Rheumatic heart disease: 15 years of clinical and immunological follow-up

Figures & data

Table 5 Cross reactive intralesional T-cell clones from RHD patients

Patients #	Cross reactive T-cell clones	CD4	CD8	Antigen recognition M5 protein peptides / Heart tissue derived proteins or Cardiac myosin (LMM) peptides
1	1.1.27		+	M5(11–25)
				Myocardium protein: >150 kDa to 44 kDa
3	3.1.79	+		M5(111–130)
				Mitral valve proteins: 54–53 kDa/pI 7.76; 52 kDa/pI 4.3
				LMM 31(1686–1703); LMM 49 (1921–1937)
4	4.1.18	+		M5(81–103) and M5(163–177)
				Aortic valve proteins: 100–115–120 kDa; 43–46–50 kDa
5*	5.1.31	+		M5(62–82)
				Myocardium protein: 90–150 kDa
				Aortic valve protein: 43–65 kDa
7	7.1.1	+		M5(81–96); M5(83–103)
				Myocardium protein: ≥150 kDa
				Aortic valve proteins : 90–150 kDa; 43–65 kDa
8	8.1.2	+		M5(81–96)
				Myocardium protein: 95–150 kDa
10	10.2.13		+	Mitral valve proteins: 58; 37 kDa/pI 5.12
				LMM14(1400–1417); LMM36(1751–1768);
				LMM38(1777–1795); LMM40(1803–1834)

Note: T-cell clones were derived from mitral or aortic valve, except T-cell clone 5.1.31 (*) that was derived from left atrium (LA). Sequences of recognized peptides: M5 protein (Manjula et al, 1985; Phillips et al, 1981; Guilherme et al, 1995, ²⁰⁰⁰ and ²⁰⁰¹): 11–25-QRAKEALDKYELENH; 62–82-LERKTAELTSEKKEHEAENDK; 81–96-DKLKQQRDTLSTQKET; 83–103-LKQQRDTLSTQKETLEREVQN; 111–130-TRQELANKQQESKENEKALN; 163–177- ETIGTLKKILDETVK. Cardiac myosin (LMM-light meromyosin) peptides (Diederich et al, 1989; Galvin et al, 2000; Fae et al, 2006): LMM14-(1400–1417) SQSELESSQKEARSLSTE; LMM31-(1686–1703) ELEELRAVVEQTERSRKL; LMM36-(1751–1768) RNAEEKAKKAITDAAMMA; LMM-38 (1777–1795) TSAHLERMKKNMEQTIKD; LMM40-(1803–1840) EQIALKGGKKQLQKLEAR; LMM 49-(1921–1937) LEARVRELENELEAEQKR.

Table 1 Clinical data of RHD patients

Number of cases	25
Gender	Male: 17
	Female: 8
Mean age at the first episode of rheumatic fever (years)	9.5 ± 3.4
Valve damaged	Mitral: (25) – 100%
	Mitral isolated: (9) – 36%
	Mitral + aortic: (8) – 32%
	Mitral + tricuspid: (1) – 4%
	Trivalvular: (7) – 28%
Functional class (NYHA)	II – (2) – 8%
	III – (7) – 28%
	IV – (16) – 64%

( ): number of cases.

Table 2 Echocardiography data before surgery and until seven days post surgical treatment

	Before surgery	Post - surgery	P
LVDD (mm)	63.60 ± 10.9	53.80 ± 11.1	0.003
LVSD (mm)	41.61 ± 8.8	37.50 ± 11	0.179
LAD (mm)	51.30 ± 12.0	41.62 ± 7.3	0.002
Shortening fraction (%)	34.32 ± 4.6	29.61 ± 7.2	0.009

Abbreviations: LVDD, left ventricle diastolic diameter; LVSD, left ventricle systolic diameter; LAD, left atrium diameter.

Figure 1 Kaplan-Meier survival curve: follow-up of 25 rheumatic valve disease patients. (A) Event-free survival (death and reoperation); (B) probability of survival.

Table 3 Histopathological data from valvar and myocardial tissues of rheumatic heart disease patients

Patients #	Tissue analyzed	Valve					Myocardium
		Inflammation	Rheumatic Activity	Neovascularization	Fibrosis	Calcification	Inflammation	Rheumatic Activity
1	AV + LA	(+)	(−)	(+)	(+++)	(+++)	(++)	(−)
2	MV + LA	(+++)	AB−PR (+) VER (+)	(−)	(++)	(−)	(+)	AB−PR (+)
3	MV + LA	(++)	AB−PR (+) VER (+)	(+)	(+)	(−)	(−)	AB−PR (+)
4	MV + LA	(+)	(−)	(−)	(++)	(−)	(−)	(−)
9	MV + PM	(++)	(−)	(+++)	(+++)	(−)	(+)	(−)
11	MV + LA + RA + PM	(+++)	VER (+)	(+++)	(−)	(−)	(++)	AB−PR (+)
12	AV + LA	(+++)	(−)	(+++)	(++)	(−)	(−)	(−)
13	AV + RA + LA	(+)	(−)	(+)	(++)	(−)	(−)	(−)
6	AV + LA	(+)	(−)	(++)	(++)	(−)	(++)	(−)
8	MV	(++)	VER (++)	(++)	(+++)	(−)
10	MV + AV	(++)	VER (+)	(++)	(++)	(−)
14	AV	(+)	(−)	(++)	(+)	(−)
15	LA						(+)	(−)
16	LA						(+)	(−)
17	LA						(++)	(−)
18	LA						(+)	AB−PR (+)
7	LA						(+)	(−)
19	LA						(+)	(−)
20	RA						(+)	AB−EX (+)
21	LA						(+)	(−)
23	LA + RA						(+)	(−)
24	RA						(+)	(−)
5	LA						(−)	(−)
25	LA						(−)	(−)

Abbreviations: RA, right atrium; LA, left atrium; MV, mitral valve; AV, aortic valve; PM, papillary muscle; AB-PR, Aschoff bodies in proliferative phase; AB-EX, Aschoff bodies in exudative phase; VER, Verrucae; (−), Absent; (+), mild; (++), moderate; (+++), severe.

Table 4 Evaluation of heart-tissue infiltrating lymphocytes subsets

Patients #	Heart tissue	B cell * cells / HPF	T-cells* Cells/ HPF		CD4/CD8
			CD4+	CD8+
2	MV	2.2	1.6	0.6	2.7
3	PM	ND	9.4	3.3	2.8
	MV	0	3.2	2.4	1.3
4	LA	ND	6.1	1.2	5.0
6	AV	0.3	0.9	0.5	1.8
7	LA	0.2	4.5	0.9	5.0
	MV	0.2	1.2	1.2	1.0
8	LA	0.01	1.4	0.5	2.8
	MV	0	3.5	0	a
9	MV	0.02	0.2	0	a
	PM	0.1	1.0	0	a
10	MV	1.5	2.4	1.7	1.4
11	MV	1.4	2.5	0.8	3.1
	PM	0.3	0.3	0.2	1.5
12	LA	0.1	0.1	0.03	3.3
13	LA	0	0.3	0.3	1.0
15	LA	2.4	3.1	0.1	31.0
17	LA	0.3	0.7	0.3	2.3
19	LA	0	0	0	0
20	LA	0.3	2.6	2.0	1.3
22	LA	ND	0.9	0.5	1.8
23	MV	2.7	1.5	0.4	3.7
25	LA	0	0	0.3	b

* Notes: Immunohistochemistry.

Abbreviations: HPF, high power microscopical field; LA, left atrium; MV, mitral valve; AV, aortic valve; PM, papillary muscle; ND, not done

a only CD4+ T-cells

b only CD8+ T-cells

Figure 2 The presence of heart tissue T lymphocytes is related with rheumatic activity. The number of infiltrating T and B lymphocytes and their relation with rheumatic activity in both valve and myocardium tissues are presented. Black bars, mean tissue with rheumatic activity and white bars, without rheumatic activity. *P = 0.03; **P = 0.03. HPF, high power microscopic field.

Figure 3 Heart infiltrating T-cells. Heart tissue biopsies were cut in small fragments and kept in culture until establishment of T-cell lines. The picture represents lymphoblasts cultivated for 15 days in Dulbecco’s modified Eagle medium supplemented with IL-2. Photograph from Immunology Laboratory, Heart Institute; original magnification ×200.

Table 6 Peripheral blood reactivity against streptococcal M5 peptides and cardiac proteins

Patients #	M5(11–25)	M5(62–82)	M5(81–96) Stimulation index (SI)	M5(83–103)	M5(163–177)	Cardiac proteins recognized
2	(−)	(−)	(−)	(−)	(−)	Negative
3	8.6	13.4	5.4	4.8	15.0	Myocardium
						12–24 kDa, SI – 3.6
4	7.7	(−)	4.8	(−)	(−)	Myocardium
						>150 kDa, SI – 6.2
						Aortic v
						>150 kDa; 90–150 kDa, 65–90 kDa, 10–30 kDa
						SI – 2.6; 3.6; 3.8; 2.4 and 6.2, respectively
5	(−)	(−)	2.6	(−)	(−)	Negative
7	(−)	(−)	(−)	(−)	(−)	Negative
9	(−)	(−)	(−)	(−)	(−)	Negative
11	2.8	3.2	4.0	4.5	3.8	Aortic v
						65–90 kDa, SI – 41.0
13	4.1	5.2	3.7	(−)	(−)	Negative
15	2.6	(−)	(−)	(−)	5.4	Aortic v
						43–50 kDa, SI – 3.6
17	20.1	(−)	(−)	(−)	(−)	Mitral v
						53 – 58, kDa/ pI 5.12, SI – 16.8
						68– 56–52 kDa/5.94, SI–5.8
						\38 kDa/pI 4.23, SI –6.9
19	(−)	(−)	(−)	(−)	(−)	Myocardium
						>150 kDa, SI – 2.4; 90–150 kDa, SI – 3.0;
						10–30 kDa, SI – 2.4 Aortic v
						65–90 kDa, SI – 2.6; 30–45kDa, SI 2.5
20	(−)	(−)	(−)	(−)	(−)	Negative

Notes: Bold typed: positive reactions when stimulation index (SI) ≥2.5; (−) negative response; Sequences of peptides are represented in Table 5.

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