Sex based relative expression of estrogen receptors and tumor necrosis factor-alpha in liver affects hepatitis C virus viral pathogenesis

Table 1 Antibodies and positive controls used for western blot

Antibody	Catalog	Host/class/isotype	Concentration	Observed size (kDa) in human liver
Anti-ERβ	Abcam; ab3576	Rabbit/polyclonal/IgG	1:1000; (1:30000 2nd)	70
Anti-TNF-α	Abcam; ab1793	Mouse/monoclonal/IgG1	1:1000; (1:10000 2nd)	45
Anti-ERα	Invitrogen; MA5-13191	Mouse/monoclonal/IgG1	1:500; (1:10000 2nd)	37
Anti-Histone H3	Abcam; ab1791	Rabbit/polyclonal/IgG	1:1000; (1:20000 2nd)	15
Anti-GAPDH (0411)	Santa Cruz; sc-47724	Mouse/monoclonal/IgG1	1:1000; (1:10000 2nd)	50
Anti-rabbit IRDye® 800CW	LI-COR; 926-32213	Donkey/IgG	Varies
Anti-mouse IRDye® 800CW	LI-COR; 926-32212	Donkey/IgG	Varies
Recombinant human TNF-α	Abcam; ab55237		10 ng/well	25

TNF-α: Tumor necrosis factor-alpha; ER: Estrogen receptor; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Table 2 Characteristics of the study population, mean ± SD

Characteristics	Normal (n = 36)		Cirrhosis (n = 32)		HCC (n = 23)		P value
	n	(%)	n	(%)	n	(%)
Age (year)
< 49	10	(27.8)	12	(37.5)	6	(26.1)	0.2593
50-59	15	(41.7)	17	(53.1)	10	(43.5)
≥ 60	11	(30.6)	3	(9.4)	7	(30.4)
Mean age (year)	52.0 ± 14.6		52.2 ± 6.8		55.5 ± 7.1		0.3087
Sex
Male	18	(50.0)	17	(53.1)	18	(78.3)	0.0765
Female	18	(50.0)	15	(46.9)	5	(21.7)
Race
White	18	(50.0)	22	(68.8)	15	(65.2)	0.4398
African American	2	(5.6)	1	(3.1)	2	(8.7)
Other/Unknown	16	(44.4)	9	(28.1)	6	(26.1)
Mean AST (U/L)			187.6 ± 389.3		114.2 ± 62.4		0.4542
Mean ALP (U/L)			158.5 ± 150.1		189.3 ± 110.4		0.0369
Mean total bilirubin (mg/dL)			8.6 ± 11.7		2.8 ± 6.8		< 0.0001
Mean creatinine (mg/dL)			1.8 ± 1.5		2.1 ± 2.9		0.3134
IFNL4-ΔG genotype
TT/TT	11	(30.6)	11	(34.4)	7	(30.4)	0.8405
ΔG/TT	18	(50.0)	13	(40.6)	9	(39.1)
ΔG/ΔG	6	(16.7)	5	(15.6)	6	(26.1)
Undetermined	1	(2.8)	3	(9.4)	1	(4.3)
IFNL4-ΔG allele frequency (%)	42.9		39.7		47.7

HCC: Hepatocellular carcinoma; AST: Aspartate aminotransferase; ALP: Alkaline phosphatase.

Table 3 Mean age of males and females in each disease group, mean ± SD

	Mean age (year)			P value
	Normal	Cirrhosis	HCC
Male	50.8 ± 18.3	50.7 ± 5.0	53.3 ± 5.9	0.0716
Female	53.1 ± 10.4	54.0 ± 8.3	63.3 ± 5.1

HCC: Hepatocellular carcinoma.

Table 4 Clinical characteristics of hepatitis C virus-positive samples, by sex

Characteristics	Female (n = 20)	Male (n = 35)	P value
Mean AST (U/L)	79.0 ± 44.7	213.8 ± 390.7	0.0186
Mean ALP (U/L)	158.3 ± 112.7	182.1 ± 156.5	0.4255
Mean total bilirubin (mg/dL)	4.8 ± 9.3	7.7 ± 11.6	0.3179
Mean creatinine (mg/dL)	1.4 ± 1.0	1.5 ± 1.0	0.6716

AST: Aspartate aminotransferase; ALP: Alkaline phosphatase.

Table 5 Spearman’s correlations between message RNA expression of estrogen receptors and tumor necrosis factor-alpha, by disease and sex

Spearman’s correlations
		Normal
		Females (n = 18)			Males (n = 18)			Together (n = 36)
		ESR1	ESR2	ESR1	ESR1	ESR2	ESR1	ESR1	ESR2	ESR1
				ESR2			ESR2			ESR2
ESR1	R value		0.01			-0.42			-0.19
	P value		0.9579			0.0861			0.2772
TNF-α	R value	0.05	0.54	-0.38	0.34	-0.11	0.35	0.2	0.18	0.01
	P value	0.8293	0.022a	0.119	0.1708	0.6508	0.16	0.2422	0.2972	0.9501
		HCV/cirrhosis
		Females (n = 15)			Males (n = 17)			Together (n = 32)
		ESR1	ESR2	ESR1	ESR1	ESR2	ESR1	ESR1	ESR2	ESR1
				ESR2			ESR2			ESR2
ESR1	R value		-0.84			0.57			-0.1
	P value		0.0002c			0.0197a			0.6038
TNF-α	R value	-0.3	0.64	-0.57	0.42	0.58	0.07	0.09	0.61	-0.19
	P value	0.2708	0.0116a	0.0286a	0.0934	0.0162a	0.802	0.6165	0.0002c	0.3017
		HCV/HCC
		Females (n = 5)			Males (n = 23)			Together (n = 28)
		ESR1	ESR2	ESR1	ESR1	ESR2	ESR1	ESR1	ESR2	ESR1
				ESR2			ESR2			ESR2
ESR1	R value		-0.1			0.14			0.08
	P value		0.95			0.5814			0.7301
TNF-α	R value	0	0.3	-0.1	-0.11	0.6	-0.56	-0.21	0.45	-0.57
	P value	> 0.99	0.6833	0.95	0.6627	0.0083b	0.0151a	0.3397	0.0293a	0.0049b

^aP < 0.05.

^bP < 0.01.

^cP < 0.001.

TNF-α: Tumor necrosis factor-alpha; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus.