Background
Methods
Study population
Collection of samples
Laboratory diagnosis
Expression of CT IncB and IncC proteins
Antibody assays
Purification and proliferation of CD4 positive T cells
Stimulants
RNA extraction and real-time RT-PCR analysis for cytokines and transcription factors
No. | Cytokine gene primers | 5'-3' sequence |
---|---|---|
1 | GM-CSF F* | AGC CCT GGG AGC ATG TGA |
GM-CSF R** | GAG TAG AGA CAC TGC TGC TGA GAT G | |
2 | IL-β1F | GCT GAT GGC CCT AAA CAG ATG |
IL-β1R | ACGAAT CTC CGA CCA CCA CTA | |
3 | IFNγ F | GAA ACG AGA TGA CTT CGA AAA GCT |
IFNγ R | ATG TCC AAC GCA AAG CAA TAC A | |
4 | IL-12 F | TCG CGT TCA CAA GCT CAA GT |
IL-12 R | CA AAC CTG ACC CAC CCA AGA | |
5 | TNF-α F | GCC CGA CTA TCT CGA CTT TGC |
TNF-α R | A ACC TTC CCA AAC GCC TCC | |
6 | IL4 F | CCA CGG ACA CAA GTG CGA TAT |
IL4 R | CGT AAC AGA CAT CTT TGC TGC C | |
7 | IL5 F | AAA GGC AAA CGC AGA ACG TT |
IL5 R | CTC TTG GAG CTG CCT ACG TGT | |
8 | IL6 F | ACT CAC CTC TTC AGA ACG |
IL6 R | AGT AGT GAG GAA CAA GCC | |
9 | IL10 F | GGG AGA ACC TGA AGA CCC TCA |
IL10 R | AAC AAG AGC AAG GCC GTG G | |
10 | T-Bet(TBX21) F | AAC ACA GGA GCG CAC TGG AT |
T-Bet(TBX21) R | ATT GTG CTC CAG TCC CTC CA | |
11 | GATA3 F | AGA TGG CAC GGG ACA CTA CCT |
GATA3 R | ATT AAG CCC AAG CGA AGG C | |
12 | B-actin F | TGT GAT GGT GGG TAT GGG TC |
B-actin R | TAC AAT GAG CTG CGT GT |
Quantification of secreted cytokines
Statistical analysis
Results
Study population
Laboratory diagnosis of CT
Detection of CT incB and incC
ELISA | PCR | Western blot | |||||
---|---|---|---|---|---|---|---|
CT IgG | IncB IgG2 | IncC IgG2 | IncB | IncC | IncB IgG | IncC IgG | |
GI (n = 25) | 1 (4) | 1(4) | 2 (8) | 0 (0) | 0 (0) | 1(4) | 1(4) |
GII (n = 14) | 12 (86)a, m | 10 (72)b, m | 11 (79)c, m | 12 (86)d, m | 12 (86)e, m | 9 (64)f, m | 11 (79)g, m |
GIII (n = 18) | 9 (50)m | 8 (44)m | 7 (39)m | 17 (94)m | 18 (100)m | 8 (44)m | 7 (39)m |
Cytokine mRNA expression levels in stimulated CD4+T cells
ELISA for cytokines in cell supernatants of stimulated CD4+T cells
GI | GII | GIII | |||||||
---|---|---|---|---|---|---|---|---|---|
IncB | IncC | EBs | IncB | IncC | EBs | IncB | IncC | EBs | |
IL-1β
|
141.8
(109.5–335.5) |
169.3
(98.8–368.5) |
243.6
(145.1–467.9) | 312.3 (109.8–489.2) |
407.1
(135.2–444.2) |
538.3
(124.8–767.3) |
692.9
a
(167–941.4) |
825.3
b
(182.8–982.3) |
900
c
(288.6–1231.3) |
IL-6
|
146.7
(74.9–400.3) |
159.4
(87.3–548.5) |
299.92
(98.67–981.32) |
604
d
(132.2–871.6) |
612.87
e
(156.5–716.9) |
751
f
(187.97–1093.2) |
900.63
a
(441.4–1054.72) |
878.41
b
(422.9–1078.78) |
1241.07
c
(656.4–1878.3) |
IL-10
|
278.4
(132.7–254.2) |
287.9
(143.66–267.5) |
313.7
(159.4–623.9) |
500.3
(267.9–702) |
587.1
(249.3–734.2) |
643.9
(338.2–742.6) |
1132.1
a
(562.6–1441.9) |
1278.3
b
(600.3–1502.4) |
1538.4
c
(768.2–1709.07) |
IL-12
|
187.6
(112.6–336.4) |
193.6
(123.2–414.1) |
265.9
(132.7–451.1) |
345.9
g
(231.4–552) |
365.2
h
(256.3–462.2) |
463.2
i
(259.7–606.3) |
200.2
(168.4–378.4) |
189.7
(179.3–334.8) |
243
(182.1–356.2) |
IFN-γ
|
443.6
j
(125.1–600.8) |
467.28
k
(141–580) |
580.3
l
(154.2–671.4) |
703.1
g
(332.6–988.61) |
571.3
h
(311.9–814.3) |
859.2
i
(342.1–1460.5) |
229.5
(110.1–300.8) |
285.4
(154.2–412.6) |
453.3
(189.5–432.9) |
TNF-α
|
156.1
(72.5–254.3) |
167.3
(73.2–269.1) |
199.3
(82.2–343.5) |
298.3
d
(138.2–400) |
271.9
e
(128.3–438.6) |
486.1
f
(188.2–569.2) |
248.6
(132.6–378.1) |
263.2
(128.97–350.6) |
411.2
(164.5–588.6) |
GM-CSF
|
181.2
(85.8–243.7) |
117.6
(93.6–229.7) |
233.1
(106.4–259.9) |
278.2
g
(127.5–333.6) |
282.5
h
(148.3–313.4) |
356.2
i
(172.3–421.6) |
172.4
(99.8–223.8) |
186.7
(103.7–245.7) |
228.9
(141.6–279.6) |
IL-23
|
59.3
(43–176.5) |
76.2
(57.2–217.4) |
126.7
(105.6–211.3) |
153.2
g
(65.7–200.1) |
134.7
h
(87.2–213.2) |
187.4
i
(148.2–231) |
75.2
(74.1–211) |
88.6
(68.9–202.6) |
147.5
(118.9–156) |