Replies to post #20639 on Avid Bioservices Inc (CDMO)

[Bulldog010]

Jazz.

Don't take this the wrong way...So far, you seem like a good guy..like CJ. YOU want the best, I understand that.

But, seriously...With all that potential you point to. Why are we where we are? Why is this stock valued less than a bankrupt construction company?

Why can't they do any deals with Pharma? (When lesser technologies are being inked for big dollars) Why are trials stalling/late etc? Why have compounds been let to decay (You are fairly new here...at one time LYM and then Cotara.. were talked of just like Bavi is today.)

Those of us holding this stock..we already see the potential in the technology. That's why we continue to hold this stock..it's our nugget, if you will. It's the one thing we hold on to...to make the years of missing out on other opportunities seem less painful. The tech is the one thing that can still save this.

You or anyone else can't possibly get upset with others, who are just as passionate about the tech as you are..and have been for years..guy's like Terry...for 15 years! That are upset with this company 5 mgt teams past..for screwin' the pooch.

We used to be the one's posting the info on the company. Terry,Investorcg,Myself,Katie,Arub4me,Webby,CJ,Larryxsc,Jasm5 etc. etc. etc. WE all knew this company had great science.

We don't need daily fighting...What we need is some understanding. That posters who have been here a long time ago..that felt like you do know..even stronger about the compounds are fed up with lousy...pathetic leadership.

These compounds are life changing...problem is...they are under the control of absolute pigs.

All of us here want this company/tech to prevail. We will all benefit. investors,patients etc. But, to let these great ideas just die off...is the fault of management. Management alone.

Even pumpers like EZ have at the very root..good intentions.

Everyone just needs to realize we all have the same goal..we are just different people with different amounts of time having to put up with the mismanagement of this life giving technology.

Volgoat is a great example. We fought early on..over is incredible exhuberance. And that is fine...what some of these new guys need to realize is that we had that passion..but, years of watching it get wasted by fools is taxing.

Volgoat now is starting to see that happening. Is/was he right about the tech..I believe so. but, what caused the problems is the back biting of posters...who passed that phase and are now just upset.

WE are all still here because we believe in this science. We just need better people managing it..or the people that are to wake the hell up.

Sorry for the rant. I hope it's at least readable..but, I had to vent (these latest options are yet another stab in the gut). Off the soapbox and offline.

Have a good day...that goes to everyone.

[ysfalconeer]

Thank you jazz! Your post is required reading for anyone who needs an explanation in easy to understand layman terms how the science works and who is behind it. I hope you finish this series of postings as soon as possible and not waste your precious time responding to the hopelessly lame and uninformed whose sole mission seems to be to try to discredit the company rather than the science.
I find it interesting that every major basher I have seen recently on this board has been out in force since Friday. To those poor souls who don't believe or don't own shares your true colors are showing now. Yes there is still risk of course, but the odds are starting to shift in favor of the longs.

Once again, thanks for making my weekend!

[jazzbeerman]

Leishmania Exploits Exposed PS.. #1



parasitic infection through "apoptotic mimicry"....



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The Journal of Immunology, 2006, 176: 1834-1839
.

Mimicry of Apoptotic Cells by Exposing Phosphatidylserine Participates in the Establishment of Amastigotes of Leishmania (L) amazonensis in Mammalian Hosts


João L. M. Wanderley Maria E. C. Moreira*, Aline Benjamin*, Adriana C. Bonomo*, and Marcello A. Barcinski2,


Signaling through exposed phosphatidylserine (PS) is fundamental for the TGFbeta1-dependent, noninflammatory phagocytosis of apoptotic cells. This same mechanism operates in the internalization of amastigotes of Leishmania (L) amazonensis (L(L)a) in a process quoted as apoptotic mimicry. Now we show that the host modulates PS exposure by the amastigotes and, as a consequence, BALB/c mice-derived amastigotes expose significantly more PS than those derived from C57BL/6 mice. Due to this difference in the density of surface PS molecules, the former are significantly more infective than the latter, both in vivo, in F1 (BALB/c x C57BL/6) mice, and in vitro, in thioglycollate-derived macrophages from this same mouse strain. PS exposure increases with progression of the lesion and reaches its maximum value in amastigotes obtained at the time point when the lesion in C57BL/6 mice begins to decrease in size and the lesions in BALB/c mice are still growing in size. Synthesis of active TGFbeta1, induction of IL-10 message, and inhibition of NO synthesis correlate with the amount of surface PS displayed by viable (propidium iodide-negative) infective amastigote. Furthermore, we also show that, similar to what happens with apoptotic cells, amastigotes of L(L)a are internalized by macropinocytosis. This mechanism of internalization is consistent with the large phagolysosomes characteristic of L(L)a infection. The intensity of macrophage macropinocytic activity is dependent on the amount of surface PS displayed by the infecting amastigote.




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Evolution has favored pathogenesis that resembles apoptosis,

j


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'Visceral Leishmaniasis, VL, also known as kala azar ("black fever"), is a fatal disease transmitted by sand flies, which spread leishmania parasites that attack internal organs. VL is endemic in 62 countries, and the number of new VL cases per year is estimated at 500,000. With the exception of malaria, VL kills more people than any other parasitic disease.'



PS: OT: people might be interested in googling:

"Gates Foundation" Neglected Diseases
for more information.


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nice pic from a paper I'll post later -





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j

[jazzbeerman]

Leishmania Exploits Exposed PS.. #2



Braz J Med Biol Res. 2005 Jun;38(6):807-12. Epub 2005 Jun 1.

Apoptotic mimicry: an altruistic behavior in host/Leishmania interplay.

Wanderley JL, Benjamin A, Real F, Bonomo A, Moreira ME, Barcinski MA.

Instituto Nacional de Cancer, 20231-050 Rio de Janeiro, RJ, Brasil.



a fascinating read this one!

j



Apoptotic mimicry: an altruistic behavior in host/Leishmania interplay.



Apoptosis is the most common phenotype observed when cells die through programmed cell death. The morphologic and biochemical changes that characterize apoptotic cells depend on the activation of a diverse set of genes. Apoptosis is essential for multicellular organisms since their development and homeostasis are dependent on extensive cell renewal. In fact, there is strong evidence for the correlation between the emergence of multicellular organisms and apoptosis during evolution. On the other hand, no obvious advantages can be envisaged for unicellular organisms to carry the complex machinery required for programmed cell death. However, accumulating evidence shows that free-living and parasitic protozoa as well as yeasts display apoptotic markers. This phenomenon has been related to altruistic behavior, when a subpopulation of protozoa or yeasts dies by apoptosis, with clear benefits for the entire population. Recently, phosphatidylserine (PS) exposure and its recognition by a specific receptor (PSR) were implicated in the infectivity of amastigote forms of Leishmania, an obligatory vertebrate intramacrophagic parasite, showing for the first time that unicellular organisms use apoptotic features for the establishment and/or maintenance of infection. Here we focus on PS exposure in the outer leaflet of the plasma membrane--an early hallmark of apoptosis--and how it modulates the inflammatory activity of phagocytic cells. We also discuss the possible mechanisms by which PS exposure can define Leishmania survival inside host cells and the evolutionary implications of apoptosis at the unicellular level.





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Evolution has favored pathogenesis that resembles apoptosis,

j


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Leishmaniasis Info -

http://www.sbri.org/diseases/leishmaniasis.asp


Impact

Leishmaniasis is a parasitic disease transmitted by the bite of a sandfly that is infected with Leishmania parasites. Currently 350 million people in 88 countries around the world are threatened, and 12 million people are affected by leishmaniasis. Of the 1.5 – 2 million new cases of leishmaniasis estimated to occur annually, most occur in the tropics and subtropics, including the Middle East. In 2002, leishmaniasis reached epidemic levels in Afghanistan, with the World Health Organization calling for more funding and research for the disease. Leishmania/HIV co-infection is emerging as a serious new disease and it is increasingly frequent. It is considered a threat in southwestern Europe, such as Spain, Italy, France, and Portugal.


Symptoms

With the bite of an infected sandfly, Leishmania parasites are passed from one infected animal or human to others. Leishmaniasis is a spectrum of diseases, each distinctly manifested and all with potentially devastating consequences – disfigurement, damage to internal organs, death. Depending on the species of the infecting parasite, the spleen, liver, bone marrow, mucous membranes, and/or skin may be attacked. Leishmania donovani, the most dangerous of these, causes Kala azar, or visceral leishmaniasis, characterized by fever, severe weight loss and anemia. If left untreated, visceral leishmaniasis can lead to death.





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j

[jazzbeerman]

Leishmania Exploits Exposed PS.. #3



Curr Biol. 2001 Nov 27;11(23):1870-3.

Apoptotic mimicry by an obligate intracellular parasite downregulates macrophage microbicidal activity.

de Freitas Balanco JM, Moreira ME, Bonomo A, Bozza PT, Amarante-Mendes G, Pirmez C, Barcinski MA.

Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo 05508-900, Brazil.



Programmed cell death by apoptosis of unnecessary or potentially harmful cells is clearly beneficial to multicellular organisms. Proper functioning of such a program demands that the removal of dying cells proceed without an inflammatory reaction. Phosphatidylserine (PS) is one of the ligands displayed by apoptotic cells that participates in their noninflammatory removal when recognized by neighboring phagocytes. PS ligation induces the release of transforming growth factor-beta (TGF-beta), an antiinflammatory cytokine that mediates the suppression of macrophage-mediated inflammation. In Hydra vulgaris, an organism that stands at the base of metazoan evolution, the selective advantage provided by apoptosis lies in the fact that Hydra can survive recycling apoptotic cells by phagocytosis. In unicellular organisms, it has been proposed that altruistic death benefits clonal populations of yeasts and trypanosomatids. Now we show that advantageous features of the apoptotic process can operate without death as the necessary outcome. Leishmania spp are able to evade the killing activity of phagocytes and establish themselves as obligate intracellular parasites. Amastigotes, responsible for disease propagation, similar to apoptotic cells, inhibit macrophage activity by exposing PS. Exposed PS participates in amastigote internalization. Recognition of this moiety by macrophages induces TGF-beta secretion and IL-10 synthesis, inhibits NO production, and increases susceptibility to intracellular leishmanial growth.





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j

[jazzbeerman]

PS & Leishmania - THE paper.....



- detailed experiments proving that exposed PS provides the advantage to facilitate infection, and proliferation, via shutting off the innate response.



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snips -


2.3 Phosphatidylserine expressing L. major promastigotes show “apoptosis-
like” morphology

3.3. Phosphatidylserine expression of L. major promastigotes determines the
amount of TGF-β1 and TNF-α release by PMN

4.1 Phosphatidylserine expression on L. major promastigotes supports the infection rate of macrophages

4.2 Annexin-V pre-treatment of stationary phase L. major promastigotes blocks TGF-β1 release and enhances TNF-α secretion by macrophages

5. Phosphatidylserine expressing apoptotic L. major promastigotes provide survival advantages for intracellular viable parasites in PMN

6.1 Phosphatidylserine expressing L. major support the disease development
in vivo



"Taken together, the results of this thesis suggest that apoptotic L. major promastigotes, via the induction of anti-inflammatory and phagocyte deactivating cytokines, assist the viable parasites to survive inside phagocytes. Apoptotic parasites “silence” the immune response of PMN and enable the survival of non-apoptotic parasites to establish a productive infection. "





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Evolution has favored pathogenesis that resembles apoptosis,

j



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Leishmania major promastigotes use phosphatidylserine
for silencing of polymorphonuclear neutrophils



http://www.students.informatik.uni-luebeck.de/zhb/ediss148.pdf


a few excerpts below,

(starting on p. 50, of the 120 page paper)


j



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Results ------------------------------------------------------------------------------------------------









1.2 Anti-phosphatidylserine antibody also detects phosphatidylserine on the surface of L. major promastigotes

In addition to staining with AV, another possibility to assess PS expression is the use of a PS specific monoclonal antibody. As a positive control for PS expressing cells, apoptotic polymorphonuclear neutrophilic granulocytes (PMN) were used. Apoptotic PMN were stained with both AV Fluos and anti-PS antibody. As depicted in fig. 8, staining with AV Fluos revealed a ratio of 83.9% PS expressing apoptotic PMN. PS staining using a monoclonal anti-PS antibody demonstrated a ratio of 64.7% PS expressing apoptotic PMN. Similar staining experiments as for apoptotic PMN were performed with L. major promastigotes. As demonstrated in fig. 8, staining with AV Fluos detected a ratio of 46.3% PS expressing parasites. PS staining using the anti- PS antibody revealed a ratio of 35.7% PS expressing L. major promastigotes.







2.3 Phosphatidylserine expressing L. major promastigotes show “apoptosis-
like” morphology

To detect “apoptosis-like” features of PS expressing L. major promastigotes, a closer look was taken on the morphology of MACS purified PS negative and PS positive parasites using electron microscopy. The presented micrographs revealed that PS negative parasites have a granularity and a nuclear morphology typical for viable cells. They also showed fully intact tubular membrane structures. On the contrary, PS positive L. major revealed less granularity and the nuclei showed a rounded shape. Furthermore, the diameters of PS positive parasites appeared smaller in comparison to the PS negative parasites. The membrane of PS positive L. major showed a disturbed tubular organisation (fig. 17).












3.3. Phosphatidylserine expression of L. major promastigotes determines the
amount of TGF-β1 and TNF-α release by PMN

Leishmanial PS does not affect the oxidative burst in PMN (see results, 3.2), but PS recognition by MФ regulates the production of anti-inflammatory TGF-β1 and pro-inflammatory TNF-α (Savill et al., 1993; Voll et al., 1997; Fadok et al., 1998, 2001; Serinkan et al., 2005). According to these data, the silencing effect of L. major promastigotes in PMN was suggested to be achieved by the regulation of TGF-β1 and TNF-α. Therefore, it was investigated whether PS expression on L. major influences the cytokine production of PMN. Freshly isolated PMN were coincubated either with stationary phase L. major (70% PS+), MACS separated L. major samples (Lm PS- (7% PS+), Lm PS+ (95% PS+)), or PMN were incubated in medium alone. Additionally, PMN were also stimulated with stationary phase promastigotes after pre-treatment with annexin-V (AV). The supernatants of these cocultures were analysed for their content of TGF-β1 and TNF-α. The release of TGF-β1 by PMN increased correspondingly to the ratio of PS expressing L. major promastigotes (fig. 20A). The secretion of TNF-α showed an inverse correlation to the ratio of PS expressing L. major (fig. 20B). In comparison to the PMN coincubation with stationary promastigotes (70% PS+), a parasite pre-treatment with AV significantly reduced the release of TGF-β1 from 349 ± 46.8 pg/ml down to 174 ± 68.3 pg/ml (fig. 20A). In contrast, AV pre-treatment of the parasites increased the TNF-α release by PMN from 23 ± 7.5 pg/ml up to 32 ± 5.5 pg/ml (fig. 20B).












4. Coincubation of phosphatidylserine expressing L. major promastigotes with macrophages

In order to investigate the ability of PS expressing L. major promastigotes to silence the infection of macrophages (MФ), MФ were coincubated with stationary phase L. major promastigotes either untreated or pre-treated with annexin-V (AV). For coincubation experiments and cytokine measurements similar experimental setups were used as described before for PMN (see 3.1 and 3.3). Again, the term “infection rate” is defined as the number of intracellularly present parasites.






4.1 Phosphatidylserine expression on L. major promastigotes supports the infection rate of macrophages

As depicted in fig. 22, MФ that were coincubated with stationary phase L. major promastigotes showed an infection rate of 69 ± 9.6%. Blocking of PS with AV resulted in a significant reduction of the infection rate down to 43 ± 10.7%.











4.2 Annexin-V pre-treatment of stationary phase L. major promastigotes blocks TGF-β1 release and enhances TNF-α secretion by macrophages

Coincubation of MФ with stationary phase L. major promastigotes induced a TGF-β1 release of 545 ± 53.4 pg/ml. Pre-treatment of the parastites with AV reduced the TGF-β1 amount in the supernatants down to 328 ± 89.5 pg/ml (fig. 23A).
The measurement of TNF-α revealed that stationary phase L. major promastigotes induced a TNF-α secretion of 55 ± 46.2 pg/ml, whereas the amount was increased up to 144 ± 34.4 pg/ml if PS was blocked on the parasites (fig. 23B).











5. Phosphatidylserine expressing apoptotic L. major promastigotes provide survival advantages for intracellular viable parasites in PMN

PS expressing apoptotic L. major promastigotes were shown to exert immunosilencing properties in PMN due to an up-regulation of TGF-β1 and a down-regulation of TNF-α (see results 3.3). These findings led to the suggestion that intracellularly viable parasites might profit from the anti-inflammatory milieu in terms of a facilitation of intracellular parasite survival. To test this hypothesis, PMN were coincubated with equal numbers of either stationary phase L. major or PS depleted stationary phase promastigotes. After 3 h, extracellular parasites were washed away and infected PMN were cultured for 18 h and 42 h. The infection rate of PMN is indicated in fig. 24. Over the time period from 3 h, 18 h to 42 h after infection, the infection rate remained almost constant in PMN infected with stationary phase L.








To investigate how many PS expressing L. major promastigotes are needed for efficient maintenance of the infection rate and support of the parasite survival inside PMN over the time, parasite samples containing a ratio of 50%, 30% or 20% PS expressing L. major promastigotes were used for coincubation experiments. As depicted in fig. 26, a coincubation period from 18 h to 42 h led to a light reduction of the infection rate if PMN were coincubated with a parasite sample with a ratio of 50% PS expressing L. major (54% (18 h) → 51% (42 h)). In case of PMN infection with a parasite sample with a ratio of 30% PS expressing promastigotes the infection rate was reduced from 49% (18 h) to 43% (42 h). However, the strongest reduction of the infection rate was detected if PMN were coincubated with a parasite sample containing 20% PS expressing promastigotes (51% (18 h) → 35% (42 h)).









Using end point titration experiments, the number of intracellularly surviving parasites was determined over a time period from 18 h to 42 h. As depicted in fig. 27, the number of viable parasites was maintained if PMN were infected with a parasite sample containing 50% PS expressing L. major (393 (18 h) → 387 (42 h) Lm/1000 PMN). PMN coincubation with a parasite sample containing a ratio of 30% PS expressing L. major showed a decrease of the parasite number from 392 Lm/1000 PMN (18 h) down to 330 Lm/1000 PMN (42 h). The strongest reduction of the viable parasite number was detected if PMN were infected with a parasite sample with a ratio of only 20% PS expressing L. major (392 (18 h) → 303 (42 h) Lm/1000 PMN).











6.1 Phosphatidylserine expressing L. major support the disease development
in vivo

Mouse infection experiments were carried out using 1 x 106 viable PS negative L. major supplemented with apoptotic PS positive parasites (Lm stat or Lm met) or 1 x 106 viable PS negative L. major (Lm stat PS- or Lm met PS-). The results demonstrated that the footpad swelling increased stronger if mice were infected with parasites containing a high ratio of PS expressing L. major. This is true for both stationary phase promastigotes (fig. 28) and purified metacyclic parasites (fig. 29).













IV Discussion

This study focussed on the identification of surface molecules on L. major promastigotes that are involved in the establishment of silent polymorphonuclear neutrophilic granulocytes (PMN) infection. In this context, the expression of the apoptotic “eat-me” signal phosphatidylserine (PS) on L. major and its influence on PMN infection was investigated. In addition, the membrane receptors on PMN that are involved in parasite uptake were studied.
It was demonstrated that L. major can express PS on their membrane. The ratio of PS expressing promastigotes increased during the culture period. In addition, stationary phase L. major promastigotes were demonstrated to enter apoptosis, as shown by TUNEL staining. TUNEL positivity increased parallel with the ratio of PS expressing parasites during the growth period from the logarithmic to the infective stationary phase. End point titration experiments demonstrated that PS expressing promastigotes do not grow in culture, thus PS expressing parasites are not viable. Moreover, electron microscopic analysis of PS expressing promastigotes revealed an “apoptosis-like” morphology, such as loss of granularity and cell shrinkage. Additionally, PS expressing L. major produced an anti-inflammatory milieu in PMN by an up-regulation of the TGF-β secretion and a down-regulation of the TNF-α release. The presence of PS expressing promastigotes promoted parasite survival inside PMN in vitro. Moreover, the presence of PS expressing parasites enhanced the disease development in vivo. In contrast, the lack of PS expressing promastigotes resulted in a reduced outcome or almost healed form of disease, which was dependent on the parasite number used for infection. The investigation of PMN receptors that can potentially interact with PS on L. major revealed three receptor candidates.



1. The role of phosphatidylserine in L. major infection

The data presented in this thesis demonstrate that PS is expressed on L. major promastigotes, which are the disease inducing form of this parasite. PS is detectable either with annexin-V (AV) in a calcium dependent manner, or using a mAb specific for PS. As shown in fig. 6, PS is arranged in “patches” on the membrane surface of L. major. Additionally, these patches were also detectable on the flagellum. These observations led to the question of the possible role of such a patchy arrangement of PS. From the literature it is known that especially membrane standing molecules need to cluster to display biological functionality (Simson and Ikonen, 1997; Brown, 2002; Zhang et al., 2005). Therefore, it is conceivable that PS might appear randomly on the parasites membrane. In order to achieve biological functionality, this initial PS externalisation might be followed by a PS reorganisation leading to an accumulation of PS in clusters. Thus, the detected patches could represent such PS clusters suggesting an active molecular organisation.
Amastigotes of the Leishmania species L. amazonensis were suggested to express PS as an active form of “apoptotic mimicry” to gain survival advantages (Freitas Balanco et al., 2001). Therefore, PS expressing L. major promastigotes were first expected to be viable. However, PS is conventionally seen as the hallmark for the onset of apoptosis. The process of apoptosis represents the transition from life to death. During this process PS flips to the outer leaflet of the cell membrane (Martin et al., 1995), but the exact stage of PS expressing cells, whether they are still alive, apoptotic or already dead, is not known. Hence, the question was addressed whether PS expressing promastigotes are viable or apoptotic. The ratio of PS expressing L. major promastigotes increased during the period of culture. As indicated in fig. 13, the ratio of TUNEL positive parasites also increases during the period of culture and correlates with the increased ratio of PS expressing promastigotes (see fig. 14). End point titration experiments demonstrated that PS expressing L. major are not viable. However, Freitas Balanco and colleagues did not demonstrate whether PS expressing L. amazonensis amastigotes are viable or apoptotic (Freitas Balanco et al., 2001). Other groups suggested the presence of an apoptotic machinery in Leishmania (Lee et al., 2002; Debrabant et al., 2003; Nguewa et al., 2004). The time dependent increase of promastigotes expressing the apoptotic marker PS suggests a general ongoing aging process of the parasites ending in apoptosis. The finding that the ratio of PS expressing purified metacyclic promastigotes also increases during the period of culture supports this conclusion. Moreover, the increase of TUNEL positive promastigotes during the culture period indicates that apoptosis is emerging in L. major promastigotes. The expression of PS as a sign for the onset of apoptosis is followed by DNA fragmentation resulting in TUNEL positivity as a sign for a later
stage of apoptosis. Thus, not all PS expressing promastigotes are already TUNEL positive. However, the suggestion that both of these apoptotic signs occur in the same promastigotes was confirmed by the inability of purified PS expressing parasites to grow in culture, whereas PS negative promastigote samples did increase in cell number. According to these data, it was shown for the first time that PS expression on L. major promastigotes is not “apoptotic mimicry”. Instead, it is clearly demonstrated that PS expressing promastigotes are indeed apoptotic. Whether PS expressing amastigotes also undergo apoptosis is not known but would certainly be interesting to investigate.
Observations using electron microscopy revealed granular structures in PS negative L. major and their nuclear morphology showed the typical shape of viable cells. In contrast, PS expressing promastigotes have lost its granular appearance and revealed a rounded and smaller nuclear morphology as well as a smaller cell size. Moreover, the electron microscopic pictures indicated that the tubular membrane structure of PS expressing L. major promastigotes appears disorganized, whereas the membrane showed intact morphology (see fig. 17).
Taken all these findings into account they point to the direction of an apoptotic process in L. major promastigotes. In this context, apoptotic processes such as the loss of membrane potential as well as chromatin condensation and caspase-like activity have been reported to occur in different Leishmania species (Lee et al., 2002; Debrabant et al., 2003; Nguewa et al., 2004). So far, a disorganized tubular membrane structure was not described as a sign for apoptosis, but it is conceivable that an organized tubular structure is needed for membrane functionalities, such as the maintenance of the membrane potential. Thus, the observed disorganisation might represent a pro-apoptotic sign. However, a closer look using electron microscopy showed that the membrane of PS expressing promastigotes appeared to be still intact. This feature might explain why a ratio of over 90 percent of stationary phase promastigotes was alive in viability staining experiments. Furthermore, the previously mentioned chromatin condensation and caspase-like activity occurring in Leishmania serve as a possible explanation for the detected formative changes and degradations in PS expressing promastigotes. In FACS analysis, if gated on PS expressing promastigotes, these parasites displayed a lower forward-angle light scatter than PS negative promastigotes (data not shown). Thus, PS positive L. major are smaller than PS negative L. major. Since cells shrink during the process of apoptosis, the shrinkage of L. major promastigotes might also represent a sign for apoptosis.
In addition, it was observed that the motility of purified PS expressing L. major is abolished. Viable L. major are mobile parasites capable of swimming in the surrounding fluid medium by a rapid movement of their flagellum. The mobility loss of PS expressing promastigotes in line with the TUNEL staining, end point titration experiments and electron microscopic investigation is concluded to be another hint for the conversion of PS expressing L. major into an apoptotic stage.
PS expression on apoptotic cells is the prerequisite for their uptake by macrophages (MФ) (Fadok et al, 1992). Recent data of our group demonstrated that PMN are also able to phagocytose apoptotic cells (Esmann et al., unpublished data). Since PMN phagocytose apoptotic bodies and the uptake of apoptotic cells by MФ exerts silencing properties in a PS dependent manner (Voll et al., 1997; Fadok et al., 1998, 2001; Huynh et al., 2002), the question was addressed, whether the engulfment of PS expressing apoptotic L. major promastigotes by PMN also results in silencing. The present data demonstrate for the first time that the infection rate of PMN, which is defined in this context as the number of intracellular present parasites, correlates with the ratio of PS expressing L. major promastigotes used for infection (see fig. 18). Moreover, the infection rate of PMN decreased if PS was specifically blocked using AV. These findings showed that PS expression on apoptotic L. major promastigotes has a functional role for parasite phagocytosis suggesting a direct involvement in PMN silencing.
L. major promastigotes need to enter PMN silently to ensure survival inside the host. Therefore, the next attempt of this thesis was to elucidate how the “silent entry” can be achieved. In this context, the leishmanial membrane constituents lipophosphoglycan (LPG) and gp63 (leishmanolysin) were described to impair the oxidative burst in MФ. However, experiments using L. major lpg 1- mutants showed that these parasites still enter MФ silently (Sorensen et al., 1994; Spaeth et al., 2003). Thus, LPG is obviously not of predominant importance to prevent host cell defence mechanisms such as the oxidative burst. The recognition of leishmanial PS was demonstrated to inhibit the NO production in MФ (Freitas Balanco et al., 2001). Recent data of our group revealed that the uptake of L. major promastigotes does not
induce an oxidative burst in PMN by L. major (Laufs et al, 2002). According to these data, it was investigated, whether a silencing capacity of PS expressing L. major promastigotes might be responsible for the prevention of the oxidative burst. As depicted in fig. 19, PS expressing L. major are not involved in the lack of the oxidative burst, because neither PS positive nor PS negative L. major populations induced this defence mechanism in PMN. Hence, other membrane standing molecules on L. major, like gp63, as suggested in MФ, might possibly play a role for the prevention of the oxidative burst in PMN. Consequently, leishmanial PS was suggested to exert other silencing functions.
The production of anti-inflammatory TGF-β correlates positively with the ratio of PS expression and controversially with the release of pro-inflammatory TNF-α during the removal of apoptotic cells by MФ (Fadok et al., 1998, 2000; McDonald et al., 1999; Serinkan et al., 2005). As mentioned before, unpublished data of our group have shown that also PMN are able to phagocytose apoptotic cells. As PMN are the first professional phagocytes that appear in high numbers at the site of infection (Laskay et al., 1997), this study investigated the role of leishmanial PS for the establishment of immunosilencing properties in PMN. The present data demonstrate for the first time that increasing ratios of PS expressing promastigotes correlate with an up-regulation of TGF-β release by PMN. Additionally, the presence of PS expressing parasites is associated with a down-regulation of TNF-α secretion (see fig. 20). Thus, PS expressing L. major direct the surrounding cytokine milieu toward an anti-inflammatory situation. Although PMN produce lower amounts of TGF-β as compared to MФ, PMN largely pre-dominate in cell number at the site of infection. Consequently, their lower production is still highly relevant. The functional relevance was further emphasized by the finding that TGF-β produced by L. major infected PMN results in the phosphorylation of the intracellular TGF-β receptor adaptor protein Smad2 in TGF-β sensitive Mv1Lu cells. This fact confirms that the released TGF-β is indeed bioactive. Possibly, secreted TGF-β acts via an autocrine loop on PMN themselves, because PMN possess a TGF-β receptor on their membrane (Brandes et al., 1991). Hence, L. major infected PMN could silence their own defence machinery that in turn might be responsible for intracellular parasite survival. Whether TGF-β might also affect L. major promastigotes, remains to be clarified.

In order to investigate whether the immunosilencing capacity of PS expressing L. major in PMN represents a more general principle, similar experiments were performed with MФ. As demonstrated in fig. 22 and fig. 23, similar influences of leishmanial PS concerning the infection rate as well as the regulation of TGF-β and TNF-α production were found. However, it is questionable whether MФ play a role for the immediate interaction with L. major, because PMN are the first cells at the site of infection. Additionally, it was demonstrated that L. major enter MФ via infected PMN (Laskay et al., 2003). Consequently, PMN represent the most relevant phagocytes during the first hours of infection with L. major.
The TGF-β secretion by MФ in response to leishmanial PS subsequently increases the susceptibility of MФ to intracellular leishmanial growth (Freitas Balanco et al, 2001; Barral et al., 1993). This thesis demonstrated that PS expressing parasites also enhance the TGF-β release in PMN. In addition, the TNF-α production increases in PMN, if PS expressing parasites are depleted. TNF-α was demonstrated to enhance the anti-microbial host cell defence in MФ, particularly against intracellular pathogens such as Leishmania (Beutler et al., 1993). Therefore, it was speculated that an enhanced TNF-α release might be responsible for parasite killing in PMN. To test this hypothesis, coincubation experiments of PMN with stationary L. major, either containing PS expressing parasites or PS depleted, were performed over a time period of 3 h, 18 h and 42 h. Subsequently, the intracellular parasite survival was investigated. Determination of the PMN infection rate, as a sign for intracellular present parasites, revealed that the infection rate is maintained over the observed time period in the presence of PS expressing promastigotes. The infection rate was decreased after 42 h of coculturing if PS was depleted. End point titration, as a sign for the presence of viable parasites, demonstrated that the presence of high amounts of PS expressing L. major supports the intracellular parasite survival. In contrast, the number of intracellular viable L. major was considerable reduced if PMN were infected with PS depleted parasite samples. These data clearly show that apoptotic PS expressing L. major support the intracellular survival of viable PS negative promastigotes in PMN. This survival advantage might be achieved by a PS dependent inhibition of the TNF-α secretion, as it was demonstrated that PS expressing parasites reduce the TNF-α secretion in PMN. The next attempt was to answer the question how many PS expressing promastigotes are needed to mediate
an efficient survival advantage for L. major in PMN. Therefore, coincubation experiments with PMN and L. major promastigotes containing a ratio of 20%, 30% or 50% PS expressing parasites were performed. Subsequently, the infection rate and the number of intracellularly surviving parasites was determined after 18 h and 42 h of coculturing. It was found that increasing ratios of PS expressing parasites contribute stepwise to the maintenance of the PMN infection rate and also to the survival of L. major promastigotes inside PMN. Thus, it is concluded that the higher is the ratio of apoptotic PS expressing promastigotes present in a L. major population, the more advantageous is the situation for viable PS negative parasites during the infection of PMN. This contribution of apoptotic L. major promastigotes to the biological fitness of viable parasites can be regarded as an altruistic mechanism, as suggested by Rittig and Bogdan (Rittig and Bogdan, 2000).
The fact that L. major gain survival advantages in PMN due to the presence of PS expressing promastigotes in vitro and the finding that PS expressing parasites down-regulate the release of TNF-α led to the suggestion that PS expressing L. major promastigotes might also support the disease development in vivo. In addition, this hypothesis was endorsed by the report that TNF is required early on to control L. major infection in mice (Murray et al., 2000; Wilhelm et al., 2001). Therefore, the regulation of TNF-α seems to be important for the final disease development. Using a cutaneous infection model in Balb/c mice, the disease development after injection of either L. major samples containing a high ratio of PS expressing promastigotes or PS depleted parasites populations was investigated. For the first three experiments, 1 x 106 PS negative promastigotes or 1 x 106 PS negative promastigotes supplemented with PS expressing L. major were injected. As depicted in fig. 28 and 29, the disease development is lower if mice were infected with PS depleted L. major samples. The difference between the infection with PS negative promastigotes or PS negative promastigotes supplemented with PS expressing L. major is especially notable if purified metacyclic parasites were used. The fourth experiment was performed using either 1 x 106 PS negative promastigotes or a total amount of 1 x 106 parasites including PS expressing L. major promastigotes. As demonstrated in fig. 30, the disease development is almost completely prevented if mice were infected with PS depleted L. major promastigotes. In contrast, parasite samples containing a high ratio of PS expressing promastigotes induce a distinct disease development.

Consequently, the presence of PS expressing parasites significantly enhances the disease development in Balb/c mice. Thus, PS expression on L. major promastigotes also has in vivo relevance. Moreover, the disease supporting effect of PS expressing promastigotes appears to be dose dependent. Possibly, this effect might even result in a stronger survival advantage for the parasite, if the total L. major number using for infection would be reduced. In this context, a reduction of the total parasite number would be closer to the natural L. major infection regarding the parasite transmission by the sandfly vector.
The relevance of PS expression on L. major promastigotes for disease development was emphasized by the finding that PS is also expressed on metacyclic promastigotes in the sandfly. In cooperation with Dr. Sacks and co-workers, the PS expression was investigated on metacyclic L. major promastigotes, which were freshly isolated from sandfly cultures. Using FACS analysis they demonstrated that metacyclic promastigotes contain a ratio of 43% PS expressing parasites (data not shown). Thus, the PS expression of L. major promastigotes is not limited to in vitro cultures but is also present under natural conditions.
The present data indicate that PS expressing L. major promastigotes are apoptotic. PS expression is present on both stationary phase and metacyclic L. major promastigotes in in vitro cultures but also on metacyclic parasites in the sandfly vector. Moreover, PS expression on L. major silence the immune response in PMN and MФ. Importantly, PS expressing parasites reduce the disease development in L. major infected mice. Consequently, the infectivity of L. major promastigotes is clearly dependent on its expression of PS. Presumably, PS is more important for the establishment of L. major infection than LPG, because PS depleted parasites samples have lost their silencing capacity, whereas L. major lpg 1- mutants still silence the immune response of MФ (Spaeth et al., 2003). Supplementation of non-infective procyclic promastigotes with PS expressing apoptotic parasites may offer a new model to investigate the contribution of PS expressing parasites to L. major infectivity. If procyclic promastigotes might subsequently show increased infectivity, an additional proof for a PS dependent infectivity would be given.



2. Investigation of the phosphatidylserine receptor on PMN

According to the data presented in the first part of this study, a PS dependent receptor might be responsible for the engulfment of apoptotic PS expressing parasites and thus for the silencing of L. major infection. On the other hand, phagocytosis of viable PS negative parasites is suggested to occur via receptor(s) other than PS receptor (PSR).
Coincubation experiments of PMN with L. major promastigotes revealed that the parasite uptake is almost completely abolished if the parasites were pre-treated with PMN membrane fragments. However, blocking of complement receptor (CR) 3 on PMN revealed a reduction of the infection rate less than 50% (Laufs et al., 2002). Therefore, it is assumed that more than only one receptor on PMN might be involved in the uptake of L. major promastigotes.
Precipitation of PMN membrane fragments with L. major promastigotes and subsequent western blot analysis revealed three distinct protein bands with a molecular weight of ~ 32, ~ 64 and ~ 84 kDa, respectively (see fig. 34). Complement receptor (CR) 3 and CR1 are known to be involved in L. major uptake by MФ (Laufs et al., 2002). Using western blot analysis it was investigated whether these receptors were present in the precipitate or in PMN membrane fragment fractions derived from column affinity chromatography with L. major membrane fragments. In both samples neither CR1 nor CR3 were detected (data not shown). However, the three protein bands shown in the precipitate suggest that at least three different proteins are present on PMN that are involved in PMN binding to L. major promastigotes. So far, it is can not be distinguished whether these potential PMN receptors might be involved in PS dependent or –independent interaction with L. major.
Experiments using different anti-PSR antibodies showed contradictory results. According to a puplication by Dr. Fadok and colleagues, the stimulation of MФ with TGF-β and β-glucan leads to expression of the suggested PS receptor (“PSR”) (Fadok et al., 2000). FACS analysis using a monoclonal mouse anti-human “PSR” IgM antibody (217) (anti-PSR IgM) (provided by Dr. Fadok) revealed that stimulation of PMN with TGF-β and β-glucan also induces “PSR” expression. Similar experiments with MФ demonstrated next to a “PSR” expression also a positive signal

with an IgM isotype control (data not shown). Consequently, this finding led to the suggestion of an aspecific binding of the anti-PSR IgM. However, stimulation of both PMN and MФ with stationary phase L. major promastigotes showed a normal surface expression of the “PSR”. In this case, the parasites themselves could possibly enhance an aspecific binding of the anti-PSR IgM through their own “stickiness”, which might also explain the higher “PSR” expression by stimulation with L. major as compared to stimulation with TGF-β and β-glucan. However, the anti-PSR IgM detected “PSR” expression on PMN could not be confirmed with a monoclonal mouse anti-human “PSR” IgG antibody (anti-PSR IgG) (provided by PD Dr. Dr. Herrmann and Dr. Voll).
In western blot experiments using PMN whole cell lysates, the situation was the other way around: The anti-PSR IgM did not detect any protein. In contrast, the anti-PSR IgG revealed a protein of the expected molecular weight of 47 kDa (fig. 36). Similar results were obtained using a whole cell lysate of MФ.
In western blot analysis the three-dimensional protein structure is changed through linearisation. As a consequence, the "PSR” epitopes recognized by the anti-PSR IgM may be to far apart from each other for sufficient binding of the antibody. This might serve as a possible explanation for the lack of anti-PSR IgM binding to its ligand in western blot analysis. In contrast, the “PSR” epitopes recognized by the anti-PSR IgG might be localized inside the three-dimensional “PSR” protein structure present on intact PMN membranes. This epitope localisation might explain why the anti-PSR IgG can not detect its ligand using FACS analysis. However, Fadok and colleagues detected PS on MФ using the anti-PSR IgM in western blotting. These conflicting results to the here presented findings might be caused by different western blot methods.
If the “PSR” detcetion using anti-PSR IgM in FACS analysis would be regarded as an aspecific binding, it is reasonable to suggest that the “PSR” is not present on the cell surface but intracellularly. This suggestion is in line with recent findings of other groups, who demonstrated that the protein encoded by the cDNA of this “PSR” is not localized on the cell membrane (Cui et al., 2003; Boese et al., 2004). A possible explanation for the misleading results achieved by the use of the “PSR” gene might be a cross reaction between the anti-PSR IgM and an epitope in phage display. A latest hint for this suggestion is a demonstrated weak cross reactivity of anti-PSR IgM
with a peptide within the protein encoded by the PSR gene (Williamson and Schlegel, 2004; Boese et al., 2004).
Taken together, the findings of this study and recent publications hint in the direction that it is more likely that the “PSR” is present exclusively intracellularly. However, the effects mediated by leishmanial PS on PMN corroborate the existence of a PSR. In case that PS might not directly interact with PSR, it is possible that PS recognition is mediated through bridging molecules. In this context, annexin-I, which is a releasable intracellular protein in PMN that mediates bivalent binding serves as a bridging molecule candidate (Francis et al., 1992). On the membrane surface annexin-I enhances phagocytosis of apoptotic cells in a PSR dependent manner (Arur et al., 2003; Fan et al., 2004; Vergnolle et al., 1995).
However, the PSR still remains to be clarified. The three protein bands obtained from the precipitate of PMN membrane fragments with L. major promastigotes possibly include a PS recognizing receptor of PMN. In order to identify a PS specific receptor, the precipitation method could be modified by using purified PS positive or PS negative parasites for precipitation experiments. If western blot analysis might detect a protein in the precipitate with PS positive L. major, which would not be present in the precipitate with PS negative parasites, this protein represents a new PSR candidate.



VI Summary

Previous studies have shown that early after infection, polymorphonuclear neutrophil granulocytes (PMN) serve as host cells for the obligate intracellular parasite Leishmania major (L. major). This finding suggests that L. major succeed to enter PMN “silently”, without the activation of antimicrobial effector mechanisms of these cells. The present study aimed to investigate the surface molecules both of the parasites and of PMN that are involved in this silent entry process. A known silent way of phagocytosis is the ingestion of apoptotic cells by professional phagocytes. During this process, phosphatidylserine (PS) on the surface of apoptotic cells serves as “eat me” signal for the phagocytes. In addition, PS is thought to mediate the silencing of the pro-inflammatory response. Since PS was found to be present on the surface of L. major, it was investigated whether PS is responsible for the silent infection of human PMN with L. major in vitro. Labeling with both annexin-V and anti-PS antibody revealed that highly infectious stationary phase L. major cultures contain a high (>60%) ratio of PS positive parasites, whereas there are only a few PS positive parasites present in non-infectious logarithmic growth phase cultures. Importantly, TUNEL staining and viability tests revealed that the PS positive parasites are apoptotic, not capable of proliferation and destined to death. Electron microscopy also demonstrated that the PS positive Leishmania are morphologically not intact.
Infectious stationary phase L. major cultures contain both PS positive apoptotic and PS negative viable parasites. Interestingly, although the PS positive parasites are not viable, depletion of these parasites from stationary phase Leishmania cultures resulted in a decreased survival rate of the parasites in PMN, as compared to the survival rate of normal stationary phase cultures containing both PS positive and PS negative parasites. Coincubation of PMN with purified PS negative L. major resulted in the release of pro-inflammatory TNF-α, whereas the additional presence of PS positive parasites in stationary phase cultures shifted the cytokine response to an anti-inflammatory milieu, characterized by increased levels of TGF-ß release and down regulation of TNF-α production.
Based on these in vitro results I propose the model that the presence of an apoptotic population of the parasites is a prerequisite of infectivity of L. major promastigotes. Experimental infection of susceptible Balb/c mice proved this hypothesis in vivo. PS negative (i.e. viable) L. major did induce a significant lower disease development as
compared to parasite populations consisting of a mixture of PS negative and a high ratio of PS positive parasites. These data clearly proved the hypothesis that apoptotic parasites assist the viable parasites to establish infection.
Natural L. major infection occurs upon a bite of an infected sandfly. The finding that L. major isolated from infected sandflies also contain a high ratio of PS positive parasites suggest the importance of PS positive parasites regarding the natural Leishmania infection.
Pre-treatment of the parasites with the PS binding protein annexin-V blocked the uptake of L. major by PMN. This observation suggested that PMN express a PS receptor (PSR). However, in spite of repeated efforts to assess PSR expression on PMN, no conclusive results were obtained. However, precipitation experiments with biotinylated PMN membrane fragments revealed three until now unidentified PMN proteins that bind to L. major. Possibly, one of these three proteins represents a PS recognizing receptor and further studies are required to identify and characterize these molecules.
Taken together, the results of this thesis suggest that apoptotic L. major promastigotes, via the induction of anti-inflammatory and phagocyte deactivating cytokines, assist the viable parasites to survive inside phagocytes. Apoptotic parasites “silence” the immune response of PMN and enable the survival of non-apoptotic parasites to establish a productive infection.


link to entire paper -
http://www.students.informatik.uni-luebeck.de/zhb/ediss148.pdf



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j

[jazzbeerman]

Malaria Exploits Exposed PS ------




Cytoadherence of Malaria-Infected Red Blood
Cells Involves Exposure of Phosphatidylserine

http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowPDF&ProduktNr=224332&Ausgabe=228865&ArtikelNr=67908&filename=67908.pdf


Introduction

Malaria, one of the most life-threatening infectious
diseases in the world, causes 1.5-2.7 million deaths
annually [1]. Of the four species of human malaria
parasites, Plasmodium falciparum causes the severest of
symptoms and the greatest number of deaths. Adherence
of red cells infected with the mature stages of the parasite
to endothelial cells (EC) lining the postcapillary venules,
especially in the deep tissues, enables the parasites to
avoid splenic clearance and parasite growth is favored
by an hypoxic microenvironment. The adherence of
P. falciparum-infected red cells also results in the
occlusion of microvessels and can lead to
unconsciousness, coma, and in some cases, death. The
virulent nature of P. falciparum has been attributed to
mechanical obstruction by adherent infected red cells [2].
In this context, an understanding of the mechanisms of
the adherence of malaria-infected red cells may lead to
the development of anti-adhesive reagents and new
therapies.

......

The asymmetry of phospholipids in the lipid bilayer
of the plasma membrane is maintained by several membrane
enzymes such as scramblase, translocase and
flippase [28]. Disruption of phospholipid asymmetry
results in the exposure of phosphatidylserine (PS)
molecules, which are normally located in the inner leaflet
of the phospholipid bilayer. We and other investigators
have shown that PS is exposed on the malaria-infected
red cell surface coincident with parasite development [29-
31]. PS liposomes or PS-exposed cells bind to CD36 [32]
and TSP [33] both of which serve as receptors for the
adherence of P. falciparum-infected red cells [34, 35].
Here, we demonstrate that PS exposure on the exofacial
surface of the P. falciparum -infected cell is involved in
the binding to CD36 and TSP.

.......

Discussion
Drastic changes in the red blood cell membrane are
induced during the intracellular maturation of the malarial
parasite, P. falciparum. These include visible changes in
the shape of the red cell, the appearance of electron-dense
protrusions (called knobs) [3, 49], increased membrane
permeability [50, 51], decreased deformability [52],
insertion of parasite-derived proteins [8-10], changes in
the composition and oxidative damage of membrane
lipids [53, 54], a reduction in cholesterol content [30],
as well as degradation of membrane proteins [55].

........

Further,
Facer et al. [63] detected elevated levels of antiphospholipid
antibodies in the serum of P. falciparuminfected
patients using an ELISA method, and found the
highest IgG and IgM binding was to PS and other anionic
phospholipids, indicating that infected red cells expose
PS on their surface in vivo. In that report, the percentage
of anti-PS IgG or IgM positive serum of P. falciparuminfected
patients as 89% and 79%, respectively. Thus,
the accumulating data support PS exposure in P.
falciparum infected red cells.

.......

The precise mechanism of PS exposure on
P.falciparum-infected red cells remains undefined.
Several investigators have demonstrated that treatment
of red cells with various oxidants, such as
phenylhydrazine [64], hydrogen peroxide [65], or
diamide [66], also results in PS exposure. And, PS
exposure has been demonstrated in red cells from patients
with sickle cell anemia [36, 67, 68], thalassemia [69],
diabetes [70], all of which may be under oxidative stress
[71-73]. The erythrocytic stages of Plasmodium as they
mature, digest hemoglobin, and release highly oxidative
iron-containing products. And, oxidized membrane lipids
have been detected in trophozoite- or schizont-infected
red cell membranes [54], coincident with the time we
and other investigators have detected PS exposure [29-
31]. It seems plausible that oxidative stress could act as
a trigger for PS exposure on the surface of the malariainfected
red cell.

.......

Since PS exposure was detected in the various strains of
falciparum malaria we tested, as well as strains used by
other workers, we contend that PS exposure can
contribute to the universal binding of infected cells to
CD36 and TSP. Our findings (i.e., PS exposure and PSmediated
infected-cell binding to TSP) is also compatible
with the suggestion that PS is involved in abnormal sickle
red cell binding to TSP [76, 77].

.......

Further investigation of the structural requirement of
inhibition of PS-mediated infected-cell binding, as well
as a determination of the relative contributions of PfEMP-
1, band 3 and PS to cytoadherence, may assist in the
development of novel and effective anti-adhesive agents.
In conclusion, PS exposure, modification in band 3
protein, and PfEMP-1 all contribute to cytoadherence/
sequestration and disease pathogenesis in P. falciparum
malaria.




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Evolution has favored pathogenesis that resembles apoptosis,

j

[jazzbeerman]

Toxoplasma Gondii Exploits Exposed PS -






Biochem Biophys Res Commun. 2004 Nov 12


Toxoplasma gondii exposes phosphatidylserine inducing a TGF-beta1 autocrine effect orchestrating macrophage evasion.


Seabra SH, de Souza W, Damatta RA.

Laboratorio de Biologia Celular e Tecidual, Centro de Biociencias e Biotecnologia, Universidade Estadual do Norte Fluminense, 28013-600 Campos dos Goytacazes, RJ, Brazil.


Toxoplasmosis is a worldwide disease caused by Toxoplasma gondii. Activated macrophages control T. gondii growth by nitric oxide (NO) production. However, T. gondii active invasion inhibits NO production, allowing parasite persistence. Here we show that the mechanism used by T. gondii to inhibit NO production persisting in activated macrophages depends on phosphatidylserine (PS) exposure. Masking PS with annexin-V on parasites or activated macrophages abolished NO production inhibition and parasite persistence. NO production inhibition depended on a transforming growth factor-beta1 (TGF-beta1) autocrine effect confirmed by the expression of Smad 2 and 3 in infected macrophages. TGF-beta1 led to inducible nitric oxide synthase (iNOS) degradation, actin filament (F-actin) depolymerization, and lack of nuclear factor-kappaB (NF-kappaB) in the nucleus. All these features were reverted by TGF-beta1 neutralizing antibody treatment. Thus, T. gondii mimics the evasion mechanism used by Leishmania amazonensis and also the anti-inflammatory response evoked by apoptotic cells.




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Toxoplasmosis-

http://www.cdc.gov/ncidod/dpd/parasites/toxoplasmosis/factsht_toxoplasmosis.htm




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Evolution has favored pathogenesis that resembles apoptosis,

j