TITLE: A high throughput method for detecting apoptosis of embryonic
stages of metazoan helminthic parasites.
FIELD OF INVENTION:
This invention relates to a flow cytometry based, assay system for
detecting apoptosis of embryonic stages of parasitic helminthes and high
throughput screening of apoptogenic antihelminthic compounds.
BACKGROUND OF THE INVENTION:
Parasitic helminthes cause chronic diseases with severe morbidity
in both humans and animals and broadly include two groups i.e.
intestinal-geo helminthes like Ascaris, Trichuris, Hook worms and tissue
invasive-systemic helminthes like Filarial and Schistosome parasites etc.
Diseases caused by these helminthic parasites have unusually high
incidence in developing countries at present e.g. Ascariasis (807 million),
Trichuriasis (604 million), Hook worm infections (574 million),
Schistosomiasis (207 million), Lymphatic filariasis (120 million) and
Onchocerciasis (37 million) etc. (Hotez, 2008, WHO 2006). These
diseases continue to pose serious challenges to the economic welfare and
public health in most of the developing countries. In the absence of any
preventive vaccine, chemotherapy remains the mainstay for treatment of
diseases caused by these metazoan helminthic parasites. Beginning with
the widespread use of DEC for the treatment of Lymphatic Filariasis (LF)
in China during the 1970s, the mass treatment of human population
with antihelminthic drugs known as mass drug administration (MDA),
has been a major approach for controlling human helminthiases in
developing countries (Hotez, et al., 2007). However, the success of MDA
programs is marred by several factors including lack of any effective
adulticidal drugs for the parasitic worms (Hotez et al., 2008), limited
effect of the existing drugs on the embryogenesis of helminthic parasites
(Awadzi et al., 1995, Plaisier et al., 1991), adverse side reactions
associated with the drug of choice for human filariasis,
Diethylcarbamazine (DEC) (Haarbrink et al., 1999), possibility of
emergence of drug resistant parasites coupled with the lack of robust
biomarkers for detection of resistance of helminthic parasites to the
mainstay drugs of MDA programs (Hotez et al., 2008) etc. The problem is
further compounded by the chances of rapid reinfection of hosts by these
parasites, post-treatment (Hotez et al., 2008, Loukas et al., 2005).
Further, in case of filarial infections the existing drugs like DEC and
Ivermectin acts rapidly to reduce the number of circulating microfilariae
for only a few months after which microfilariae reappear at levels of 20%
or more of pretreatment numbers within a year (Awadzi et al., 1995).
This microfilarial density is presumed to be sufficient for continuation of
transmission of the parasite (Alley et al., 2001). The reason for this
rather limited effect of chemotherapy in filarial infections is that it does
not kill the long lived adult worms and that its embryocidal activity
seems to be mainly restricted to the final embryonic stages i.e.
microfilariae, leaving early embryogenesis intact (Plaisier et al., 1991).
Studies using Albendazole have also revealed very little effect on
embryogenesis of filarial nematodes (Awadzi et al., 1995). In such a
scenario, worldwide elimination of diseases caused by metazoan
helminthic parasites including lymphatic filariasis may remain a distant
goal, not achievable without development of better drugs. Hence, new
approaches for chemotherapy with well defined mode of action are an
acute necessity for controlling the rising helminthic infections in human
and animal communities (Hotez et al., 2008, Hoerauf et al., 2002;
Melrose, 2003).
Being obligate parasites with no intermediate animal hosts,
persistence of these helminthic parasites in their mammalian hosts
becomes central to their survival. This is materialized by release of a
large number of embryonic stages called larval stage-1/L-1/ microfilariae
into peripheral circulation of the infected hosts (in case of systemic
helminthic parasites) or fertilized eggs in to the soil/water (in case of
parasitic geo-helminthes) by adult female worms which are subsequently
transmitted into new susceptible hosts by insect vectors in case of former
and through contaminated food and water in case of later. Thus,
successful embryogenesis in adult female parasites is a critical rate
limiting step required for survival and propagation of the parasitic worms
in host communities. Hence, agents that can induce apoptosis in the
fertilized eggs or embryonic stages of helminthic parasites can be
expected to have immense potential for elimination of such pathogens
from human and animal communities by blocking their embryogenesis
and subsequent transmission. However, currently there are no
established reports available in literature regarding apoptosis in the
embryonic stages as well as drugs blocking embryogenesis in parasitic
worms. Further, microscopy, the only tool used to score apoptosis in the
free living soil nematode C. elegans permits analysis of only limited
number of apoptotic features and cells/embryonic stages. In this context
the current study demonstrating high throughput assays for apoptosis in
fixed as well as live embryonic stages of a metazoan helminthic parasite
filarial parasite S.digitata is a quantum improvement in the study of
embryogenesis of parasitic helminthes and can be used for high of anti-
helminthic compounds having apoptogenicity towards embryonic stages
of parasitic worms.
OBJECTS OF THE INVENTION:
An object of this invention is to propose assays for detecting apoptosis of
embryonic stages of parasitic helminthes;
Another object of this invention is to demonstrate the possibility of high
throughput screening and identification of compounds having
apoptotogenic and antihelminthic activity against the embryonic stages
of a metazoan helminthic parasite using three different agents namely
Plumbagin, H2O2 and Staurosporine;
Still another object of this invention is to propose assays to detect and
quantify multiple conserved features of apoptosis such as externalization
of phosphatidyl serine
Yet another object of this invention is to propose assays to detect and
quantify multiple conserved features of apoptosis such as depolarization
of mitochondria;
Further, object of this invention is to propose assays to detect and
quantify multiple conserved features of apoptosis such as enhanced
cytosolic presence of cytochrome-c;
Still further object of this invention is to propose assays to detect and
quantify multiple conserved features of apoptosis such as increased intra
cellular expression of nematode apoptosis related proteins CED-3, CED-4
& CDE-9;
Yet further object of this invention is to propose assays to detect and
quantify multiple conserved features of apoptosis such as activation of
caspase family of cysteine proteinases;
Still further object of this invention is to propose assays to detect and
quantify multiple conserved features of apoptosis such as cleavage of
intracellular caspase substrate PARP;
Another object of this invention is to propose assays to detect and
quantify multiple conserved features of apoptosis such as fragmentation
of chromosomal DNA and formation of sub-diploid nuclei in the
embryonic stages of filarial parasite S.digitata using 3 known apoptosis
inducing agents namely Plumbagin,H2O2 and Staurosporine .
BRIEF DESCRIPTION OF THE INVENTION:
According to this invention there is provided a method for detecting
apoptosis of embryonic stages of helminthes comprising:
isolating Intra Uterine embryonic stages from adult female parasite;
culturing in vitro and treating said embryonic stages;
subjecting the said embryonic stages to the step of flow cytometric
analysis;
developing assays for apoptosis in the embryonic stages
and screening of three compounds for their apoptogenicity against
embryonic stages of helminthic parasite S.digitata.
and screening of compounds for their apoptogenicity against embryonic
stages of helminthic parasite S.digitata.
DETAILED DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
Figure 1 Externalisation of phosphatidyl serine in embryonic stages of
S.digitata a: Dot Plots for embryonic stages of S.digitata: R-1
represents microfilariae while R-2 and R-3 represent early and late egg
stages respectively, b: Externalization of Phosphatidyl serine (PS) in MF
stages of S.digitata by Annexin-V- PE staining. Embryonic stages were
harvested, treated in-vitro with Plumbagin for 24 hrs and stained with
Annexin -V-PE. Overlayed histogram shows PS exposure on MFs after
treatment with 10µM (Red) or 50 µM (Green) of Plumbagin over Annexin-
V-PE stained untreated controls (Blue).
Figure 2 Mitochondrial depolarization in MF stages of S.digitata. After
24hr treatment with Plumbagin, the embryonic stages were harvested,
subjected to Mitoscreen JC-1 staining and gated for MFs and analyzed.
a,b,c: Dot Plots showing dose dependent induction of depolarization of
mitochondria in Mfs on treatment with 10µM(b) or 50µM(c)
concentrations of Plumbagin as compared to the Mitoscreen JC-1 stained
untreated controls(a); Percentage of events in gate R-2 displaying
reduced fluorescence as an expression of depolarization d,e,f: Histogam
showing dose dependent induction of depolarization of mitochondria in
Mfs, on treatment with 10µM(e) or 50µM(f) Plumbagin as compared to
Mitoscreen JC-1 stained untreated controls(d). Reduction of GMI of
fluorescence is shown as an expression of depolarization.
Figure 3. Enhanced cytosolic presence of Cytochrome c in cytosol, intra
cellular activation of caspase-3 like proteases and cleavage of caspase
substrate PARP in MF stages of S.digitata. Embryonic stages treated with
Plumbagin for 24 hrs were fixed with para formaldehyde and stained
with anti cytochrome c-FITC or PE-Conjugated Polyclonal antibody
against active caspase-3 or PE- conjugated polyclonal antibody against
cleaved PARP in separate experiments and gated for MFs. a: Overlayed
histogram for increased cytosolic presence of Cytochrome c in MFs
treated with 10µM(Pink), 50µM(Blue),100µM (Red) Plumbagin; Green -
untreated control(stained intra cellularly with anti cytochrome c- FITC).
b,c: Dot plots showing activation of Caspase-3 like proteases in MF upon
treatment with 50µM of Plumbagin (c) shown as percentage of
population shifting right into the gate in comparison to the untreated
control (stained intra cellularly with anti active caspase-3-PE)(b); d,e,f:
Dot plots showing dose dependent induction of PARP cleavage in MF
upon treatment with 10µM(e) or 50µM(f) of Plumbagin in terms of
percentage of population shifting right into the gate compared to the
untreated control(stained intra cellularly with anti cleaved PARP-PE) (d)
respectively.
Figure 4 Demonstration of intracellular expression of CED-3,CED-4 and
CED-9 in embryonic stages and Sub cellular localization of CED-4 in
eggs of S.digitata: a,b,c: Intra cellular expression of CED-3, CED-4 and
CED-9 in MF stages of S.digitata by flow cytometry. Embryonic stages
were treated for 24 hrs with Plumbagin, fixed, permeabilised and stained
with goat antibodies to CED-3, CED-4and CED-9 respectively, probed
with anti goat IgG-PE conjugate and gated for MFs for analysis; Intra
cellular expression of CED-3(a), CED-4(b) and CED-9(c) in Mfs treated
with 10µM(Green) or 50µM(Red/Pink) of Plumbagin. Untreated control
(stained intra cellularly with anti CED-3/CED-4/CED-9 followed by
probing with anti goat IgG-PE) is shown in Blue lines. d,e: CED-4
staining in a Control egg showing web like cytoplasmic staining
pattern(d) and Plumbagin treated egg displaying diffused cytoplasmic
staining pattern(e).
Figure 5 Fragmentation of chromosomal DNA and formation of sub-
diploid nuclei: Embryonic stages were treated for 24 hrs with Plumbagin,
fixed, permeabilised stained and gated for MFs for analysis, (a):
Overlayed histogram for fragmentation of chromosomal DNA in MFs by
TUNEL assay - treated with 10µM(Green), 50µM(Red) or 100µM (Violet) of
Plumbagin; untreated control (Blue); b,c: Fluorescence image of
control(b) or treated(c) embryonic stages of S.digitata; d,e,f: Histogram
plots showing dose dependent formation of sub-diploid nuclei in MF after
treatment with 10µM (e) or 25µM(f) of Plumbagin, showing a smaller
peak with reduced fluorescence for sub-diploid nuclei behind the normal
peak representing diploid nuclei (d): Controls showing a single peak for
normal diploid nuclei in stained MF.
DETAILED DESCRIPTION OF THE INVENTION:
In the present invention we report standardization and evaluation
of 10 novel quantitative flow cytometry based assays to detect and
quantify multiple conserved features of apoptosis such as externalization
of phosphatidyl serine, mitochondrial depolarization, enhanced cytosolic
presence of cytochrome c, increased expression of nematode apoptosis
related proteins CED-3, CED-4 and CED-9, activation of caspase family
of cysteine proteinases, cleavage of intracellular caspase substrate PARP,
fragmentation of chromosomal DNA and formation of sub-diploid nuclei
etc. in the embryonic stages of filarial parasite S.dgitata using three
known apoptosis inducing agents (e.g. Plumbagin, H2O2 and
Staurosporine). These assays constitute the first ever report on
development and evaluation of flow cytometery based assays for
apoptosis of embryonic stages in helminthic parasites. These assays also
offer opportunities for development of automated high throughput
screening assays for identifying apoptosis inducing agents /drugs to
block embryogenesis in parasitic helminthes which can potentially affect
their transmission and survival in host communities.
Apoptosis of embryonic stages of parasitic helminthes has not been
reported earlier in literature primarily due to lack of sensitive assay
system. The current invention describes 10 different flow cytometry
based assays for detecting and quantifying apoptosis in the embryonic
stages of a filarial nematode S.digitata.
• Method-1. Isolation of Intra Uterine Embryonic stages from Adult
Female parasites.
• Method-2. In-vitro culture and Treatment of Embryonic stages.
• Method-3. Flow cytometric analysis of the Embryonic stages.
• Method-4. Assays for Apoptosis in the Embryonic stages
(a) Detection of externalization of Phosphatidyl Serine
(b) Analysis of Mitochondrial depolarization
(c) Detection of increased intra cellular expression of CED-3 protein -
a member of the caspase family of cystiene proteinases.
(d) Detection of increased intra cellular expression of CED-4 protein -
a homologue of mammalian Apaf-1
(e) Detection of increased intra cellular expression of CED-9 a
homologue of mammalian Bcl2 protein.
(f) Demonstration of enhanced cytosolic presence of cytochrome-c in
the cytoplasm.
(g) Demonstration of intra cellular activation of caspase-3 like cysteine
proteinases.
(h) Demonstration of intra cellular cleavage of caspase substrate
PARP.
(i) Detection of Fragmentation of Chromosomal DNA.
(j) Demonstration of the presence of Sub-diploid Nuclei.
• Method -1- Isolation of Intra Uterine Embryonic stages from
Adult Female parasite.
Adult female filarial worms Setaria digitata were collected from the
peritoneum of cattle, slaughtered at a nearby abattoir in sterile Hanks
Balanced Salt Solution (HBSS) medium (Sigma H 2387-1L) and
transported to the laboratory in a sterile container.The medium
containing 1% glucose (Sigma G 7528), Penicillin 100 units/ml,
Streptomycin 100µg/ml (Sigma P 4333). And Amphotericin - B
0.25ug/ml (Sigma A 2942) was buffered with NaHCO3 (Sigma S 5761).
About 5 to 7 worms were taken in a petridish, washed three times in
medium, dissected into small pieces in 10 ml of medium under sterile
conditions and incubated at 37°C for 30 minutes to allow the release of
embryonic stages (eggs and microfilariae) into the medium. The
embryonic stages were harvested into sterile 15 ml. centrifuge tubes and
washed three times by centrifuging at 300g for 10 minutes each with
medium and the final pellet was suspended in 1ml. of RPMI-1640
medium (Sigma R 8005) supplemented with 10% FBS (Sigma F 2442)
• Method-2-In-vitro culture and Treatment of Embryonic stages.
The number of embryonic stages of S.digitata in the above preparation
were counted using light microscope, and the suspension was taken in
RPMI-1640 medium containing 10% FBS at 1x105 embryonic stages/ml.
One ml. each was dispensed into individual wells of a 24 well tissue
culture plate. One set of wells was taken as untreated control containing
only medium with 10% FBS. Other sets of wells were subjected to
treatment with agents like H2O2 (Sigma H 1009) or Plumbagin (Sigma P
7262) at a concentration ranging from 10-100um while treatment with
Staurosporine (Sigma S 3939) was performed at a concentration range of
0.5-5.0µM for 24 hr. at 37°C in a 5%CO2 incubator. Motility of the Mf
was scored under an inverted microscope.
• Method -3-Flow cytometric analysis of the Embryonic stages
After appropriate treatment and incubation, each set of the cultured
embryonic stages of S. digitata were harvested from the 24 well tissue
culture plate separately in 15 ml. sterile centrifuge tubes, washed with
PBS twice and stained as per requirement following manufacturer's
instructions as described below. This was followed by two times washing
with PBS and final suspension of the stained embryonic stages was
taken in 0.5ml of sheath fluid (BD Biosciences 342003) before their
acquisition in the flow cytometer (BD FACS Calibur, Becten and
Dickinson, USA). Analysis was performed on 10,000-acquired events
depicted as Dot Plots and Histogram Plots using Cellquest Pro soft ware.
All plots were representative of at least 3 experiments. The distinctly
clustered populations obtained consistently in the Dot plots for the intra
uterine embryonic stages of S.digitata (Figure 1 a) were gated for Mfs
(R-l) and eggs (R-2 and R-3) and analyzed for different assays for
apoptosis as described below.
• Method-4. Assays for Apoptosis in the Embryonic stages
(a)-Detection of externalization of Phosphatidyl Serine.
The externalization of phosphatidyl serine was detected by staining the
embryonic stages with Annexin-V-PE (BD Biosciences 556421) as per the
instructions of the manufacturer. Briefly, the embryonic stages of
S.digitata were subjected to various treatments in culture for 24 hrs.
After the said period of incubation the embryonic stages were harvested,
washed with ice cold PBS twice and resuspended in lOOul of IX
Annexin-V binding buffer (BD Biosciences 556454) at 1x10s embryonic
stages per ml. to which 5 ul of Annexin-V-PE was added. After 15
minutes of Incubation in dark at room temperature another 400ul of IX
Annexin-V binding buffer was added to the above suspension. The
resulting 500ul suspension of Annexin-V-PE stained embryonic stages
was then acquired and analyzed by flow cytometer.
(b) Analysis of Mitochondrial depolarization.
The mitochondrial membrane potential (AWm) was measured using JC-1
probe (a cataionic lipophilic fluorochrome that accumulates in
mitochondria in proportion to mitochondrial membrane potential)
supplied in mitoscreen JC-1 kit (BD Biosciences 551302). Briefly a
suspension of in-vitro cultured embryonic stages at lx 105 embryonic
stages/ml. was mixed with 0.5 ml of freshly prepared JC-1 working
solution for 10-15 minutes at 37°C in a CO2 incubator followed by two
times washing and final suspension of the embryonic stages in lx assay
buffer (supplied with the kit) before acquisition and analysis by flow
cytometry. Reduction of fluorescence intensity of the mitoscreen JC-1
stained embryonic stages was taken as the degree of depolarization of
mitochondria.
(c) Detection of increased intra cellular expression of CED-3 protein.
After in vitro culture for 24 hours with apoptosis inducing reagents the
embryonic stages were harvested in 15 ml, sterile centrifuge tubes and
fixed for 60 minutes at 0°C in 1% paraformaldehyde (Sigma P 6148) in
PBS. The suspension was washed first with PBS and then with IX
permeabilisation buffer (eBioscience 00-8333-56) and finally
resuspended in lOOul of IX permeabilisation buffer at 1x10s embryonic
stages per ml. The intra cellular expression of CED-3 was scored by
incubation of the fixed and permeabilised embryonic stages with primary
goat antibodies - anti CED-3 (Santacruz sc-9192) followed by probing
with PE conjugated secondary anti goat IgG-PE (Santacruz sc-3747)
antibodies. Incubation of both primary and secondary antibodies (both
1/100 diluted) was done successively in IX permeabilisation buffer
(e Bioscience 00-8333-56) for lhr and 45 minutes respectively. The
suspension of stained embryonic stages was then washed with PBS twice
and finally reading was taken by flow cytometry.
(d) Detection of increased intra cellular expression of CED-4 protein.
After in vitro culture for 24 hours with apoptosis inducing reagents the
embryonic stages were harvested in 15 ml. sterile centrifuge tubes and
fixed for 60 minutes at 0°C in 1% paraformaldehyde (Sigma P 6148) in
PBS. The suspension was washed first with PBS and then with IX
permeabilisation buffer (eBioscience 00-8333-56) and finally
resuspended in 100 µl of IX permeabilisation buffer at 1x105 embryonic
stages per ml. The intracellular expression of CED-4 was scored by
incubation of the fixed and permeabilised embryonic stages with
primary goat antibodies - anti CED-4 (Santacruz sc-9193) followed by
probing with PE conjugated secondary anti goat IgG-PE (Santacruz sc-
3747) antibodies. Incubation of both primary and secondary antibodies
(both 1/100 diluted) was done successively in IX permeabilisation buffer
(e Bioscience 00-8333-56) for lhr and 45 minutes respectively. The
suspension of stained embryonic stages was then washed with PBS twice
and finally reading was taken by flow cytometry or confocal microscopy,
(e) Detection of increased intra cellular expression of CED-9 protein.
After in vitro culture for 24 hours with apoptosis inducing reagents the
embryonic stages were harvested in 15 ml. sterile centrifuge tubes and
fixed for 60 minutes at 0°C in 1% paraformaldehyde (Sigma P 6148) in
PBS. The suspension was washed first with PBS and then with IX
permeabilisation buffer (eBioscience 00-8333-56) and finally
resuspended in 100 µl of IX permeabilisation buffer at 1x10s embryonic
stages per ml. The intracellular expression of CED-9 was scored by
incubation of the fixed and permeabilised embryonic stages with primary
goat antibodies - anti CED-9 (Santacruz sc- 9202) followed by probing
with PE conjugated secondary anti goat IgG-PE (Santacruz sc-3747)
antibodies. Incubation of both primary and secondary antibodies (both
1/100 diluted) was done successively in 1X permeabilisation buffer (e
Bioscience 00-8333-56) for lhr and 45 minutes respectively. The
suspension of stained embryonic stages was then washed with PBS twice
and finally reading was taken by flow cytometry or confocal microscopy.
(f) Demonstration of enhanced presence of cytochrome-c in the
cytoplasm.
After in vitro culture for 24 hours with apoptosis inducing reagents the
embryonic stages were harvested in 15 ml. sterile centrifuge tubes and
fixed for 60 minutes at 0°C in 1% paraformaldehyde (Sigma P 6148) in
PBS. The suspension was washed first with PBS and then with 1X
permeabilisation buffer (eBioscience 00-8333-56) and finally
resuspended in 100 µl of 1X permeabilisation buffer at 1x10s embryonic
stages per ml. This suspension was then incubated for 45 minutes with
20 ul of anti cytochrome c-FITC (eBioscience 11-6601-82). This was
followed by washing with PBS twice and analysis by Flow cytometer.
(g) Demonstration of intra cellular activation of caspase-3 like cystiene
Proteinases.
After in vitro culture for 24 hours with apoptosis inducing reagents the
embryonic stages were harvested in 15 ml. sterile centrifuge tubes and
fixed for 60 minutes at 0°C in 1% paraformaldehyde (Sigma P 6148) in
PBS. The suspension was washed first with PBS and then with 1X
permeabilisation buffer (eBioscience 00-8333-56) and finally
resuspended in 100 ul of 1X permeabilisation buffer at 1x10s embryonic
stages per ml. This suspension was then incubated for 45 minutes with
20 µl of anti active caspase-3-PE (BD Biosciences 559762). This was
followed by washing with PBS twice and analysis by Flow cytometer.
(h) Demonstration of intra cellular cleavage of caspase substrate PARP.
After in vitro culture for 24 hours with apoptosis inducing reagents the
embryonic stages were harvested in 15 ml. sterile centrifuge tubes and
fixed for 60 minutes at 0°C in 1% paraformaldehyde (Sigma P 6148) in
PBS. The suspension was washed first with PBS and then with 1X
permeabilisation buffer (eBioscience 00-8333-56) and finally
resuspended in 100 µl of 1X permeabilisation buffer at 1x105 embryonic
stages per ml. This suspension was then incubated for 45 minutes with
20 ul of anti cleaved PARP-PE (BD Biosciences 552933). This was
followed by washing with PBS twice and analysis by Flow cytometer.
(i) Detection of Fragmentation of Chromosomal DNA.
Presence of fragmentation of chromosomal DNA was detected by TUNEL
staining. Briefly, the treated embryonic stages were harvested, fixed with
1% Para formaldehyde followed by washing with PBS and stored in 70%
ethanol at -20°C. After 18 hrs they were washed and subjected to TUNEL
staining at lxlO5 embryonic stages per ml. of suspension using the APO-
Direct apoptosis detection kit (BD Biosciences 556381) and analyzed by
flow cytometer.
(j) Demonstration of the presence of sub-diploid Nuclei.
Presence of sub-diploid nuclei was detected by PI/RNase staining.
Briefly, the treated embryonic stages were harvested, fixed with 1% Para
formaldehyde followed by washing with PBS and stored in 70% ethanol
at -20°C. After 18 hrs they were washed and subjected to PI/RNase
staining at lxlO5 embryonic stages per ml. of suspension using the APO-
Direct apoptosis detection kit (BD Biosciences 556381) and analyzed by
flow cytometer.
Initially externalization of phosphatidyl serine, mitochondrial
depolarization, cytosolic presence of cytochrome c, activation of caspase
family of cysteine proteases and break down of caspase substrate-PARP,
fragmentation of chromosomal DNA and formation of sub-diploid nuclei
etc. were analyzed in the embryonic stages of the pathogenic filarial
nematode S. digitata. Apoptosis induction was further confirmed by
quantifying apoptosis related nematode proteins viz. CED-3, CED-4 and
CED-9. Three compounds namely Plumbagin, H2O2 and Staurosporine
were screened for their apoptogenicity against embryonic stages of filarial
parasite S.digitata.
Externalization of Phosphatidyl Serine
Phosphatidyl serine is a membrane phospholipid, usually confined to the
inner bilayer of the membrane. In mammalian cells the inner bilayer
phospholipid has been demonstrated to get translocated to the outer
bilayer during apoptosis (Elmore et al., 2007). To assess if such
externalization of phosphatidyl serine takes place in nematodes,
embryonic stages of S.digitata were subjected to Annexin-V-PE staining
and scored by flow cytometry. A dose dependent externalization of
Phosphatidyl serine was observed in Microfilariae (Mfs) (Fig. lb) as well as
in eggs (Table-1) upon treatment with Plumbagin and H2O2 while
Staurosporine did not mediate such effects on Phosphatidyl serine
externalization. The externalization of PS upon treatment was relatively
more evident in treated egg stages when compared to Microfilariae
(Table-1).
Disruption of Mitochondrial trans membrane potential
Mitochondria is known to be one of the important regulators of metazoan
apoptosis (Arnoult et al., 2002) as the signals generated by various death
promoting agents converge in mitochondria leading to reduction in trans
membrane potential (??m) in several models of apoptosis (Zamzami et
al., 2001). Embryonic stages of S. digitata were subjected to mitoscreen
JC-1 staining. A differential and dose dependent induction of
mitochondrial depolarization was observed both in Mfs (Fig. 2) and eggs
(Table-1) on treatment with Plumbagin and H2O2 but not with
Staurosporine. Mf stages displayed significantly more mitochondrial
depolarization upon treatment in comparison to eggs (Table-1).
Intra cellular profile of Proteins related to apoptosis
Intra cellular staining of the fixed embryonic stages of S.digitata with
antibodies to cytochrome c, active caspase-3 and cleaved PARP revealed
enhanced cytosolic presence of cytochrome c (Fig. 3a), activation of
mammalian caspase -3 like proteases (Fig. 3b, c) and cleavage of the
substrate of caspase family of proteases viz. PARP (Fig. 3d, e, f) in Mf as
well as in treated eggs (Table-1) undergoing apoptosis. Studies in the free
living soil nematode C. elegans had identified three nematode specific
proteins such as CED-3, CED-4 and CED-9 associated with the process
of apoptosis. Homolog of the three proteins have been found in genomes
of all animals and were shown to be involved in apoptosis in all systems
studied so far (Zamsek et al., 2007). The status of these three proteins
CED-3, CED-4 and CED-9 was investigated in embryonic stages of
pathogenic nematode S.digitata and increased intracellular expression of
all the three proteins was observed in Mfs (Fig.4 a,b,c) as well as in eggs
(Table-1). Further, shedding of CED-4 from mitochondria could also be
demonstrated (Fig.4 d, e). The degree of expression of the three proteins
was more prominent in Mfs in comparison to egg stages. However, the
proportionate increase of proapoptotic proteins CED-3 and CED-4 was
found to be more compared to anti apoptotic protein CED-9 (Table-1)
which satisfies the biological condition required for induction of
apoptosis.
Fragmentation of chromosomal DNA and Sub- diploid nuclei.
Demonstration of caspase family of cysteine proteinases as described
above was followed by investigations on analysis of nuclear features of
apoptosis i.e. fragmentation of chromosomal DNA in embryonic stages of
S.digitata by TUNEL assay and PI (propidium iodide)/RNase staining.
TUNEL assay is generally considered to be a sensitive and confirmatory
assay to detect chromosomal DNA fragmentation in apoptotic cells and
the results are shown in Fig. 5 a-c. A dose dependent induction of DNA
fragmentation in Mf stages (Fig. 5a) and Eggs (Table-1) on treatment with
Plumbagin and H2O2 but not with Staurosporine (at indicated
concentrations) was observed. Moreover, Plumbagin was found to be
more efficient than H2O2 in induction of apoptosis. PI is a fluorogenic
compound that binds stoichiometrically to nucleic acids (Ormerod et al.,
2002) so that fluorescence emission from PI stained cells is proportional
to the DNA content of the cells (provided RNA is removed by RNase
treatment). When apoptotic cells are stained with PI/RNase and analyzed
by flow cytometery a distinct hypo-diploid peak (representing the
apoptotic nuclei) adjacent to the normal diploid peak (representing
healthy nuclei) is displayed in a Histogram plot (Riccardi et al., 2006). In
the present study flow cytometric analysis after PI /RNase staining
yielded similar results (Fig.5d-f)
Table 1. GMI (Geometric Mean Intensity) of Fluorescence for each of the
9 assays (except PI/RNase staining for sub-diploid nuclei out of the 10
assays described here)

*The values in the table represent GMI (Geometric Mean Intensity) of
Fluorescence for each of the 9 assays
** Mitochondrial Depolarisation assay by Mitoscreen-JC-1-Staining-
expressed in terms of reduction in GMI of fluorescence
WE CLAIM:
1. A method for detecting apoptosis of embryonic stages of helminthes
comprising:
isolating of Intra Uterine embryonic stages from adult female parasite;
in- vitro culture and treatment of said embryonic stages;
subjecting the said embryonic stages to flow cytometric analysis;
developing assays for apoptosis in the embryonic stages;
and screening of compounds for their apoptogenicity against embryonic
stages of helminthic parasite S.digitata.
2. The method as claimed in claim 1, wherein the embryonic stages were
harvested into sterile centrifuge tubes and washed at least 3 times by
centrifuging for 10 minutes each time with medium and the final pellet
was suspended in medium supplemented with 10% FBS, subsequent to
the counting the embryonic stages under microscopy about, 1x10s
embryonic stages/ml each was dispensed into individual wells of a sterile
tissue culture plate, one set of well was left untreated ( taken as control)
and the other sets of wells were subjected to the step of treatment with
three different agents like H2O2, Plumbagin and Staurosporine
separately.
3. The method as claimed in claim 1, wherein the cultured embryonic
stages of S.digitata were harvested from different sets of wells separately,
washed with PBS, variously stained for apoptosis and the final
suspension of the stained embryonic stages was then subjected to flow
cytometric analysis.
4. The method as claimed in claim 1, wherein the assays for apoptosis in
embryonic stages of filarial nematode S.digitata are:
a) detecting externalization of phosphatidyl serine;
b) analyzing mitochondrial depolarization;
c) detecting increase in intra cellular expression of CED-3 protein;
d) detecting increase in intra cellular expression of CED-4 protein;
e) detecting increase in intra cellular expression of CED-9 protein;
f) demonstrating enhanced presence of cytochrome-c in the
cytoplasm;
g) demonstrating intra cellular activation of caspase-3 like cystiene
proteinases;
h) demonstrating intra cellular cleavage of caspase substrate PARP;
i) detecting fragmentation of chromosomal DNA and
j) demonstrating the presence of hypo-diploid nuclei.
5. The method as claimed in claim 1, wherein the assays for apoptosis
that are used for screening of apoptogenic activity of three agents i.e.
H2O2, Plumbagin and Staurosporine towards the embryonic stages of
filarial nematode S.digitata can be used for high throughput screening
and identification of broad spectrum antihelminthic compounds having
apoptogenic activity.

A method for detecting apoptosis of embryonic stages of parasitic helminthes comprising: isolating of Intra Uterine embryonic stages from adult female parasite; culturing in vitro and treating said embryonic stages; subjecting the said embryonic stages to the step of flow cytometric analysis; developing assays for apoptosis and high throughput screening and identification of compounds having apoptogenic activity towards the embryonic stages of helminthic parasites.