE PAPER" are you referring to?
Duke/bavi PPHM CEO quotes -
Please note,as mentioned below, Duke is working with more of Peregrine's stuff than just Bavituximab. Personally, I would expect them to be looking into EVERYHING Peregrine's got, which includes, off the top of my head:
Bavi
humanized Bavi
other earlier anti-PS as mentioned in Thorpe patents, (9D2, etc.)
3G4
2aG4
the Betabodies
Chen's mabs, (IS1, etc.)
anti-PS conjugated to IFN
anti-PS conjugated to IL-2
mabs created through their collaboration with Affitech
mabs and conjugates created through their collaboration with Biotecnol
PS-binding peptides
PE-binding peptides
PE-targeting mabs
PE-binding peptide conjugates (DLB etc...)
likely several others I've forgotten...
j
9-11-06 QTLY. CONF. CALL (KING/LYTLE): http://tinyurl.com/k7uo6
SK: “BAVI HIV INITIATIVE: HIV is currently our primary area of interest for potential new AV indications... We have already generated positive data supporting the fact that Bavi recognizes both HIV virus and HIV infected cells. In order to fully evaluate the potential of Bavi in HIV therapy, we have expanded our collaborations in the HIV area. Our curr. collaborators include investigators at Tulane National Primate Research Center, Duke Univ., as well as contract research labs.
10-24-06 ANNUAL SHM, incl. Thorpe presentation on Bavi AC/AV http://tinyurl.com/vmasl
SK: “On the pre-clinical front, we are continuing our collab’s for AV applications, with the primary focus on HIV. Those studies are ongoing at Tulane & Duke, as well as at contract labs.”
3-12-07 QTLY. CONF. CALL (KING/LYTLE): http://tinyurl.com/2dtmca
SK: “Expanding the patient population we are treating to potentially include HCV/HIV co-infected individuals is a 3rd focus area… Our interest in this patient population has been further stimulated by solid evidence, thru our collab’s at Duke, that Bavi binds to HIV virus, binds to HIV-infected cells, and may have potent neutralizing effects on the virus.
3-12-07 QTLY. CONF. CALL (KING/LYTLE): http://tinyurl.com/2dtmca
SK: “ the funding that's coming out of there [Duke] is actually funding a lot of studies that are being done with bavituximab as a model..."
"The collaboration is going extremely well, the data we're generating is really helping us in the way we think about our development of bavituximab for HCV & HIV,"
7-11-07 QTLY. CONF. CALL (KING/LYTLE): http://tinyurl.com/2c9kwr
SK: “We have also been collaborating with researchers at Duke and other institutions to better understand the potential of Bavituximab in an HIV setting. Significant findings of these studies include data supporting Bavituximab binding to mult. strains of HIV and binding to HIV-infected cells
9-10-07 QTLY. CONF. CALL (KING/LYTLE): http://tinyurl.com/38lky9
SK: “Our collab. with HIV researchers at Duke remains strong and is making good progress. The collab. is providing exciting, new insights into the potential of Bavituximab.."
"we are happy with the collaboration and are getting a lot of data from the studies they are doing,"
"Our role in the studies is primarily to provide materials that are then being tested"..
"We are working with a group at Duke. In particular, we participated in a pgm that has been funded through the Gates Foundation [CAVD]. The CHAVI pgm is actually a separate pgm. They are both studying phospholipids as potential targets for the dev. of vaccines. So, in order to pursue that, they’ve been testing Bavituximab.."
There are add’l studies being planned. It’s a very large pgm that’s being run, again, simultaneously through these 2 different pgms – the CHAVI and then the Gates pgm [CAVD]. We are as anxious as anyone else to be able to get some of this info. out there. I know the guys at Duke will want to get it out there at the appropriate time. We are getting a lot of insight into, not just Bavituximab, but other antibodies that we have generated through our various collaborations. Over the long run, that’s going to be some extremely valuable info. that is only going to strengthen our overall antiviral pgm.”
2-10-07 QTLY. CONF. CALL (KING/LYTLE): http://tinyurl.com/393mau
SK: DUKE – “In addition to the clinical study, we are continuing a significant amount of preclinical work in the antiviral area. Our leading collaboration is with researchers at Duke University and a number of other institutions, including Harvard. This collaboration is progressing very nicely and we are highly encouraged by the results we have seen. As a reminder, we are exploring the potential of Bavituximab and several other anti-PS antibodies for their potential in the treatment and prevention of HIV infections. This collaboration is particularly important because without the collaboration we would not be able to conduct this informative research into the potential of our anti-PS platform for the treatment of HIV. Both we and our collaborators believe we are getting close to being able to share results of these studies either through publications or presentations in the upcoming months.”
the first and last slides of Rolf Brekken's Bavi preclinical studies ppt presentation -
BTW- Please review Brekken's eye-opening Bavituximab presentation in it's entirety -
http://presentations.cancerconferences.com/7th_monoclonal/files/2007_0817/ws02_brekken.pdf
-------
j
Kryan,
Perhaps because IS1 is Pojen Chen's?
Did you read my post?....
Here, again....
-------------
Kryan,
The person you should take note of in the 'recent' additions to Peregrine's SAB is Pojen Chen of UCLA....
please review the following, in this order:
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30709208
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30709411
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30710341
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30710526
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30711002
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30711900
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30712206
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30712838
-----------
j
moby, if you want to see the Haynes patent application that is referring to Peregrine's mab as HIV therapy, you need to look here -
1. A method of treating HIV comprising administering to a patient in need thereof an antibody derivable from a normal subject or from an autoimmune disease subject that binds to a lipid on the surface of HIV or on the surface of HIV-infected cells and thereby neutralizes HIV-1, wherein said antibody is administered in an amount sufficient to effect said treatment.
2. The method according to claim 1 wherein said antibody is derivable from an anti-phospholipid syndrome subject.
3. The method according to claim 1 wherein said antibody is non-pathogenic.
4. The method according to claim 1 wherein said antibody is IS1, IS4 or IS6, or binding fragment thereof.
5. The method according to claim 1 wherein said antibody is IS1, or binding fragment thereof.
[0105] ....That IS1 neutralized HIV evidences the facts that: a) humans can make non-pathogenic anti-lipid antibodies that neutralize HIV, and b) IS1 is an antibody that can be safely used as a therapeutic Mab for treatment of HIV infected subjects or in the setting of post-exposure prophylaxis of subjects following needle, sexual or other exposure to HIV or HIV infected materials.
[0108] Alving and colleagues have made a mouse mab against phosphatidyl inositol phosphate and have shown that it neutralizes HIV in a PBMC assay. What the present studies show is that humans can spontaneously make anti-lipid antibodies and that these antibodies can broadly neutralize HIV in an unprecedented manner.
[0109] Summarizing, autoimmune disease patients can make antibodies that bind to virus-infected cells and, presumably, to budding HIV virions by virtue of their reactivity to HIV membranes and host membranes. Certain anti-lipid antibodies from autoimmune disease patients can also react with the Envelope trimer (such as IS6) but not all of the antibodies react also with the trimer (i.e., IS1 and IS4 do not react). Therefore, reactivity with the HIV envelope is not a prerequisite for neutralization in these antibodies.
AND MY FAVORITE "broad implication" point mentioned by Haynes -
[0051] It will be appreciated from a reading of the foregoing that if HIV has evolved to escape the host immune response by making the immune system blind to it, other infectious agents may have evolved similarly. That is, this may represent a general mechanism of escape. That being the case, approaches comparable to those described herein can be expected to be useful in the treatment of such other agents well.
http://appft1.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2&p=1&f=G&l=50&d=PG01&S1=%28%22haynes+Barton%22.IN.%29&OS=in/%22haynes+Barton%22&RS=IN/%22haynes+Barton%22
--------
j
Dr. Seth Berkley, president of the International AIDS Vaccine Initiative, said, "The decision does not reflect paralysis in the AIDS vaccine field, or lack of direction forward. In fact, it reflects the opposite – the dynamic learning that is the scientific process, that is pharmaceutical product development. The decision reflects leadership on the part of NIAID."
---------
j
Kryan,
The person you should take note of in the 'recent' additions to Peregrine's SAB is Pojen Chen of UCLA....
please review the following, in this order:
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30709208
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30709411
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30710341
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30710526
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30711002
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30711900
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30712206
http://investorshub.advfn.com/boards/read_msg.aspx?message_id=30712838
-----------
j
Kryan,
If you do your reading, you'll find that the patent application I posted today is mostly about a way of testing a HIV-infected person to measure the amount of (PS-exposing) microparticles in their blood. (This came from the results of experiments that are discussed in the most recent paper, in the August Journal of Virology). The PS-exposing microparticle measurement can then help to predict the timing of the progression to AIDS. (The current measurement used to assess whether therapy is needed is the number of CD4+ T cells).
Interestingly, vaccine and therapeutic implications are also mentioned again. The most detail regarding Haynes latest opinions on those two topics are the two previous recent Haynes patent applications I've posted, which were:
* One about what any future successful HIV vaccine must do, and what Haynes says it must do is induce antibodies that interupt PS-mediated immunosuppressive signaling.
* The other was specifically about one of PEREGRINE'S licensed mabs as a "SAFE HIV THERAPEUTIC".
(see my previous posts on the topic).
The patent application I posted today also goes further into detail about how PS-exposing microparticles are responsible for the pathogenesis of many other diseases.
(see my many previous posts on this topic).
Evolution has favored pathogenesis that resembles apoptosis.
j
most important bits ------------------
The time of appearance of antibodies in the development of acute HIV infection has been recently mapped and it has been shown that most of the antibodies arise after a delay in the peak response to HIV envelope epitopes of approximately two to three weeks. Indeed, the most protective antibodies, those that neutralize autologous virus, can be delayed for up to a year.
To begin to understand the "delay" in induction of antibodies at the time of HIV transmission, the first question to be addressed was whether there are immunosuppressive events, such as massive apoptosis, with release of phosphatidylserine microparticles at the time of viral load ramp up during acute HIV infection.
Apoptotic microparticles are the products of either activated or apoptotic cells, that are increased in the plasma of a number of diseases, including autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, Crohn's disease, coronary artery disease and other forms of heart disease, and chronic HIV-I infection.
Apoptotic microparticles can bind to non-apoptotic cells and induce apoptosis, are procoagulant, proinflammatory, and can be immunosuppressive for T and B cell responses to specific antigen.
Microparticle levels correlate with the levels of IL-6 in healthy adults, are increased in acute coronary syndromes and correlate with severity of angiographic coronary lesions. CD31/annexin V+ apoptotoc microparticles correlate with coronary endothelial function in patients with coronary artery disease.
Thus, the massive apoptosis that occurs with acute HIV infection with resulting release of TRAIL, mediation of apoptosis via FAS-FASL interactions, and release of PS containing viral and other particles, all conspire to initially immuno suppress the host, preventing rapid protective B cell responses.
Of importance, most of the microparticles from patient 6246 are phosphatidylserine (PS) positive (Fig. 13).
The time from HIV-I transmission to establishment of the latently infected pool of CD4 T cells has been termed the window of opportunity within which a preventive HIV-I vaccine has to extinguish HIV-I. The latent pool is established at least by the time of symptomatic acute HIV-I infection at the time of seroconversion (-25 days after transmission), although the exact earliest time of establishment of the latent CD4 T cell pool is not known. Adaptive CD4, CD8 and B cell antibody responses to HIV-I do not appear during the eclipse or viral load ramp-up phases of HIV-I infection, but rather appear coincident with the fall in viral load (VL) and appearance of acute infection symptoms at the end of the window of opportunity. Thus, study of the events that transpire from transmission until the onset of plasma viremia (the eclipse phase) and during the viral load ramp-up phase of acute HIV-I infection are critical to understanding why immune responses do not occur earlier after HIV-I transmission, and to define what a successful vaccine must overcome to extinguish HIV-I.
In the study described below, the hypothesis is raised that, in addition to gut CD4 T cell loss, delay in HIV-I protective immune responses early on after HIV-I transmission may involve the production of elevated levels of immunosuppressive moieties such as TRAIL, TNFR2 and Fas ligand as well as plasma mi croparticles. If elevations in immunosuppressive molecules, coupled with early CD4+ T cell death, occur early on after HIV-I transmission, then this would define a protected time for HIV-I to replicate while anti-HIV-1 T or B cell responses were suppressed.
While the average peak HIV-I VL level was 1,421,628 copies/ml, the average peak of total MPs was 606,881,733/ml. Thus, there was an average of 427-fold more MP than virions present in plasma at their peaks during acute HIV- 1 infection.
MP-Induced B Cell Suppression In Vitro.
While plasma MPs have potent known suppressive effects on macrophages and DCs, only one study has suggested MP may inhibit B cell activation. There was particular interest in MP effects on human memory B cell activation, since what is desired is a rapid virus-induced memory B cell response after transmission. To determine if PBMC-derived or tonsil leukocyte-derived MP could be suppressive for memory B cell activation, a memory B cell Ig induction assay was used using pokeweed mitogen (PWM) + class B oCpG . The addition of MP in PWM-stimulated tonsil cell cultures reduced total IgG and IgA production by 70.8 % +/- SEM for IgG (p=0.0064) and 94.2% +/- SEM for IgA (p=0.00004) (Fig. 24A); B cell suppression by MPs was dose-dependent (Fig. 24B). Similar results were observed when MP were generated from autologous tonsil leukocytes or from the Jurkat T cell line.
In summary, a major finding in this study is the early appearance of a peak of TRAIL at 17 days of transmission in plasma donors, and implies the TRAIL/DR5 in a key pathway in HIV-I induced cell death immediately following transmission. An IFN-α, TRAIL, DR5 pathway of CD4+ T cell apoptosis has been proposed for chronic HIV-I infection based on in vitro studies and on studies in HIV-1+ progressor tonsillar tissues. CD4+ T cells in infected subjects are more sensitive to TRAIL-mediated apoptosis than are CD4+ T cells from uninfected subjects due to upregulated TRAIL receptor DR5.
An important question is why do plasma TRAIL levels peak earlier after HIV-I transmission than do plasma Fas ligand, TNFR2 and MP? Plasma elevations of TRAIL, Fas ligand and TNFR2 occur in chronic HIV-I, and can be induced by immune cell activation, cell death, or both. Stacy et al have found a burst of IFN-α in the same plasma donors that coincides with the timing of the TRAIL peak seen in this study . Thus, the plasma TRAIL peak that precedes the VL plasma peak may be due either to early apoptosis, but may also result from immune activation and pDC production of IFN-αin response to rising VL. It is hypothesized that the later appearance of elevated plasma Fas ligand, TNFR2 and microparticles maybe the result of, or in response to, massive cell death, as this peak comes at an analogous time to the cell death peak documented in experimental SIV infection in rhesus macaques.
Thus, the production of high levels of biologically active plasma mediators and byproducts of cell death during the first two to three weeks of HIV- 1 transmission raises the notion that the window of opportunity for a preventive vaccine to work may be shorter than previously thought, ie within the first 14-17 days of transmission, placing considerable constraints on the time available for development of robust anti-HIV-1 immunity following transmission. Preventive vaccine candidates may need to target HIV-I molecules that induce cell death and be designed to induce protective immune responses to HIV-I that will either be at maximum inhibitory levels at the time of transmission, or be boosted within hours to days as a secondary immune response to extinguish HIV-I before HIV-I- induced immunosuppression occurs.
Inhibition of cell death and immunosuppressive MP mediated effects by a vaccine for HIV or other infectious agents may be important as well. This could be accomplished, for example, by an HIV vaccine component inducing anti-lipid antibodies or antibodies against other components of microparticles to facilitate clearance of microparticles and/or to block microparticle toxic effects.
Another use of the data herein is as a rationale for the treatment of HIV-I. For example, antibodies against TNFR or TNF-α, antiphosphatidylserine antibodies or other inhibitors of cell death (Fas-Fc as an inhibitor of FAS-FAS ligand interactions and DR5-Fc as an inhibitor of TRAIL DR5 interactions) can be used to inhibit cell death in HIV as a therapy.
------------
j
snips
BACKGROUND
The time of appearance of antibodies in the development of acute HIV infection has been recently mapped and it has been shown that most of the antibodies arise after a delay in the peak response to HIV envelope epitopes of approximately two to three weeks. Indeed, the most protective antibodies, those that neutralize autologous virus, can be delayed for up to a year (Wei et al, Nature 422:307-12 (2003); Richman et al, Proc. Natl. Acad. Sci. USA 100:4144-9 (2003)) (Figure 1).
Fiebig et al (AIDS 17:1871-1879 (2003)) have studied plasma panels from plasma donors in US Blood Banks and have found that the plasma panels represent the earliest time points sampled surrounding HIV transmission (Figure 2). The time course of these panels begins before any detectable virus is present, and then continues through the viral ramp-up stages, or Eclipse phase, through the
first and second states of HIV, when seroconversion has not yet occurred. Figure 3 shows the viral loads of 11 such panels of plasma.
To begin to understand the "delay" in induction of antibodies at the time of HIV transmission, the first question to be addressed was whether there are immunosuppressive events, such as massive apoptosis, with release of phosphatidylserine microparticles at the time of viral load ramp up during acute HIV infection (Mattapallil et al, Nature 434:1093 (2005), Veazey et al, Science 280:427 (1998), Guadalupe et al, J. Virol. 77:11708 (2003), Benchley et al, J. Exp. Med. 200:749 (2004), Mehandru et al, J. Exp. Med. 200:761 (2004), Esser et al, J. Virol. 75: 6173-6182 (2001), Aupelx et al, J. Clin Invest. 99:1546-1554 (1997), Callahan et al, J. Immunol. 170:4840-4845 (2003)). Apoptotic microparticles are the products of either activated or apoptotic cells, that are increased in the plasma of a number of diseases, including autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (Distler et al, Arth. Rheum. 52:33337-3348 (2005), Tesse et al, Arterioscler. Thromb. Vase. Biol. 25:2522-2527 (2005), Cerri et al, J. Immunol. 177:1975-1980 (2006)), Crohn's disease (Chamouard et al, Dig. Dis. Sci. 50:574-580 (2005)), coronary artery disease and other forms of heart disease (Boulanger et al, Cardiovas. Res. 67:1-3 (2005)), and chronic HIV-I infection (Esser et al, J. Virol. 75:6173-6182 (2001), Aupelx et al, J. Clin Invest. 99:1546-1554 (1997)). Apoptotic microparticles can bind to non-apoptotic cells and induce apoptosis (Distler et al, Apoptosis 10:731- 741 (2005)), are procoagulant (Distiller et al, Apoptosis 10:731-741 (2005)), proinflammatory (Tesse et al, Arterioscler. Thromb. Vase. Biol. 25:2522-2527 (2005), Cerri et al, J. Immunol. 177:1975-1980 (2006)), and can be immunosuppressive for T and B cell responses to specific antigen (Esser et al, J. Virol. 75:6173-6182 (2001), Fadok et al. J. Immunol. 174:1393 (2005)).
Microparticle levels correlate with the levels of IL-6 in healthy adults (Chirinos et al, Amer. J. Card. 95:1258-1260 (2005)), are increased in acute
coronary syndromes and correlate with severity of angiographic coronary lesions (reviewed in Mezentsev, Am. J. Physiol. Heart Circ. Physiol. 289:H1106-H11 14 (2005)). CD31/annexin V+ apoptotoc microparticles correlate with coronary endothelial function in patients with coronary artery disease (Werner et al, Arterioscler. Throm. Vase. Biol. 26:112-116 (2006), Epub Oct. 2005). Aupelx et al (J. Clin Invest. 99:1546-1554 (1997)) have suggested that measuring levels of apoptotic microparticles in plasma may provide information regarding the severity of immune cell destruction in HIV, and also provide an indicator of the responsivness to anti-retroviral drugs. The present invention provides a method of a method of predicting the course of HIV infection in a patient during acute HIV infection (AHI), a method of determining the degree of potential damage to the immune system in AHI, a method of determining the need for anti-retroviral treatment in AHI and a method of monitoring the course of that infection by measuring plasma levels of microparticles coupled with tests of cell activation and/or apoptosis.
........Thus, the massive apoptosis that occurs with acute HIV infection with resulting release of TRAIL, mediation of apoptosis via FAS-FASL interactions, and release of PS containing viral and other particles, all conspire to initially immuno suppress the host, preventing rapid protective B cell responses. Next, flow cytometry phenotypic analysis of the microparticles in AHI sample 6246-15 was performed (Fig. 10). Fig. 11 shows the forward and side scatter of background with phosphate buffered saline, pH 7, purified microparticles from staurosporine treated Jurkat T cells, and the microparticles in patient 6246 plasma on day 11 at the time of peak viremia. Fig. 11 shows the microparticles in plasma with each panel counted for 2 minutes. Fig. 12 shows the phenotype of either purified Jurkat microparticles (pMP) or patient 6246 MP and shows that the microparticles can be phenotypically analyzed and that the AHI patient microparticles are 78.6 % CD3+, 53% CD45 +. CD45 is a surface molecule of T and monocyte cells that is not incorporated into HIV virions (Esser et al, J. Virol. 75:6173-6182 (2001)). Thus, about one half of the microparticles are likely virions in Fig. 12 and one half of the particles are apoptotic particles from HIV-uninfected cells or do not contain HIV genetic material. Of importance, most of the microparticles from patient 6246 are phosphatidylserine (PS) positive (Fig. 13). Similarly, HIV-infected cells are PS+, as shown in Fig. 14.
...................
The time from HIV-I transmission to establishment of the latently infected pool of CD4 T cells has been termed the window of opportunity within which a preventive HIV-I vaccine has to extinguish HIV-I (Johnston et al, N. Engl. J.
Med. 356:2073-2081 (2007, Wong et al, Biology of Early Infection and Impact on Vaccine Design, pgs. 17-22 (Caister Academic Press, Norfolk, UK (2007)). The latent pool is established at least by the time of symptomatic acute HIV-I infection at the time of seroconversion (-25 days after transmission), although the exact earliest time of establishment of the latent CD4 T cell pool is not known (Wong et al, Biology of Early Infection and Impact on Vaccine Design, pgs. 17- 22 (Caister Academic Press, Norfolk, UK (2007), Chun et al, Proc. Natl. Acad. Sci USA 95:8869-8873 (1998)). Adaptive CD4, CD8 and B cell antibody responses to HIV-I do not appear during the eclipse or viral load ramp-up phases of HIV-I infection, but rather appear coincident with the fall in viral load (VL) and appearance of acute infection symptoms at the end of the window of opportunity (Reynolds et al, J. Virol. 79:9228-9235 (2005), Abel et al, J. Virol. 79:12164-12172 (2005), Fiebig et al, AIDS 17:1871-1879 (2003)). Thus, study
of the events that transpire from transmission until the onset of plasma viremia (the eclipse phase) and during the viral load ramp-up phase of acute HIV-I infection are critical to understanding why immune responses do not occur earlier after HIV-I transmission, and to define what a successful vaccine must overcome to extinguish HIV-I.
In the study described below, the hypothesis is raised that, in addition to gut CD4 T cell loss, delay in HIV-I protective immune responses early on after HIV-I transmission may involve the production of elevated levels of immunosuppressive moieties such as TRAIL, TNFR2 and Fas ligand as well as plasma mi croparticles. If elevations in immunosuppressive molecules, coupled with early CD4+ T cell death, occur early on after HIV-I transmission, then this would define a protected time for HIV-I to replicate while anti-HIV-1 T or B cell responses were suppressed.
To study the eclipse and early viral load ramp-up phases of acute HIV-I infection, archived plasma of plasma donors with samples available before, during, and after HIV-I viral load ramp-up have been used (Fiebig et al (AIDS 17:1871-1879 (2003)). An initial burst of soluble TRAIL was found in plasma soon after the appearance of HIV-I in plasma, corresponding to ~17 days following transmission. Also observed were later elevated plasma TNFR2, Fas ligand and plasma microparticles (MP) levels around the peak of plasma VL. These data implicate TRAIL as an early mediator of cell death in acute HIV-I infection, and demonstrate a narrow window of opportunity in which a HIV-I vaccine must extinguish the transmitted virus.
................
While the average peak HIV-I VL level was 1,421,628 copies/ml, the
15 average peak of total MPs was 606,881,733/ml. Thus, there was an average of 427-fold more MP than virions present in plasma at their peaks during acute HIV- 1 infection.
.................
MP-Induced B Cell Suppression In Vitro. While plasma MPs have potent known suppressive effects on macrophages and DCs (Hoffmann et al, J. Immunol. 174:1393-1404 (2005), Huynh et al, J. Clin. Invest. 109:41-50 (2002)), only one study has suggested MP may inhibit B cell activation (Koppler et al, Eur. J. Immunol. 36:648-660 (2006)). There was particular interest in MP effects on human memory B cell activation, since what is desired is a rapid virus-induced memory B cell response after transmission. To determine if PBMC-derived or tonsil leukocyte-derived MP could be suppressive for memory B cell activation, a memory B cell Ig induction assay was used using pokeweed mitogen (PWM) + class B oCpG (Crotty et al, J. Immunol. Methods 286:11 1-122 (2004)). The addition of MP in PWM-stimulated tonsil cell cultures reduced total IgG and IgA production by 70.8 % +/- SEM for IgG (p=0.0064) and 94.2% +/- SEM for IgA (p=0.00004) (Fig. 24A); B cell suppression by MPs was dose-dependent (Fig. 24B). Similar results were observed when MP were generated from autologous tonsil leukocytes or from the Jurkat T cell line (data not shown).
In summary, a major finding in this study is the early appearance of a peak of TRAIL at 17 days of transmission in plasma donors, and implies the TRAIL/DR5 in a key pathway in HIV-I induced cell death immediately following transmission. An IFN-α, TRAIL, DR5 pathway of CD4+ T cell apoptosis has been proposed for chronic HIV-I infection based on in vitro studies and on studies in HIV-1+ progressor tonsillar tissues (Lum et al, J. Virol. 75:11128- 11136 (2001), Herbeuval et al, Clin. Immunol. 123:121-128 (2007), Herbeuvel et al, Blood 106:3524-3531 (2005)). CD4+ T cells in infected subjects are more sensitive to TRAIL-mediated apoptosis than are CD4+ T cells from uninfected subjects due to upregulated TRAIL receptor DR5 (Lum et al, J. Virol. 75:11128- 11136 (2001), Herbeuval et al, Clin. Immunol. 123:121-128 (2007), Herbeuvel et al, Blood 106:3524-3531 (2005), Jeremias et al, Eur. J. Immunol. 28:143-152
(1998)). In vitro, HIV-I gpl20 (Herveuval et al, Blood 105:2458-2464 (2005)) induces monocyte and plasmacytoid dendritic cell IFN-α, which in turn induces CD4+ T cell and monocyte/macrophage TRAIL (Lum et al, J. Virol. 75:11128- 11136 (2001), Herbeuval et al, Clin. Immunol. 123:121-128 (2007), Herbeuvel et al, Blood 106:3524-3531 (2005)). HIV-I Tat has also been reported to induce TRAIL as a mechanism of bystander killing of CD4+ T cells (Yang et al, J. Virol. 77:6700-6708 (2003)).
An important question is why do plasma TRAIL levels peak earlier after HIV-I transmission than do plasma Fas ligand, TNFR2 and MP? Plasma elevations of TRAIL, Fas ligand and TNFR2 occur in chronic HIV-I, and can be induced by immune cell activation, cell death, or both (Herveuval et al, Blood 105:2458-2464 (2005), Aukrust et al, J. Infect. Dis. 169:420-424 (1994), Hober et al, Infection 24:213-217 (1996), Hosaka et al, J. Infect. Dis. 178:1030-1039 (1998)). Stacy et al (Stacey et al and the NIAID Centre for HIV/ AIDS Vaccine Immunology. Elevations in plasma levels of innate cytokines prior to the peak in plasma viremia in acute HIV-I infection (2007)) have found a burst of IFN-α in the same plasma donors that coincides with the timing of the TRAIL peak seen in this study . Thus, the plasma TRAIL peak that precedes the VL plasma peak may be due either to early apoptosis, but may also result from immune activation and pDC production of IFN-αin response to rising VL. It is hypothesized that the later appearance of elevated plasma Fas ligand, TNFR2 and microparticles maybe the result of, or in response to, massive cell death, as this peak comes at an analogous time to the cell death peak documented in experimental SIV infection in rhesus macaques (Veazey et al, Science 280:427-431 (1998), Haase, Nat. Rev. Immunol. 5:783-792 (2005), Li et al, Nature 434:1148-1152 (2005), Mattapallil et al, Nature 434:1093-1097 (2005)).
Veazey (Mattapallil et al, Nature 434:1093-1097 (2005)) noted the onset of CD4+ gut T cell loss as early as 7 days after SIV infection . In humans,
Guadalupe et al (J. Virol. 77:11708-11717 (2003), Mehandru et al, J. Exp. Med. 200:761-770 (2004) and Mehandru and colleagues (Brenchley et al, J. Exp. Med. 200:749-759 (2004)) have studied 2, 1 and 9 patients, respectively, during the first month of HIV-I infection and found depletion of gut CD4+ T cells. The eclipse phase of HIV-I infection is the time from transmission until the appearance of plasma viremia, and is estimated to be 10 days with a range of 7-21 days (Clark et al, N. Engl. J. Med. 324:954-960 (1991), Gaines et al, BMJ 297:1363-1368 (1988), Littl et al, J. Exp. Med. 190:841-850 (1999), Schacker et al, Ann. Intern. Med. 125:257-264 (1996)). The time from appearance of HIV-I viremia until the first antibody response and symptomatic HIV-I infection (and therefore establishment of the latent pool) is approximately 14 days (Cooper et al, J. Infect. Dis. 155:1113-1118 (1987), Daar et al, N. Engl. J. med. 324:961-964 (1991), Gaines et al, Lancet 1 :1249-1253 (1987)). Thus, the maximal window of opportunity for preventive HIV-I vaccine efficacy without cell death-induced immune suppression is approximately 24 days. With mediators of apoptosis and immune suppression present as early as day 17 following transmission (10 days average eclipse phase + onset of TRAIL 7 days after T0), the window of opportunity is narrowed to ~14-17 days.
The presence of TRAIL, TNFR2 and elevated MP during this early period of acute HIV-I infection suggests at least four potential mechanisms of immunosuppression. First, direct HIV-I 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 ((Guadalupe et al, J. Virol. 77:11708-11717 (2003), Brenchley et al, J. Exp. Med. 200:749-759 (2004), Mehandru et al, J. Exp. Med. 200:761-770 (2004), Fiebig et al, AIDS 17:1871-1879 (2003)). Second, in uninfected CD4+ T cells, TRAIL induces bystander killing ((Lum et al, J. Virol. 75:11128-1 1136 (2001), Herbeuval et al, Clin. Immunol. 123:121-128 (2007), Herveuval et al, Blood 105:2458-2464 (2005)). In this regard, Miura et al (J. Exp.
Med. 193:651-660 (2001)) have shown that administration of an anti-TRAIL mAb in HIV-I infected hu-PBL-NOD-SCID mice markedly reduces CD4+ T cell apoptosis.
Third, suppression of immune responses can be mediated by T cell MP (Huang et al, J. Immunol. 177:2304-1313 (2006), Distler et al, Arth. Rheum.
52:33337-3348 (2005), Krysko et al, Apoptosis 11:1709-1726 (2006)). CXCR4+ and CCR5+ MP can transfer co-receptors to co-receptor negative cells making them susceptible to HIV-I (Mack et al, Nat. Med. 6:769-775 (2000), Rozmyslowicz et al, AIDS 17:33-42 (2003)). Phagocytosis of MP by macrophages releases TGF- βprostaglandin E2 and IL-10 that can inhibit antigen-specific T and B cell responses (Huang et al, J. Immunol. 177:2304-1313 (2006), Hoffmann et al, J. Immunol. 174:1393-1404 (2005), Huynh et al, J. Clin. Invest. 109:41-50 (2002)). In this regard, Estes et al (J. Infect. Dis. 193:703-712 (2006)). have demonstrated dramatic increases in lymph node TGF-β and IL-10 on day 12 following SIV infection. Importantly, it has been directly shown that PBMC and tonsillar cell MP can directly inhibit memory B cell activation (Fig. 24).
Finally, both Fas ligand and TRAIL are incorporated into MP (Huynh et al, J. Clin. Invest. 109:41-50 (2002), Koppler et al, Eur. J. Immunol. 36:648-660 (2006), Crotty et al, J. Immunol. Methods 286: 111-122 (2004)). Fas ligand expressing MP can directly induce apoptosis in nearby cells (Huang et al, J. Immunol. 177:2304-1313 (2006), Jodo et al, J. Biol. Chem. 276:39938-39944 (2001), Monleon et al, J. Immunol. 167:6736-6744 (2001)) activated T cells can be the target of Fas ligand mediated proapoptotic microvesicles (Monleon et al, J. Immunol. 167:6736-6744 (2001)). Salvato et al (Clinical and Developmental Immunology (2008)) 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.
Thus, the production of high levels of biologically active plasma mediators and byproducts of cell death during the first two to three weeks of HIV- 1 transmission raises the notion that the window of opportunity for a preventive vaccine to work may be shorter than previously thought, ie within the first 14-17 days of transmission, placing considerable constraints on the time available for development of robust anti-HIV-1 immunity following transmission. Preventive vaccine candidates may need to target HIV-I molecules that induce cell death and be designed to induce protective immune responses to HIV-I that will either be at maximum inhibitory levels at the time of transmission, or be boosted within hours to days as a secondary immune response to extinguish HIV-I before HIV-I- induced immunosuppression occurs.
Inhibition of cell death and immunosuppressive MP mediated effects by a vaccine for HIV or other infectious agents may be important as well. This could be accomplished, for example, by an HIV vaccine component inducing anti-lipid antibodies or antibodies against other components of microparticles to facilitate clearance of microparticles and/or to block microparticle toxic effects.
Another use of the data herein is as a rationale for the treatment of HIV-I. For example, antibodies against TNFR or TNF-α, antiphosphatidylserine antibodies or other inhibitors of cell death (Fas-Fc as an inhibitor of FAS-FAS ligand interactions and DR5-Fc as an inhibitor of TRAIL DR5 interactions) can be used to inhibit cell death in HIV as a therapy.
-----------
j
Jake, spell it "Tarvicin".....
- not that that's the important part...
BTW - "Bavituximab" was also in his other recent US patent application.
- not that that's the important part...
That's a minor thing compared to Haynes' overall hypothesis that is emerging!
WHAT IS IMPORTANT is that he is so convinced that PS IS THE CULPRIT THAT MUST BE DEALT WITH in both HIV vaccine design and HIV therapeutics!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
LOL - AND he goes on to touch on some other diseases that also exploit overwhelming abundance of PS-exposing microparticles to facilitate their pathogenesis!
My god man, can't you see what's happening here?
j
WOW!!! JULY 24 - HAYNES NEW WIPO PATENT APPLICATION -----------
http://www.wipo.int/pctdb/en/fetch.jsp?SEARCH_IA=US2008000412&DBSELECT=PCT&C=10&TOTAL=37&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41223109-KEY&QUERY=%28IN%2F%22haynes+barton%22%29+&RESULT=1&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=1444025&IA=US2008000412&LANG=ENG&DISPLAY=DESC
ENJOY!!!!!!!!!!!!!
IT BE HAPPENIN :)
j
djohn,
For some reason I can't get to the article with the Haynes quote you refer to.
Could you paste the text into a post?
tia
but since we're on the topic,
Here is the last Haynes quote in the article I had posted -
"These and other studies that recently revealed more about the singular nature of HIV-1 have given us valuable information that is helping us move closer to establishing a basic science foundation that can lead to novel technologies for vaccine design, Haynes said. Haynes. "It is becoming clearer why we have failed in our efforts to date, and what we need to confront to succeed in the future."
http://www.sciencedaily.com/releases/2008/07/080718092221.htm
--------------
And of course, the paper that the article is referring to, and the new cutting-edge work that Haynes is talking about, is how
an overwhelming amount of exposed PS must be dealt with for any future vaccine to work!
it's happening,
j
Big T ---------
"the Duke publications will introduce a global paradigm shift"
Yes, they will, period.
"As Jazz keeps pointing out, Barton Haynes would not have filed a patent that relies heavily on anti-PS mabs to clean up apoptotic debris unless he already had strong data on the efficacy of anti-PS mabs to do just that."
Again- yes, of course. I think we'll hear all about it, and yes, it will be seen as a paradigm shift, (for those who understand the current/(previous!) immunological/immunotherapeutic paradigm! LOL :)
"Since PPHM and Chen are the only ones making anti-PS mabs, sounds like Bavi is going to be a centerpiece of the government’s new approach to HIV as well as hemorrhagic fever."
Well, the various therapeutic anti-PS mabs are ALL Peregrine's.
Whether they're from Thorpe, Avanir, Schroit, Chen, Biotecnol, Affitech, etc.,
they're all Peregrine's!
and THAT'S what will be talked about - IN DETAIL - in the upcoming "Duke paper(s), - PEREGRINE'S anti-PS mabs.
- and their promise in treating HIV (and other viral) infections.
Hey, JMHO,
j
HIV Conquers Immune System Faster Than Previously Realized
- thanks to freethemice for the find-
This is a great layman's summary of the Gasper-Smith / Haynes paper that I've been posting. The work showed how PS-exposing microparticles shut down immune responses, and that exposed PS must be dealt with for any hope of a successul HIV vaccine. Basically, the new research shows how any successful HIV therapy or vaccine MUST deal with the abundant PS-exposing apoptotic debris, in the form of microparticles, which ramps up significantly- right before the viral count ramps up significantly.... (which is understandable to any who have been reading my posts and "5 requird reading papers" about how PS is immunosuppressive, which Haynes and Gates/CHAVI are now, as evidenced by their recent papers and patent applications which cite the same papers, fully in agrement with). This led to the Haynes patent application I posted which specifically mentions induction of anti-PS abs as an essential component of future successful HIV vaccines. Most interesting, is the recent Haynes patent application that discusses the use of anti-PS mabs as HIV THERAPY....
I expect to read all about WHY Haynes is suggesting anti-PS mabs as promising HIV therapy - soon! :)
j
---------------------------
HIV Conquers Immune System Faster Than Previously Realized
http://www.sciencedaily.com/releases/2008/07/080718092221.htm
ScienceDaily (July 19, 2008) — New research into the earliest events occurring immediately upon infection with HIV-I shows that the virus deals a stunning blow to the immune system earlier than was previously understood. According to scientists at Duke University Medical Center, this suggests the window of opportunity for successful intervention may be only a matter of days -- not weeks -- after transmission, as researchers had previously believed.
Appearing in the August issue of the Journal of Virology, the finding may make the challenge of designing an effective HIV/AIDS vaccine appear daunting. But researchers say the study has also yielded a blueprint for what a successful vaccine should like, and moreover, when such a vaccine would need to work.
Until now, scientists believed that the window of opportunity to intervene in the process of HIV-1 infection lay in the three to four weeks between transmission and the development of an established pool of infected CD4 T cells. HIV-1 cripples the immune system by invading and killing CD4 T cells, key infection-fighters in the body.
"But this new study shows that HIV-I does a lot of damage to the immune system very early in that time frame, and now we feel that the opportunity to intervene most effectively may range from about five to seven days after infection," said Barton Haynes, M.D., the senior author of the study and director of the Center for HIV/AIDS Vaccine Immunology (CHAVI) at Duke University Medical Center.
Haynes said the findings suggest that an optimal vaccine strategy would have to pack a double punch: First, establishing as much immunity as possible before infection, much as classic vaccines do, and then following a few days later with a mechanism to provoke a strong, secondary, broad-based antibody response. "Vaccine candidates to date have pretty much followed a single strategy. Now we know that we need to activate multiple arms of the immune system and we have a better idea of when to do it."
The conclusion comes from the study of 30 people who were newly-infected with HIV-1. Plasma from these individuals was sampled every three days for several months -- before, during, and after the "ramp-up" phase of infection, when HIV-1 is multiplying rapidly and heading toward its peak viral load. In measuring the levels of four products of CD4 T cell death during this period in these samples, they were able to track and establish a timetable of the virus's destructive path.
The four byproducts of CD4 T cell death include TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), Fas ligand, TNF receptor type 2 and plasma microparticles, tiny bits of cell membrane that are broken up and left floating around in the plasma when the cell dies and breaks apart.
The researchers found that TRAIL levels increased significantly a full week (7.2. days) before peak viral load, which is approximately 17 days after HIV-1 transmission, suggesting that during the earliest period of infection, called the eclipse phase, TRAIL may actually initiate or hasten HIV-1's destruction of CD4 T cells. In contrast, they found that the levels of the other three cell death products were most significantly elevated during peak viral load.
"What this demonstrates is that significant T cell death is occurring much earlier during this period than we previously believed, and that TRAIL itself may be a co-conspirator in enhancing cell death," Haynes said. "This leads us to believe that the time frame for successful intervention has to move even close to the point of infection."
Researchers also examined the effects of cell death products upon B cells, another arm of the immune system responsible for the creation of antibodies. Previous studies have shown that the antibody response to HIV-1 is "too little, too late" -- appearing after the virus has peaked and after the reservoir of infected T cells has already been established.
Through a series of in vitro laboratory experiments with peripheral blood cells, scientists found that microparticles suppressed levels of IgG and IgA, two classes of antibodies that normally would protect a person against infection. "This is important because many scientists believe that a fast-acting memory B cell response as well as a T cell response will be necessary to fight HIV-1" said Nancy Gasper-Smith, PhD, the lead author of the study.
Daniel Douek, M.D., PhD, chief of the Human Immunology Section of the National Institutes of Health, said the study sheds new light on key events in the earliest phase of infection. "The cohort is a gem. It is clear from the raised levels of TRAIL that the body senses the virus before plasma viral loads have peaked. This suggests that the virus begins to cause damage in ways that may be unrelated to the well-described massive depletion of gut CD4 T cells that becomes apparent around peak viral load. For clinical practice, this means the window of opportunity in which antiviral therapies and vaccines must act is becoming ever narrower."
"These and other studies that recently revealed more about the singular nature of HIV-1 have given us valuable information that is helping us move closer to establishing a basic science foundation that can lead to novel technologies for vaccine design, Haynes said. Haynes. "It is becoming clearer why we have failed in our efforts to date, and what we need to confront to succeed in the future."
The study was supported by grants from the National Institutes of Health
Colleagues from Duke who contributed to the research include Deanna Crossman, John Whitesides, Nadia Mensali, Janet Ottinger, Steven Plonk, M. Anthony Moody, Guido Ferrari, Kent Weinhold, Sara Miller and Thomas Denny. Additional co-authors are David Pisetsky and Charles Reich, from the Durham Veterans Administration Hospital; Li Qin and Stephen Self, from Fred Hutchinson Cancer Research Center and the Statistical Center for HIV-AIDS Research and Prevention; George Shaw from the
Journal reference:
1. Gasper-Smith et al. Induction of Plasma (TRAIL), TNFR-2, Fas Ligand, and Plasma Microparticles after Human Immunodeficiency Virus Type 1 (HIV-1) Transmission: Implications for HIV-1 Vaccine Design. Journal of Virology, 2008; 82 (15): 7700 DOI: 10.1128/JVI.00605-08
Adapted from materials provided by Duke University Medical Center.