RESULTS

STUDY POPULATION

The study area yvas selected from few villages under Khurda district of Orissa

State that are highly endemic for bancroftian filariasis and the study group

comprised of permanent residents of these villages who volunteered to

participate in the study. The characteristics of the study population are given in

the Table 4. After the initial physical examination by a clinician, we used the

following criteria for categorizing the asymptomatic human volunteers in our

detailed study: 1- MF status by microscopic examination of night blood, 2- level

of CFA by Og4C3 ELISA, 3- antigen-specific IgG4 titer, and 4- longitudinal follow

up. From a starting population of 510 individuals (mean age 25, range 15-65, M:

F= 2.4), we finally zeroed down to 10 individuals for each category i.e. ASM and

EN. The clinical, parasitological and immunological status of individuals selected

for the detailed studies are presented in Table 5. To summarize, for all detailed

studies our population is as following:

ASM individuals: MF positive, CFA positive and high antigen-specific IgG4 titer.

EN individuals: MF negative, CFA negative and low antigen-specific IgG4 titer.

ANTIGEN ANALYSIS BY SDS-PAGE AND 20-PAGE:

For the immunological studies we have used somatic antigens from B. malayi

adult parasites (BmA). The somatic antigen preparations from adult male, female

and MF of were analyzed by gel electrophoresis and the antigen profiles are

presented (Fig 6). Densitometric scanning of the polyacrylamide gels were

carried out using Gel Base/Gel Blot™ Pro, Gel Analysis Software, UVP. SDS-PAGE

resolved the male and female adult soluble antigens and MF sonicate in to 20, 24

and 23 bands, respectively, as seen by Coomassie blue staining (Fig 6a). When

gels were Silver/Coomassie double stained, rv 45 protein bands in case of both

64

TABLE 4: CHARACTERISTICS OF STUDY POPULATION-I

Total no. of CH/Ac individuals screened

510 65

Male [360] 50

Female [150] 15

Age group· 15-65

Mean age 25

ASM

96

72

24

15-40

23

EN

CPA -ve CF A+ve

250 99

185 53

65 46

17-60 20-55

26 31

*Finger prick blood, collected between 10.00 PM-1.00 AM, was screened for microfilaria. CH- Chronic, Ac-Acute, ASM-Asymptomatic microfilaraemic, EN­Endemic normal, CF A-Circulating W. banrofti filarial antigen

TABLE 5. CLINICAL, PARASITOLOGICAL AND IMMUNOLOGICAL STATUS

OF INDIVIDUALS SELECTED FOR THE DETAILED STUDY

21 2 52 -ve 400 3 53 -ve .200 4 55 M -ve

34 M -ve 6 EN 35 M -ve

19 F -ve 15.2 200

F -ve F M

1073 1330 2431 1648

5 22 M +ve 2786 ASM 18 M +ve 1757

7 32 M +ve 1224 33 F 1521

F 1 10 19 F

* 100 U of CF A per ml was taken as the limit for positivity.

EN- Mean age= 32.7 (range: 18- 55) (M/F = 7:3) [EN individuals were longitudinally followed for atleast 3 years]

ASM- Mean age= 26.8 (range: 18- 40) (MIF = 7:3)

a

205 ~

116 ~ 97~ 84 .... 66 ~

55 .... 45 ....

36~

29~

24 .... 20 .... 14 ....

b

M 2

2 3

3

M

~ 205

.. 11 6 - .. 97 - ~84

- . .. 66

- .. ~5 5 ...... ~ 45

-- ~3 6

~29

~24

Fig. 6. 50S-Polyacrylamide gel electrophoretic profile (Laemmli

System, 10°/o gel) of Brugia malayi antigens.

Lane 1- soluble antigens from adult male parasites,

Lane 2- soluble antigens from adult female parasites,

Lane 3- soluble antigens from Mf sonicates

Lane M- Wide range markers (Sigma)

a. Coomassie-stained gels

b. Silver and Coomassie double-stained gels

a

... _

b ... pi

+ 4.5 5.1 5.5 5.9 6.6

MW 20 5 116 97 84 66

55 45

36 29

24

20

14

Fig.7. Two-dimensional (2D) electrophoretic analysis of the Brugia malayi adult (male) soluble antigens. (BmAM) Isoelectric focussing in the vertical dimension followed by SDS- polyacylamide gel electrophoresis (Laemmli system, 10% acrylamide) in the horizontal dimension separated the antigens . The proteins/ polypeptides were visualized by staining with a. CBB, b. silver /CBB double stained

a 5 .5 5-9 5-5 5.1 f. . 5 I

~--~--~----~--4-~-------p-~

•

b 5-9 5.5 5.1 4.5

----

MW

97 8 L. 66

55

L. 5 36 29

2L.

20

ll.

Fig.S .Two-dimensional (2D) electrophoretic analysis of the Brugia malayi adult (female) soluble antigens (BmAF). Isoelectric focussing in the vertical dimension followed by SDS- polyacylamide gel electrophoresis (Laemmli system, 10% acrylamide) in the horizontal dimension separated the antigens. The proteins/ polypeptides were

visualized by staining with a. CBB, b. silver/CBB double stained.

29 24

5.1 4. 5

M W rr---p-1 _6...:.·16_5--!.i9 __ 5.L·5---'""--5..l....1_4.L·5-=--..:..__

205

Fig.9. Two-dimensional (20) electrophoretic analysis of the Brugia malayi microfilarial sonicate. Isoelectric focussing in the first dimension followed by 50S-polyacrylamide gel electrophoresis (Laemmli system, 10% acrylamide) in the second dimension separated the antigens. The proteins/polypeptides were visua lize by staining with a. CBB, b. Silver/CBB double staining.

0 z .....

12

10

8

c 6 Q) ~ nl a..

4

2

0 0

Antigen (BmA)-Specific lgG Titer

§ N

2000 4000 _6000 ---8000 _j_()()QO 12000 --+4000

lgG

Fig.lO . Brugia malayiadult soluble antigen-specific IgG antibody titers in ASM (n=lO) and Ef\1 (n=lO) individuals' sera measured by EUSA

0 z .......

12

10

8

c 6 Q) +J ro a..

4

2

0

Antigen (BmA)-Specific lgG4 ~sotype Titer

~ N

::::::l

:::J

i

~

p

::::::l '

:::J

::::::l

:::J

0 1 000 2000 3000 4000 5000 6000 7000 8000 9000 1 0000

lgG4 Titer

Fig.ll. Brugia malayi adult soluble antigen-specific IgG4 isotype antibody titers in ASM (n=lO) and EN (n=lO) individuals' sera measured by ELISA.

male and female adults and rv49 bands in MF sonicate could be visualized (Fig

6b ). Similarity in protein profile could be observed between somatic Ag extracts

of male and female adult parasites and there was not much difference in the

protein profile of the MF with adult worms. However, In the mol wt range of

tv 72- 64 kd few-closely spaced, diffused bands were seen that are about 4-5 in

number in the MF sonicate and rv2-3 and tvl in male and female parasites,

respectively. These are possibly glycoproteins as they stain well with Periodic

acid- Schiffs reagent (data not shown).

By 20-PAGE separation and Silver/Coomassie double staining, the B.

malayi adult male and female parasite soluble antigens were resolved into > 85

discrete spots and MF sonicate showed tv 140 bands (Fig 7, 8 & 9). Similar to

observations made by SDS-PAGE analysis, the somatic antigen profiles of both

male and female parasites appeared very similar. The microfilarial sonicate

showed a different protein profile, although some antigens present in the adult

worm extracts were also found to be present.

BRUGIA MALAY! ADULT SOLUBLE ANTIGEN-SPECIFIC IgG ISOTYPE

RESPONSES

Titers of B. malayi adult soluble antigen-specific total IgG and IgG4 isotype

antibody present in the sera of EN and ASM individuals were determined by

indirect ELISA (Fig 10-11). The total IgG titre was higher in case of ASM

individuals in comparison to the EN individuals. The IgG4 titer in the sera of ASM

patients was very high (> 80% of the total IgG) and the same was very low in

the sera of EN individuals (Fig 11).

DETECTION OF B. malayi ADULT SOLUBLE ANTIGENS RECOGNIZED BY

DIFFERENT IgG ISOTYPES IN IMMUNOBLOT

Binding patterns of IgGl, IgG2, IgG3, and IgG4 isotype antibodies, in the sera of

both EN and ASM individuals, to adult filarial antigens separated by SDS-PAGE

65

ASM EN

2 3 4 2 3 l. MW 205- . 116-97-

84-

Fig. 12

45-

36-

29-24-

ASM EN

2 3 l. 2 3 l. MW 205~ ~¥-,. -~ ., __ 11 j ~

Fig. 13 97-l _ I l

84- l

55- r' I

Immunoblot showing lgGl reactivity (Fig.12)and IgG2 reactivity (Fig.13Jof sera from the ASM and EN individuals at a l/50 dilution with Brugia malayi adult soluble antigens (BmA). Lanes- ASM-1 , 2, 3, 4-antigens probed with ASM sera ; Lanes­EN- I, 2, 3, 4-antigens probed with EN sera .

MW 205-

84-

55 -

36-29-

20-

-

ASM EN

2 3 4 2 3 4

Fig.14 Immunoblot showing IgG4 reactivity of sera from the ASM and EN individuals at a 1/50 dilution with Brugia malayi adult soluble antigens (BmA). Lanes- ASM-1 , 2, 3, 4-antigens probed with ASM sera; Lanes-EN-1 , 2, 3, 4-antigens probed with EN sera .

a Bas ic end Acid ic e nd ~~~~--------------------~MW

--

b Basic end Aci di c e n d

· .. (

29

- 24

- 20

14

MW

29

Fig.15 2D-immunoblot. 2D-PAGE was carried out as described in fig .7. After 2D­

PAGE, the proteins were transferred to NCP and probed with a. ASM sera, b. EN sera, as detailed in figl6 to fmd out the IgG4 antibody isotype reactive antigens in BmA.

and blotted to NCP, were qualitatively analyzed. All sera were tested at a dilution

of 1/50. The antigen binding patterns for the IgG subclasses are presented (Fig

12-14). For IgG1 and IgG2, the binding was predominantly directed towards the

higher molecular weight components. IgG3 showed the least or no visible

binding among tbe four IgG subclasses (data not shown). IgG4 antibodies were

most immunoreactive and showed binding to most of the proteins in case of ASM

sera while EN sera showed reactivity towards antigens in the medium mol wt

range.

IgG4 reactivity was also analyzed by 20-immunoblot. For this, the 20-gels

after the second dimension run were processed for immunoblotting. The

immunoblot of the 20-resolved proteins with ASM and EN sera showed marked

differences in the reactivity pattern of these two groups (Fig 15 a, b). As

observed in the 20-gel, many of the proteins lighted up seem to have several

isoforms as they appear as a row of bands.

ANTIGEN FRACTIONATION FOR STIMULATION OF PBMCs AND CELl

CULTURE STUDIES

On the basis of 10- and 20- immunoblot, the IgG4 reactivity pattern could be

grouped into 5 antigen fractions or immunoreactive zones (Fr 1: > 205-rv90kd,

Fr 2: < 90-rv53kd, Fr 3: < 53-rv38kd, Fr 4: < 38-rv24kd and Fr 5 < 24-rv14kd)

(Fig 16). The fractions 1 and 4 showed reactivity with both ASM and EN sera

while the Fr 2 and Fr 3 reacted preferentially with the ASM sera. Fr 4 contained

many of the isozymic proteins that are picked up both by ASM and EN sera. This

IgG4 immunoreactivity pattern was the criteria, basing on which the B. malayi

adult soluble antigens were fractionated by SOS-PAGE and transferred to NCP.

The NCP-bound antigens were converted into fine antigen-bearing particles,

following method described by Abou-Zeid et a/. (1987) for lymphocyte

stimulation and study of cytokine profile in the ASM and EN individuals.

66

MW

205 ... Fr. 1

116 ... 97 ... SL..,.. 66 ...

Fr. 2

55 ... 45 ...

Fr. 3

36 ...

29 ... Fr. 4

24 ...

20 ... Fr. 5

1 L. ....

Fig. 16. Fractionation of Brugia malayi adult soluble antigens (BmA).The BmA were SDS-PAGE fractionated and then transferred electrophoretically to NCP. The NCP was cut in to fivefractions (Fr.l-5) on basis of the IgG4 reactivity of the BmA with ASM and EN sera . These NCP-bound antigens were used to study their ability for induction of cytokine secretion or expression of ce ll surface markers in PBMC culture of the · ASM and EN individuals.

CELL CULTURE: STANDARDIZATION

OPTIMAL ANTIGEN CONCENTRATION REQUIRED: FOR STIMULATION

OF PERIPHERAL BLOOD LYMPHOCYTES

For determining the antigen concentration at which the cells are optimally

stimulated, we cultured the PBMC collected from endemic normal individuals in

the presence of different concentrations of SDS fractionated B. malayi antigens,

B. malayi total somatic antigen (BmA) and H37Rv for 5 days following 20 h

pulsing with 3H -Thymidine. The optimal concentration for different Ags are as

stated: BmA - 5r.-tg/ml, Fr 1 -10~g/ml, Fr 2 - 51Jg/ml, Fr 3 - 101Jg/ml, Fr 4 -

51Jg/ml, Fr 5 -51Jg/ml (Fig 17); and H37Rv whole lysate (irrelevant Ag)- 101Jg/ml.

Therefore, in all subsequent experiments the optimal concentrations of various

antigens were used to stimulate PBMC proliferation and cytokine secretion. The

quantity of IL-2 secreted and SI data are presented (Table 6).

KINETICS OF INDUCTION OF CYTOKINE PRODUCTION IN PERIPHERAL

BLOOD LYMPHOCYTES BY OPTIMAL CONCENTRATION OF DIFFERENT

ANTIGENS:

PBMC collected from the ·endemic normal individuals were cultured individually in

the presence of optimal cone. of different antigens, viz. SDS-PAGE fractionated &

NCP-bound B malayifractions (1-5), the adult soluble antigen extract (BmA) and

H37Rv. (NCP control was taken to account for any base line stimulation in

absence of any bound antigens), for 24h, 48h, 72h and 96h and secreted

cytokines in culture supernatants were quantified by sandwich ELISA. Kinetics of

mean IL-2, IL-4, IL-12 and IFN-y secretion by the lymphocytes from the EN

individuals in response to B. malayi adult soluble antigens (Fig 18), fractionated

BmA antigens: Fr.1 (Fig 19), Fr.2 (Fig 20), Fr. 3 (Fig 21), Fr.4 (Fig 22) and Fr.5

67

TABLE §: IL-2 IN pglml SECRETED AND LYMPHOCYTE PROLIFERATION INDUCED BY FILARIA PARASITE ANTIGENS AND H3,Rv TOTAL LYSATE IN PBMC FROM ENDEMIC NORMAL (EN) AND ASYMPTOMATIC MICROFll..ARIAE CARRIER (ASM) INDIVIDUALS.

Antigen

Fr-1

Fr-2

Fr-3

Fr-4

Fr-5

BmA

H31Rv

NCP control

EN

Mean IL-2 (Range) inpglml

79.6 (67.0-91.0)

205.6 (177.0-247.0)

217.4 (169.0-276.0)

272.8 (198.0-331.0)

238.0 (174.0-295.0)

225.8 (207.0-263.0)

373.8 (308.0-46 1.0)

76.6 (55.0-91.0)

S.I

1.07 (0.2-1.5)

5.43 (4.4-6.4)

4.2 (2.9-4.9 )

4.31 (3.7-5.7)

3.05 (2.1-4.0)

5.26 (3.9-6.3)

20.34 (16.1-24.9)

0.95 (0.4-1.7)

ASM

Mean IL-2 (Range) inpglml

61.71 (31.0-98.0)

223.14 (196.0-287.0)

189.14 (107.0-233.0)

222.42 (182.0-264.0)

225.14 (176.0-256.0)

208.14 (104.0-305.0)

397.14 (312.0-478.0)

80. 71(63.0-93.0)

S.l

0.87 (0.1-1.9)

4.04 (2.5-6.7)

4.13 (2.2-5.9)

2.68 (1.2-4.8)

4.61 (2.7-6.4)

3.81 (2.1-6.3)

13.82 (9.4-19.1)

0. 77(0.2-1.2)

Fr-1, 2, 3, 4 and 5 are SDS-PAGE fractionated and NCP bound antigens of Brugia malayi adult

soluble antigens having different molecular weight range <Mr): Fr-1, >205-90 kd; Fr-2, <90-53 kd;

Fr-3, <53-38 kd; Fr-4, <38-24 kd; Fr-5, <24-14 kd BmA, B. malayi adult soluble antigen

(unfractionated) H37Rv; a strain of Mycobacterium tuberculosis.

7

6

5

~ "C c

4 c 0 :-=-ro '"5 3 E

:-=-en 2

1

0 0 2 4 6 8 10 12 14

Antigen concentration (J.LQ I ml)

Fig 17. Stimulation index of lymphocytes taken from EN individuals (n=6) when cultured in the prese-nce-of different cone. of Brugia malayiwhole lysate and SDS-PAGE fractionated and NCP- bound B. malayi adult antigen fractions (Fractions 1, 2, 3, 4 & 5).

-e- Bm Adult Whole Lysate • Fraction 1

-e- Fraction 2 ___.._ Fraction 3 _._ Fraction 4 -Fraction 5

16

1200

1000

800

E 600 -0> a.

400

B. malayi adult solubte-antigens -{unfractionated)

~IL-2

-e- IL-4 ..,..,._,_ IL-12 ~ IFN-y -e- IL-10

24 48 72

Time in hours

Fig 18. Kinetics of secretion of IL-2, IL-4, IL-12, IFN-y and IL-10 by the lymphocytes of EN Individuals (n=6) when cultured with optimal con of Brugia malayi adult soluble antigens.

96

E -0> a...

BmA Fr.1

150

---- IL-2

125 __..... IL-4 ~ IL-12 --e- IFN-r --+-- IL-10

100

75

50

25

o~,-------------.-------------.,-------------.-~

24 48 72

Time in hours

Fig 19. Kinetics of secretion of IL-2, IL-4, IL-12, IFN-y and IL-10 by the lymphocytes of EN individuals (n=6) when cultured with optimal cone of SDS-PAGE fractionated and NCP­bound Brugia malayi adult soluble antigen Fr. 1.

96

1200

800

E -0) c..

400

. BmA Fr.2

=e= IL-2

----- IL-4 --- IL-12 =e= IFN-y

--- IL-10

24 48 72

Time in hours

Fig 20. Kinetics of secretion ofll-2, lL-4, lL-12, 1FN-y and ll-10 by the lymphocytes of EN individuals (n=6) when cultured with optimal cone of sos~PAGE fractionated and NCP­bound Brugia malayi antigen Fr. 2.

-96

E 0> a.

BmA Fr.3

1000.---------------~~----------------------------.

-e-= IL-2 ~IL-4

800 ,...__ IL-12 ---.= IFN-y

- IL-10

24 48 72

Time in hours

Fig 21. Kinetics-of secretion of IL-2, IL-4, IL-12, IFN-y and IL-10 by the lymphocytes of EN individuals (n=6) when cultured with optimal cone of SDS-PAGE fractionated and NCP-bound Brugia malayi adult antigen Fr. 3.

96

E 0> 0..

BmAFr. 4

1000.-------------~-------------------------------.

800

600

400

200

=e= IL-2 __._ IL-4 ___...., IL-12 =-= IFN-r ---+-- IL-10

24 48 72 96

Time in hours

Fig 22. Kinetics of-soor-et-ioo-ofJL-2. JL-4, IL-12, IFN-y and JL-1 0 by the lymphocytes of EN individuals (n=6) when cultured with optimal cone of SDS-PAGE fractionated and NCP-bound Brugia ma/ayi adult soluble antigen Fr. 4.

BmA Fr.5

1200.-----------------------------------------------~

1000

800

~ 600 a.

400

200

e IL-2 -e- IL-4 .,..,...._.. IL-12 .....,...IFN-y -e- IL-10 ·

24 48 72

Time in hours

Fig 23. Kinetics of secretion of IL-2, IL-41 IL-121 IFN-y and IL-10

96

by the lymphocytes of EN individuals (n=6) when cultured with optimal cone of SDS-PAGE fractionated and NCP-bound Brugia malayiantigen Fr.S.

(Fig 23) are presented. It was observed that IL -2 and IFN-y were secreted

maximally at 72h and 96 h, respectively, and IL-4 and IL -12 were secreted

maximally at 48h and IL-10 was secreted maximally at 24h. Therefore, in all

subsequent experiments we measured secretion of IL-10.-at 24h; IL-4 and IL-12

at 48h; IL-2 at 72h; and IFN-y at 96h in the culture supernatants.

INDUCTION OF DIFFERENTIAL IL-2 SECRETION AND PROLIFERATION

OF PERIPHERAL BLOOD MONONUCLEAR CELLS BY B. malayi ADULT

ANTIGEN FRACTIONS:

Level of cytokine IL-2 and 51 are measures of T cell proliferation. We have

compared these two values to find out the ability of the different antigens to

stimulate T cell proliferation and secretion of IL-2 in PBMC of the two groups

under in vitro culture conditions (Table 6). SI of EN individuals for all the

antigens tested are higher than that of ASM individuals and the SI is highest for

H37Rv whole cell lysate.

It was observed that BmA Fr.1 caused negligible stimulation of PBMC as

seen by the low IL-2 level and SI (Fig 19). This fraction contained few high mol

wt proteins that are present in very low cone. The low stimulation of cells by this

fraction may be due to t~e poor immunogenecity of the proteins in this fraction,

for which reason the Fr.1 was excluded from all further analysis.

CD4/CD8 POPULATION IN EN AND ASM INDIVIDUALS AGAINST

DIFFERENT ANTIGENS BY FLOW CYTOMETRY

The PBMCs were collected and CD3+ cells were purified on MACS column and

they were stained and visualized to see the CD4+ (T helper, Th) and cos+ (T

cytotoxic, Tc) population. Few unstained cells were also analyzed to check the

68

g r-----~----~~~----~--------0

0 0 N

. : .-: ':· .

: .. .. - ....

FSC~

Fig. 24. Forward vs. Side scattergram obtained upon flow cytometric analysis of unsta ined human PBMC population.

Rl Lymphocytes R2 IVJonocytes R3 Gra nulocytes R4 Dead ce ll s

population of viable cells (Fig 24). When plotted the kinetics of appearance of

these membrane surface molecules against the different antigens, the peak was

found to be at 96hr, at which all data are shown. All the analysis done in ASM

(n=3) and EN (n=3) showed similar results and the· results presented are

representative ones (Fig 25). With no antigens, there is more expansion of CD4+

in ASM individuals as seen from the contours. With Fr.S the ASM individuals

show expansion of a small number of double positive cells. However with BmA,

the expansion of CD4+. cells is not seen as expected.

CELL ACTIVATION ANALYSIS- STUDYING EXPRESSION OF CD4/CD25

AND CD4/ CD4SRO BY FLOW CYTOMETRY

To show T-cell activation, CD4+CD25+ and CD4+CD45RO+ population was

analyzed. CD45RO is also an effector/ memory cell activation marker and we

looked at the CD4+ cells for the expression of CD4RO+ ((Fig. 26). With Fr.S

antigen, there is much better activation of CD4+ cells in EN population in

comparison to ASM. This antigen population is capable of better expansion of

CD45RO+ and CD4+ cells.

The expansion of CD4.CD25 is similar in both EN and ASM groups in

absence of any antigenic stimulation. With all the three antigens, viz. H37Rv,

BmA and BmA- Fr.S, there is higher expansion of CD4+ cells and there is more

expansion of double positive cells in EN as compared to the ASM individuals (Fig

27).

DIFFERENTIAL INDUCTION OF CYTOKINES IN PBMC CULTURE OF EN

AND ASM INDIVIDUALS BY DIFFERENT ANTIGENS

69

EN

~~----~--------~

M 0

~0 __._ u. l------+---------;

0

~~-----.-------~

~0~~ ~-~-----r---------; -0

~~----~-------~

M 0

~~-----.--------~

("')

0

ASM

~~-----.--------~

M 0

~0 ~-~-----+---------;

0

~~----,--------~

~0 ~-~~--~--------~

-0

~~-----.--------,

("')

0

~ ~s;;~ N C>

~-~----~--------;

C>

~~-----.--------,

0 ~ ~~ov NO

~ - '1--------t------------1

-C>

NoAg

H37 Rv

BmA

Fr-5

Fig.25. Representative data of Flow Cytometry analysis of CD3+ (MACS purified) lymphocyte population expressing CD4 and/ or CDS in EN (n= 3}, and ASM (n= 3) individuals in response to different antigens. PBMCs were collected after 96h of culture and stained for CD4 and CD 8. K-S statistics was applied and the values given. H37Rv- H37Rv whole cell lysate; BmA- Brugia malayi adult soluble antigens; Fr. 5- SDS-PAGE fractionated and NCP-bound antigen Fr. 5 (tv <24-14kd) of BmA.

EN

~------~------~

~~----~-------,

M 0

=f<\1 (\10

Li - ~----=d'7""7'19'ft'-"'"'-----j

CD45RO

-0

~~----~------~

M 0

ASM

NoAg

~~----~-------.

H37 Rv

~~-----.--------·

BmA

~~-----.--------.

Fr-5

Fig.26. Representative data of Flow cytomeby analysis of lymphocyte population expressing CD4 and/or CD4SRO in EN (n~ 3), and ASM (n= 3) individuals in response to different antigens. PBMCs were collected after 96h of culture and stained for CD4 and CD45RO. K-5 statistics was applied and the values given. H37Rv- H37Rv whole cell lysate; BmA- Brugia malayi adult soluble antigens; Fr. 5- 50S­PAGE fractionated and NCP-bound antigen Fr. 5 (.v <2~14~d)of BmA. ·

EN ~~-----.--------,

(")

0

~ NO

~-~--~~~~----~ 0

~~~~~ NO

~ - ~----;;;i!i!~---::;'"'rMT--------1

ASM ~~----~--------,

~~----~--------~

~~-----.--------,

~~----~--------~

(")

0

~ NO

~-~~~~~~----~

-0

NoAg

H37 Rv

BmA

Fr-5

Fig.27. Representative data of Flow cytometry analysis of lymphocyte population expressing CD4 and/or CD4 CD25 in EN (n= 3),and ASM (n= 3) individuals in response to different antigens. PBMCs were collected after 96h of culture and stained for CD4 and CD25. K-5 statistics was applied and the values given. H37Rv- H37Rv whole cell lysate; BmA­Brugia malayi adult soluble antigens; Fr. 5- SDS-PAGE fractionated and NCP­Bound antigen Fr. 5 (tv <24-14kd) of BmA.

To characterize the patterns of cellular responses in each group, levels of IL-2,

IFN-yD, Il-4, IL-10 and IL-12 were measured in supernatants or PBMC culture of

individuals belonging to EN and ASM category. As it is known that the cytokines

IL-4/IFN-y and IL-10/IL-12 are antagonistic, we have plotted them against each

other in 2-D plots in EN and ASM individuals for comparing the secretory

cytokine induction profile, using different antigens viz. fractionated B. malayi

antigens (Fr. 1-5), unfractionated adult worm lysate and H37Rv (Fig 28- 39).

H31Rv: H31Rv is a prototype strain of the M. tuberculosis and it is well

established that Mycobacterial antigens and PPD are Th1 inducer. In the present

study, it was found that both the EN and ASM categories of individuals showed

higher amount of IFN-y and IL-12 secretion (Th1 response) in comparison to IL-4

and IL-10. The IL-4 vs. IFN-y and IL-10 vs .. IL-12 (Fig 28, 29) show that except a

few individuals who produce less IFN-y most of the EN and ASM individuals show

a similar Th1 response against H37Rv whole cell lysate, as expected.

BmA {Adult Soluble Antigen, Unfractionated): The BmA (unfractionated,

adult worm soluble antigen) induced differential secretion of IFN-y and IL-12 and

IL-4 in EN and ASM individuals; the EN individuals showed a differential Thl

response (higher IFN-yD and IL-12) whereas the ASM individuals showed Th2

responses (higher IL-4) ~ith the unfractionated lysate (BmA) which can be easily

marked from the 2-D plots; IL-4 vs. IFN-y (Fig 30) and IL-10 vs. IL-12 (Fig 31).

Fraction 2: The BmA Fr 2 contains proteins of molecular weight from < 90 to rv

53 kd. The antigen fraction showed a mixed response when plotted as IL -4 vs.

IFN-yD (Fig 32) and IL-10 vs. IL-12 (Fig 33).

70

..........

E -C) a. -1"

....J

H37Rv-whole:ceH~ysate

~~------------------------------------------~

40

30

20

A

• 10 A • •

~ ~

• ~.

A - • • • A •

0+-------~--------~------~--------~------~

0 400 800 1200 1600

IFN-y (pg I ml)

Fig 28. IL-4 vs. IFN-y plot: IL-4 and IFN-y secreted by the lymphocytes of ASM (n= 10) and EN (n= 10) individuals in response to H37Rv whole ceU Jysate. [Ib.-4: P=0.5967, IEN-y: R=O.Q022].

2000

800

700

600

""""' 500 E -0> a. 400 ......... 0 T"""

I ...J 300

200

100

0 0

H37Rv whole cell lysate

.. § .A A M

~ • ~ ~ • -.A • •• • ~

• • • • ••

•

100 200 300 400

IL-12 (pg/ml)

Fig 29. IL-10 vs. IL-12 plot: IL-10 and IL-12 secreted by the lymphocytes of ASM (n=10) and EN (n=10) individuals in response to H37Rv whole cell lysate [IL-10: P= 0.0013, IL-12: P=0.2568].

500

::::-E -0> a. .........

""" I ...J

Brugia malayladuJt soluble antigen -(-BmA}

350.-----------------------------------------~~

280

210

140

,A

70 • • • •

el ••

~­~

•

0+-------~--------~------~--------~------~

0 200

Fig. 30.

'

400 600 800

IFN~ (pg I ml)

IL-4 vs. JEbl"f'_plot:..J.l4 and IFN--y secreted by the lymphocytes of ASM and EN individuals in response to Brugia malayi ad•1lt soh 1ble antigen (optimal conc.5~g/mi)[IL-4: ~< 0.0001, IFN-y: P=0.0017].

1000

1000

800

.E 600 rn a. -0 ...--

I 400 ...J

200

0 0

Brugia malayi adult soluble antigen (BmA)

'- § ~

M

:!. £

a. r il.

-· ~ •

•• • - • • • ·- • 50 100 150 200 250 300 350

IL-12 (pg/ml)

Fig 31. IL-10 vs. IL-12 plot: IL-10 and IL-12 secreted by the lymphocytes of ASM (n=10) and EN (n=10) individuals in response to Brugia malayi adult soluble antigen (optimal conc.S~g/ml) [IL-10: P<0.0001, IL-12: P= 0.0452].

400

E -0> a.. .._... "':t

I ...J

BmA Fr.2

150

§ 120

M

• 90 • • ... • • • • • .. 60

• .. • 30

0+-----~----~----~----~------~----~--~

0 100 200 300 400 500 600

tFN-y (pg/ml}

Fig 32. IL -4 vs. IFN-y plot: IL -4 and IFN-y secreted by the lymphocytes of ASM (n=lO) and EN (n-10) individuals in response to SDS-PAGE fractionated and NCP-bound, Brugia malayi adult antigen Fr. 2 ( <90-53kd, optimal cone Smg/ml) [IL-4: 0.9097, IFN-y: 0.4057].

700

350

300

250

• E 200 -0> Q.

............

0 ..- 150 I ...J

100

50

0 0

BmA Fr.-2

~- § .6. M

~ .. A

~ :I.

~

• -· .. • -.o-

• • ' 50 100 150 200 250 300 350

IL-12 (pg/ml)

Fig 33. IL-10 vs. IL-12 plot: IL-10 and IL-12 secreted by the lymphocytes of ASM (n=10) and EN (n=10) individuals in response to SDS-PAGE fractionated and NCP-bound, Brugia malayi adult antigen Fr.2 (90-53 kd, optimal conc.S!lg/ml) [IL-10: P= 0.0001, IL-12: P=0.8501].

Fraction 3: Fr 3 contains proteins in the molecular weight range of< 53-38kd.

This cytokine induced by the antigens of this fraction shows a mixed response as

seen from the plots of IL-4 vs. IFN-y (Rg 34) and IL-10 vs. IL-12 (Fig 35).

Fraction 4: This fraction contains some highly immunogenic proteins in the

molecular weight range of < 38 kd to 24 kd that are recognized by the IgG 4

isotype antibody of both ASM and EN sera (Fig. 14, 15). These antigens induce a

differential immune response (Th1 type in EN vs. Th2 type in ASM) as seen from

the IL-4 vs. IFN-y and IL-10 vs. IL-12 plots (Fig 36, 37). It is noteworthy that

even the antigen-specific IgG4 titer is quite low in EN individuals compared to

the very high titer in ASM, both the groups strongly recognize the antigens in Fr.

4 (which typically appear as a row of bands) in 2-D immunoblot (Fig 15).

Fraction 5: The fraction 5 contains the low molecular weight protein < 24 to

14kd, which are uniquely recognized by the IgG4 isotype antibodies in ASM sera.

This fraction also shows a highly polarized Th1{Th2 response in EN/ASM

individuals like the Fr.4. The EN individuals produce high level of Th1 cytokines

(IFN-y and IL-12, P< 0.0001 for both) and ASM individuals secrete higher Th2

cytokines (IL-4 and IL-10, P< 0.0001 for both) (Fig 38, 39).

INTRACELLULAR STAINING OF CELLS PRODUCING CYTOKINES IFN-y,

IL-4 AND IL-10 & CELL FREQUENCY ANALYSIS

The typical Th2 response by the ASM individuals (to Fr. 5 and Fr. 4) are filarial

antigen specific and do not imply any inherent inability of these individuals to

produce Th1 cytokines, as they produce comparable (with EN) amounts of IFN-y

and IL-12 against H37Rv whole cell lysate. Thus, this response is filarial antigen

driven and clearly there is a bias to these filarial antigens in EN/ ASM individuals.

· With these interesting findings from secretory cytokine studies, we wanted to

focus on Fr. 5 for further analysis. The BmA Fr.S was analyzed further for their

71

BmA Fr. 3

150.----------------------------------------------.

. 120

E' go -0> a.

::!; 60 A

30

• • • • • -. •

••

•• •

f

~§~·--- • "'l:·N.· : .. : .. : . ~ . . . & ASM·:

. . . . . .

0+--------,,--------.--------.---------.-------~

0 200 400 600 800

IFN;.;.y (pg tml)'

Fig 34. IL -4 vs. IFN-r plot: IL -4 and IFN-r secreted by the lymphocytes of ASM.(h=lO}and.EN (n=lO)ihdivi­duals in response to SDS-PAGE fractionated an NCP­bound B. malayi adult antigen Fr. 3 ("' 53- 38 kd, optimal cone 10J.LQ/ml). [IL-4: P = 0.0022, IFN-r: P=0.1405].

1000

600

500

400

E -0> a. 300 .......... 0 ..-

I ...J

200

100

0 0

BmA Fr.-3

~-A § M

~ .i.

A A

~

a

• • ~ e

• ~- • • • e

50 100 150 200

IL-12 (pg/ml)

Fig 35. IL-10 vs. IL-12 plot: IL-10 and IL-12 secreted by the lymphocytes of ASM (n=10) and EN (n=10) individuals in response to SDS-PAGE fractionated and NCP-bound Brugia malayi adult antigen Fr.3 ( "'53-38 kd, optimal cone. 10J..Lg/ml) [IL-10: P=0.0001, IL-12: P=0.0211].

250

~

E -0> Q_

......... "\t

I _J

BmA·hA

200~--------------------------------------------~

160

120

80 a

40

0 0

Fig36.

~ A a

a.

• 200

A

• A\ • • • "' •• • 400 600

IFN-y (pg I ml)

A

-~­~

800

IL-4 vs. IFN-y plot: IL-4 and IFN-y secreted by theo lymphocytes of ASM (n=10) and EN (n=10) individuals in response tQ·SDS-PAGE fractio'nated andNCP­bound Brugia malayi adult soluble antigen fraction 4 (-38-24 kd, optimal conc.5!lg/mi)[IL-4:P=0.0001, IFN-y: P=0~00011--_

1000

800

700

600

..--.. 500 E -Ol

~- 400 0 .......

I ...J 300

200

100

0 0

BmA Fr.-4

A § A

M

A

A A

~

• ,!.

j.

A • • - . • . - - . • • •

I

50 100 150 200 250 300

IL-12 (pg/ml)

Fig 37. IL-10 vs. IL-12 plot: IL-10 and IL-12 secreted by the lymphocytes of ASM (n=10) and EN (n=10) individuals in response to SDS-PAGE fractionated and NCP-bound, Brugia malayiadult antigen Fr.4 (38-24kd, optimal cone. Sllg/ml) [IL-10: P<O.OOOl, IL-12: P<0.0001].

350

BmA F.r.5 - .-_

200.--------------------------------------------.

150

E -~ 100 -

50 •

•

• •• • - . . - . •

0+-------~--------.--------.--------,--~--~

0 200 400 600 aoo

IFN-y (pg I ml)

Fig. 38 IL-4 vs. IFN-y plot: IL-4 and IFN-y secreted by the lymphocytes of ASM and EN individuals in response to SDS- PAGE fractionated and NCP bound, Brugia malayi adult antigen fraction 5 (-24-14 kd, optimal cone. Sllg/ml). P=0.0025.

1000

aoo

700

600

........... 500 E -0> c.. 400 -0 ...-

I ....J 300

200

100

0 0

BmA Fr.5

:r § M !

A .. ••

A ~

~

• • • • .. • • • •e

50 100 150 200 250

IL-12 (pg/ml)

Fig 39. IL-10 vs. IL-12 plot: IL-10 and IL-12 secreted by the lymphocytes of ASM (n=10) and EN (n=10) individuals in response to SDS-PAGE fractionated and NCP-bound, Brugia malayiadult antigen Fr.S (24-14kd, optimal cone. 51-ig/ml) [IL-10: P<0.0001, IL-12: P<0.0001].

300

ability to produce the polarized immune response by flow cytometric analysis of

internal cytokines and cell surface receptors/molecules.

The internal cytokines viz. IFN-y, IL-4 and IL-10,: were analyzed by flow

cytometry with BmA Fr.S, H37Rv and BmA, in both EN and ASM individuals. IFN­

Y levels in EN individuals against both BmA and its Fr. 5 was appreciably higher

in comparison to ASM individuals and between the two antigens Fr.S induced

better response (Fig 40). H37Rv induced high IFN-y in both the groups, which is

quite expected. Population of IL-4 producing cells was quite high in ASM

individuals in comparison to EN against both BmA and Fr.S and against Fr.S

antigens IL-4 producing cell density is higher than that of BmA. IL-4 producing

lymphocyte population was almost similar in both the groups against H37Rv (Fig

41). Analyses of IL-10 levels indicate that density of lymphocyte population

against both BmA and Fr.S is quite high in comparison to the ASM group (Fig

42). Proper controls were also included in the study (Fig 43).

EXPRESSION OF IL-4R, IL-10R AND IL-12R IN PBMCs OF EN AND ASM

INDIVIDUALS

We then correlated the resurts. of secretory cytokine and internal cytokine

produced by PBMC population in EN and ASM individuals with expression of the

receptors for IL-4, IL-10 and IL-12 on cell membrane, by flow cytometry. When

lymphocytes were stimulated with Fr.S and BmA total lysate, IL-4 receptor

expression was very low in EN individuals (Fig.44). Similarly, the turn over of IL-

10 receptor expression is higher in the ASM individuals (Fig.45). IL-12 receptor

expression is higher in EN individuals (Fig.46). Thus, secretory cytokine and

internal cytokine levels are well reflected in the profile of their receptor

expression.

72

0 ~

0

~

0 .. "' c :3 0

O o

"'

0 M

0 ~

C>

~

0

"' .. c :3 0 O o

"'

C> M

C>

0 0

8 .23 ~ 5.15 ~ 5 .32

0 0

~ ~ Ml Ml Ml

0 0 .. "' .. "' c c :3 6 0 O o O o

"' "' 0 ~ M

0 j '*\.. __

0 ~ ~-103 104 10° 101 102 103 10" 10° 101 102 103 10"

Fl1~ '. FL1~

C> C>

4.08 ~ 2. 19 ~

1.6 8

0 C>

Ml ~ ~ Ml Ml

0 0

"' "' .. .. c c :3 :3 0 0 O o O o

~ "' "'

C> C>

l) M M

~ C>

) l C>

j \. 10° 101 ,oz 103

FL1~

H37Rv

104 100 101

BmA

I so type Control

No Antigen

102 103 104 10° 101 102 103 104 Fl1~ Fl1~

Fraction-S

Fig.40. Representative data of Flow cytometric analysis of Internal cytokine- IFN-y in -lyntphocytes of EN (n= 3), -and ASM (n= 3) individuals in response to different antigens. K-5 statistics was applied and the values given. H37Rv- H37Rv whole cell lysate; BmA- Brugia malayi adult soluble antigens; Fr. 5-SDS-PAGE fractionated and NCP-bound antigen Fr. 5 (rv <24-14kd) of BmA.

EN

ASM

0 0 ..,

0 1.01

.., 1.48

.., 1.19

0 0 "' "' 0

"' 0

0 "' "' 0 Ml "' Ml

"' Ml i:o .. .. :>M i:o i:o 0 :>M EN (.) 0 :3M

(.) 0

..

0 N

~ \_ 0

10° 101 102 103 104 FLH-t

~....------------,

0 .... Ml

1·53

0 N

~

0

100

0 ..,

:il

0

"'

(.)

0 N

~ L 0

100 101 102 103 104 FL3~

0

4.26 ..,

4.49

0 ..,

Ml Ml 0 .... ASM

§~ .. "' i:o i:o :>M :3M 0

(.)

0 N

0 (.)

0 N

0 (.)

0 N

0 l 0 ~.

10° 101

H37Rv --BmA

Isotype Control

No Antigen

-.ICractien 5

Fig.41. Representative data of Flow cytometric analysis of Internal cytokine- IL-4 in lymphocytes of-EN (n= 3), end ASM (n= 3) individuals in response to different antigens. K-S Statistics was applied and the values given. H37Rv- H37Rv whole cell lysate; BmA- Brugia malayi adult soluble antigens; Fr. 5 505-PAGE fractionated and NCP-bound Ag Fr. 5 ("' <24-14kd) of BmA.

0

~

0 .. 0 oD ..

;: " 0

<> o ..,.

0 N

0

0

~

0 00

0 oD .. ;:

::s 0 <>o ..,.

0 N

5.64

Ml

~ ~.A 100 101 102

FL2~

Ml

102

FL2~

H37Rv

103 104

6. 8 4

103 104

lsotype -Control

0

~

0 00

0 oD ..

;: ::s 0 u o

. ..,.

0 N

0

~

0 00

0 oD

"' ;: ::s 0 <>o ..,.

0 N

0

10°

BmA

No Antigen

101

0

1 .11 ~ 1. 96

0 00

Ml Ml

0 oD ..

§ EN 0 <>o ..,.

., . 0 N

0 \.

103 104 10° 101 102 103 104

FL2~

0

4-95 ~

H> 6

0 .. Ml Ml ASM

0 oD

"' ;: ::s 0 <>o ..,.

0 N

102 103 104 103 104 FL2~

Fraction-s

Fig.42. Representative data of Flow cytometric analysis of internal cytokine- IL-10 in lymphocyteS--Qf E~ -{n= 3), .aRd-A~M -{n= 3) individuals in response to different antigens. K-5 statistics was applied and the values giv~-H37Rv- H37Rv whole cell lysate; BmA-~i adult soluble antigens; Fr. 5-SDS-PAGE fractionated and NCP-bound antigen Fr. 5 (""' <2Zl-=Mkt1) ufi3m"A.

0 00

0 ~

0 .0

0 In

VI co :3'11" 0 u

0 M

0 N

0

0 0 00 00

0 0 ~ ~

0 0 .0 I .0

.;-: Ml

I 0

fl, 0

In In

Ml VI VI co co ~v :3'11"

( 0 u Ml u

0 0 M M

0 0 N N

0 0

0

to3 to3 to4 103 to4 to0 tot to2

FL3-H

IFN-g positive IL -1 0 positive IL-4 positive tonttol control control

Fig.43. Overlay histograms of isotype control, recombinant cytokine neutralized control, and positive control of internal cytokine staining for IFNr (~, IL-10 (B), and IL-4 (C).

to4

C> C> C> ~

1. 03 ~ 0.4 8 ~ 1.07

C> C> C>

"" "" "" Ml Ml Ml

C> C> C> .. "' "' "' .. .. c c c EN " " " 0 0 0 V o V o Vo .... .... ....

C> C> C> "' j_ "' "'

C> \,.. J \... IJ \.

to0 tOt to2 103 to4 C> C>

tOO tot to2 to"3 to4 tOO tot to2 to3 to4 FLHi Flt+t Flt+t

g....----------, g-r---------, §-.---------. 6.96

C>

""

121

Ml

H37Rv

C>

""

-BmA

lsotype Control

No Antigen

6.26

Ml Ml

ASM

--F..r.action 5

Fig.44. Representative data of Flow cytometric analysis of lymphocyte population expressing IL-4R in EN (n= 3), and-ASM {n= 3) individuals in response to different antigens.J{::S....statistics was applied and the values given. H37Rv- H37Rv whole cell lysate; BmA- Brugia malayi adult soluble-antigens; Fr-.5- 505-PAGE fractionated and NCP-bound antigen Fr. 5 (rv <24-14kd) of BmA.

0

S!

0 00

0 .., .. c " 0 O o ....

0 N

0

S!

0 00

0 .., .. c " 0 Oo ....

0 N

0 0 S! S! 0 -97 1.30 O.B9

0 0 00 00

Ml Ml Ml

0 0 .. .., .., EN c .. "

c 0 " <> o

~ 0

A <>o .... ....

0 0 N N

0 \. \. 102 103 104

0

10° 101 102 103 104 100 101 102 103 104 Fl2.Jol Fl2.Jo! Fl2.Jol

0

~ J .JJ S! 4.46 4 .9 B

~ 0 00 ASM Ml Ml Ml

0 0 .., .. .. .., c ~ " " 0 0 Oo Oo ....

~ ....

0 0 v N N

~ ~ 0 0

103 104 10° 101 102 103 104 100 101 102 103 10'1 Fl2.Jol Fl2.Jol

H37Rv -SmA Fraction 5

lsotype - Control

No Antigen

Fig.45. Representative data of Flow cytometric analysis of lymphocyte pepulation expressiftg ll. lOR iR 91--(n= 3}, and ASM {n= 3) individuals in-.response to different antigens. K-5 statistics was applied and the values given. H37Rv- H37Rv whole cell lysate; BmA- Brugia malayi ad••lt soh •ble antigens; Fr. 5- -SDS-PAGE fractionated and NCP-bound antigen Fr. 5 ("' <24-14kd) of BmA.

0 0 0 ~ 4.28 ~ 3·39 ~ 5-56

0 0 0 co 00 00

Ml I j Ml Ml 0 0 0 .. "' .., "' <: .. .. EN <: <: " " " 0

<> o 0 0 V o <> o v

0 N

103 104

8 ..,-------,

0 00

H37Rv

3.8 8

Ml

Isotype Control

No Antigen

v

104

8 ..,------.....,

0 00

Ml

1 59

...

0 N

103 104

8 ...------- -,

0 00

Fraction 5

1.07

Ml ASM

Fig.46. Representative data of Flow cytometric analysis of lymphocyte population-expressing IL-l~R in EN (n= 3), and ASM {n= 3) individualsJn response to different antigens. K-S statistics was applied and the values given. H37Rv- H37Rv whole cell lysate; BmA- Brugia mafa,vi ad••lt soh •ble antigens; Fr~ 5- __$DS::EAGE fractionated and NCP-bound antigen Fr. 5 (rv <24-14kd) of BmA.

EXPRESSION OF CELL SURFACE MOLECULES CD40/ CD40L

Representative flow cytometric analysis data of lymphocytes expressing CD40

(Fig.47) and CD40L (Fig.48) in EN and ASM individuals ·in response to H37Rv,

BmA and BmA Fr.S are presented. CD40 expression is significantly higher in ASM

individuals in comparison to EN against both BmA and fractionS of BmA. Between

the two antigens, BmA Fr.S causes higher expression of CD40 and the expression

is appreciably high in ASM individuals. However, there is not much difference the

expression of CD40 between both the groups against H37Rv whole cell lysate.

CD40 L expression was studied in both the groups against the same set of

antigens. CD40L expression pattern correlates well with the membrane CD40

molecules. The expression is higher in the ASM individuals in comparison to that

of the EN individuals against all the three different antigens. Moreover, similar to

CD40 expression, CD40L expression was higher against the Fr.S of BmA,

followed by BmA. Against H37Rv also, the CD40L was appreciable in the ASM

individuals, although CD40 expression was low in ASM group.

0

~

0 Q)

C) .., "' c :> 0 U o ....

C) N

C)

~

C) Q)

C) .., VI c :> 0 U c ....

C) N

0 0

].42 ~

1.88 ~ ].28

0 C) Q) Q)

I C) I "' Ml C)

Ml EN Ml "' ..,

c "' :> c 0 :> U o 0

.... U o ....

C) N C)

N

103 104 103 104

0 C)

1.54 ~ 2.6 3 ~ 3.22

C) 0 Q) Q)

I I I C) C) Ml ASM Ml "' Ml "' "' .. c c "' . :> 0 0 U c Uc .... ....

C) C) N N

103 104 103 104

103 104

H37Rv -BmA Fraction 5

Isotype Control

No Antigen

Fig.47. Representative data of Flow cytometric analysis of lymphocytes expressing CD40 in EN (.n= 3), and ASM (n= 3) individuals in response to different antigens. K-5 statistics was applied and the values given. H37Rv­H37Rv whole cell lysate; BmA- Brugia malayi adult soluble-antigens;_..&. 5-SDS­PAGE fractionated and NCP-bound antigen Fr. 5 (I'V <24-14kd) of BmA.

0 ~

0 (X)

0

"' .. c " 0 Uo

v

0 N

0

~

0 (X)

) ~ ..

c " 0 Uo

v

0 N

1-4 7

Ml

103 104

5 .4 7

Ml

H37Rv

0 ~

0 (X)

0

"' .. c " 0 Uo

v

0 N

0

~

0 (X)

0

"' .. c "' 0 uo

v

0 N

lsotype Centro I

No Antigen

0

1.44 ~ 101

0 (X)

Ml Ml

0

"' .. EN c " 0 Uo

v

0 N

104

0

5-92 ~

7. 75

0 (X)

Ml Ml ASM 0

"' .. c "' 0 uo

v

0 N

Fraction-s

Fig.48. Representative data of Flow cytometric analysis of lymphocytes expressing -<:-D46L in EN (n= 3),---and--ASM (n -3) individuals · in response -to .different .antigens. K-S statistics .was applied and the values given. H37Rv- H37Rv whole cell lysate; BmA­Brugia ma!ayi pdult soluble-ant.igens.;-n. 5- -SDS-P.AGE -f.rac-t.iooated and NCP-bound antigen Fr. 5 (tv <24-14kd) of BmA.