the gamma - aminobutyric acid ( gaba ) , the major inhibitory neurotransmitter in the mammalian central nervous system , causes changes in a polarization of cell membrane acting through the activation of gaba receptors .
the most prevalent , gaba type a ( gaba a ) , receptors tend to exist as pentameric structures consisting of various combinations of six major subunits :  ,  ,  ,  ,  , and  .
recent data indicate that gabaergic activity is not restricted to the central nervous system , but also involves cells of different origin that , like hepatocytes , possess the peripheral type of gaba a receptors .
as was shown in several studies , an activation of gaba a receptors ( especially the 3 subunit ) leads to the hyperpolarization of cell membrane , which , in turn , causes a rapid decrease in cell proliferation [ 3 , 4 ] .
this feature seems to be interesting with regard to the study that demonstrated the impact of cell polarization on the efficiency of hepatitis c virus ( hcv ) entry .
an elevated gabaergic activity was found to be responsible for the impaired hepatocyte proliferation in regenerating livers after partial hepatectomy [ 6 , 7 ] . on the other hand ,
it has been known since the early 1980s that the serum level of gaba may be elevated in case of acute or chronic hepatocellular failure , and the gaba neurotransmitter system is involved in the pathogenesis of hepatic encephalopathy ( he ) in humans .
recently , it has been suggested that he - dependent ammonia may be developed due to the modification of the gaba a receptor affinity .
other findings suggest that increased inhibition through gaba a receptors may represent an important pathophysiological mechanism of fatigue in chronic hcv infection .
this multifunctionality of gabaergic action in numerous liver failures has drawn our attention to the possible role of the modulation of gaba a receptors expression in the course of hcv infection and in the response to the antiviral treatment .
although the liver is the main place of hcv replication , gathered data , including our own [ 12 , 13 ] , indicate that hcv can persist and replicate efficiently in extrahepatic tissue , including peripheral blood mononuclear cells ( pbmcs ) .
hcv rna can persist in pbmcs long after spontaneous or treatment - induced viral elimination from sera , but the relevance of this phenomenon is still unknown .
it has been documented recently that pbmcs originated from the healthy human population express functional 1 and 3 subunits of the gaba a receptor .
the aim of the current study was to investigate whether the comparable expression of gaba a subunits can be observed in pbmcs from chronic hepatitis c ( chc ) patients that have undergone anti - hcv treatment .
consequently , not only did we succeed to show 1 and 3 expression in pbmcs from hcv - infected patients , but our results also demonstrated the substantial differences in 3 and , less manifested , in 1 subunits expression in pbmcs between healthy donors and post - treatment hcv patients .
we then speculate on how the alterations in the expression of gaba a subunits may be of special importance for hcv rna persistence .
blood samples were collected , after the informed consent had been obtained , from ten healthy donors ( 6 males , 4 females : age 1826 years ) and from 22 chronically infected with hcv patients ( 12 males , 10 females : age 1622 years ) within 2  weeks after the cessation of antiviral treatment ( ifn alfa2b  +  ribavirin ) .
neither patients nor healthy controls were taking medication known to alter gaba a receptor expression or activity .
pbmcs and sera were isolated by blood centrifugation in density gradient ( histopaque 1077 , sigma ) .
total rna was extracted from pbmcs by a modified guanidinium thiocyanate / phenol / chloroform technique .
hcv rna presence in sera was determined by reverse transcription polymerase chain reaction ( rt - pcr ) ( cobas , amplicor hcv 2.0 monitor , roche ) .
briefly , 6  g of total rna were reverse - transcribed and amplified by masteramp tth dna polymerases ( epicentre biotechnologies ) with external hcv - specific primers ( table  1 ) in a reaction as follows : 20  min of rt at 70c and 3  min at 94c , followed by 35 cycles of 94c for 20s , 50c for 20s , 72c for 20s , and 72c for 7  min .
the resultant amplicon was used in the second - round pcr ( 2  min at 94c , 30 cycles of 94c for 40s , 55c for 40s , 72c for 40s , and 72c for 10  min ) .
after pa gel electrophoresis , an hcv - specific product of 278  bp was visualized with ethidium bromide staining.table 1list and sequences of primers used in the reverse transcription polymerase chain reaction ( rt - pcr ) analysis and size of pcr productsspecificity of primerspcr product ( bp)sequence of forward and reverse primershcv rna ( external)321f : ccaccatgaatcactcccctgtr : gctcatggtgcacggtctacgagaccthcv rna ( internal)278f : gtcttcacgcagaaagcgtctagccr : cactcgcaagcaccctatcaggcaggaba a 1(external)241f : cggtcaattttgctgacactr : ggttatgcatgggatggcgaba a 1(internal)95f : gacctctttaaggttctatggr : gctccaacagcaaccagcgaba a 3 ( external)319f : cacatcggttagatcaggr : caaggcaaagaatgaccggaba a 3 ( internal)110f : cgctggaagttcacaatgr : cgaggcatgctctgtttc-actin434f : caaagacctgtacgccaacacar : aaccgactgctgtcaccttcac list and sequences of primers used in the reverse transcription polymerase chain reaction ( rt - pcr ) analysis and size of pcr products for the detection of mrnas specific for gaba a receptor subunits 1 and 3 , random cdna was synthesized according to the manufacturer s instructions by using the transcription high fidelity cdna synthesis kit ( roche diagnostics , mannheim , germany ) and then amplified in  nested
pcr assays ( each of 35 cycles ) were performed as follows : 5  min of preliminary heating at 94c , 94c for 40s , 50c ( for gaba a 1/3)/55c ( for actin ) for 40s , 72c for 40s , and 72c for 10  min .
pcr 1- and 3-specific products of 95  bp and 110  bp , respectively , were analyzed as described above .
protein lysates ( 20  g / lane ) were separated on 10% sds - pa gel .
resolved proteins were electroblotted into nitrocellulose and incubated with gaba a 1 , 52  kda ( gtx 30204 , dilution 1:12,000 ) and 3 , 55  kda ( gtx 261302 , dilution 1:2,000 ) specific antibodies purchased from gene tex , inc . the reactions with the goat antibodies against -actin ,
43  kda ( sc-1615 , santa cruz biotechnology ) were carried out at the dilution of 1 to 300 .
the bound antibodies were visualized using the enhanced chemiluminescence ( ecl ) western blotting reagent ( sc-2048 , santa cruz biotechnology ) with signals captured on film . in order to quantify the density of signals ,
the bio - rad quantity one system was used . to estimate the comparative levels of gaba a 1 and 3 subunits expression , all immunoreactivities were normalized to -actin expression before statistical analysis .
cells were collected from healthy donors and chc patients who had undergone antiviral treatment . according to the hcv rna status in sera and in pbmcs
, three subgroups of patients were distinguished : 0/0 with sera and pbmcs free from hcv rna ( n  =  10 ) ; 0/1 with sera negative for hcv rna , but with hcv rna - positive pbmcs ( n  =  6 ) ; and the 1/1 group , where sera and pbmc samples contained hcv rna ( n  =  6 ) . to analyze the gene expression of gaba a receptor subunits 1 and 3 ,
pcr amplification produced single 1- and 3-specific pcr products , of 95 and 110  bp , respectively .
our results showed that all pbmc samples contained mrna specific for the 1 and 3 subunits of gaba a receptor .
the expression of -actin was used as the endogenous control of gene expression . as shown in fig .
1 , the gene expression of gaba a receptor subunits differs among pbmc samples , even among the same group .
no significant differences were found in gene expression level for either 1 or 3 between groups .
we could only observe that 3 gene expression showed a margin tendency to be elevated in pbmcs from healthy donors in comparison to the hcv - infected patients .
in contrast , the gene expression of 1 subunit in pbmcs tends to be slightly higher in the group of chc patients compared to the healthy controls.fig .
1gamma - aminobutyric acid type a ( gaba a ) receptor , 1 and 3 genes expression in peripheral blood mononuclear cells ( pbmcs ) .
the figure shows several examples of the results obtained by reverse transcription polymerase chain reaction ( rt - pcr ) analysis performed in the groups as follows : a healthy donors ( hd ) and ( b  d ) anti - hepatitis c virus ( hcv)-treated chronic hepatitis c ( chc ) patients , who : b eliminated hcv rna from both sera and pbmcs ( 0/0 ) , c only from sera ( 0/1 ) , or d neither from sera nor from pbmcs ( 1/1 ) . as a control of gene expression , the analysis of -actin
was performed gamma - aminobutyric acid type a ( gaba a ) receptor , 1 and 3 genes expression in peripheral blood mononuclear cells ( pbmcs ) .
the figure shows several examples of the results obtained by reverse transcription polymerase chain reaction ( rt - pcr ) analysis performed in the groups as follows : a healthy donors ( hd ) and ( b  d ) anti - hepatitis c virus ( hcv)-treated chronic hepatitis c ( chc ) patients , who : b eliminated hcv rna from both sera and pbmcs ( 0/0 ) , c only from sera ( 0/1 ) , or d neither from sera nor from pbmcs ( 1/1 ) . as a control of gene expression ,
the analysis of -actin was performed the presence of the protein expression of gaba a 1 and 3 subunits was confirmed using western blot in all of the tested pbmc samples .
figure  2 shows several examples of western blot analysis performed on lysates obtained from the pbmcs of healthy donors and chc patients .
the quantitative immunoblotting based on a density analysis , described in the materials and methods section , was utilized in order to determine the possible differences in the gaba a 1 and 3 subunits expression between pbmc samples . as indicated in fig .
3a , the gaba a 1 protein expression was somewhat higher ( p  =  0.047 ) in the group of anti - hcv - treated chc patients than in the group of healthy controls . however , no statistical differences were found between individual subgroups of patients.fig .
2expression of 1 and 3 subunits of the gaba a receptor in pbmcs as revealed by western blot analysis .
representative images of western blot analysis come from groups as follows : a healthy donors ( hd ) and ( b  d ) anti - hcv - treated chc patients , who : b eliminated hcv rna from both sera and pbmcs ( 0/0 ) , c only from sera ( 0/1 ) , or d neither from sera nor from pbmcs ( 1/1 ) .
3differences in gaba a 1 and 3 subunits expression in pbmcs evaluated by western blot analysis .
the results are shown after normalization to the -actin expression . a relative expression level of gaba a 1 protein in pbmcs from healthy donors and from post - treatment chc patients .
b relative expression level of gaba a 3 protein expression in pbmcs from healthy donors and three groups of anti - hcv - treated chc patients ( 0/0 , 0/1 , 1/1 ) expression of 1 and 3 subunits of the gaba a receptor in pbmcs as revealed by western blot analysis .
representative images of western blot analysis come from groups as follows : a healthy donors ( hd ) and ( b  d ) anti - hcv - treated chc patients , who : b eliminated hcv rna from both sera and pbmcs ( 0/0 ) , c only from sera ( 0/1 ) , or d neither from sera nor from pbmcs ( 1/1 ) .
the analysis of -actin was performed as a control of gene expression differences in gaba a 1 and 3 subunits expression in pbmcs evaluated by western blot analysis .
the results are shown after normalization to the -actin expression . a relative expression level of gaba a 1 protein in pbmcs from healthy donors and from post - treatment chc patients .
b relative expression level of gaba a 3 protein expression in pbmcs from healthy donors and three groups of anti - hcv - treated chc patients ( 0/0 , 0/1 , 1/1 ) an opposite relation was found for the expression of 3 subunit . in pbmcs from anti - hcv - treated chc patients , the 3 protein showed decreased levels in comparison to the group of healthy donors .
as presented in fig.3b , significant differences ( p  =  0.0038 ) in the 3 expression among healthy donors and the three subgroups of chc patients were observed as a result of the 3 expression analysis .
the lowest expression level of 3 protein , compared to the healthy donors , was observed in pbmcs from patients , who , despite the antiviral treatment , have retained hcv rna in both sera and pbmcs ( p  =  0.00047 ) .
cells were collected from healthy donors and chc patients who had undergone antiviral treatment . according to the hcv rna status in sera and in pbmcs
, three subgroups of patients were distinguished : 0/0 with sera and pbmcs free from hcv rna ( n  =  10 ) ; 0/1 with sera negative for hcv rna , but with hcv rna - positive pbmcs ( n  =  6 ) ; and the 1/1 group , where sera and pbmc samples contained hcv rna ( n  =  6 ) . to analyze the gene expression of gaba a receptor subunits 1 and 3 ,
pcr amplification produced single 1- and 3-specific pcr products , of 95 and 110  bp , respectively .
our results showed that all pbmc samples contained mrna specific for the 1 and 3 subunits of gaba a receptor .
the expression of -actin was used as the endogenous control of gene expression . as shown in fig .
1 , the gene expression of gaba a receptor subunits differs among pbmc samples , even among the same group .
no significant differences were found in gene expression level for either 1 or 3 between groups .
we could only observe that 3 gene expression showed a margin tendency to be elevated in pbmcs from healthy donors in comparison to the hcv - infected patients .
in contrast , the gene expression of 1 subunit in pbmcs tends to be slightly higher in the group of chc patients compared to the healthy controls.fig .
1gamma - aminobutyric acid type a ( gaba a ) receptor , 1 and 3 genes expression in peripheral blood mononuclear cells ( pbmcs ) .
the figure shows several examples of the results obtained by reverse transcription polymerase chain reaction ( rt - pcr ) analysis performed in the groups as follows : a healthy donors ( hd ) and ( b  d ) anti - hepatitis c virus ( hcv)-treated chronic hepatitis c ( chc ) patients , who : b eliminated hcv rna from both sera and pbmcs ( 0/0 ) , c only from sera ( 0/1 ) , or d neither from sera nor from pbmcs ( 1/1 ) . as a control of gene expression , the analysis of -actin
was performed gamma - aminobutyric acid type a ( gaba a ) receptor , 1 and 3 genes expression in peripheral blood mononuclear cells ( pbmcs ) .
the figure shows several examples of the results obtained by reverse transcription polymerase chain reaction ( rt - pcr ) analysis performed in the groups as follows : a healthy donors ( hd ) and ( b  d ) anti - hepatitis c virus ( hcv)-treated chronic hepatitis c ( chc ) patients , who : b eliminated hcv rna from both sera and pbmcs ( 0/0 ) , c only from sera ( 0/1 ) , or d neither from sera nor from pbmcs ( 1/1 ) . as a control of gene expression ,
the presence of the protein expression of gaba a 1 and 3 subunits was confirmed using western blot in all of the tested pbmc samples .
figure  2 shows several examples of western blot analysis performed on lysates obtained from the pbmcs of healthy donors and chc patients .
the quantitative immunoblotting based on a density analysis , described in the materials and methods section , was utilized in order to determine the possible differences in the gaba a 1 and 3 subunits expression between pbmc samples . as indicated in fig .
3a , the gaba a 1 protein expression was somewhat higher ( p  =  0.047 ) in the group of anti - hcv - treated chc patients than in the group of healthy controls .
2expression of 1 and 3 subunits of the gaba a receptor in pbmcs as revealed by western blot analysis .
representative images of western blot analysis come from groups as follows : a healthy donors ( hd ) and ( b  d ) anti - hcv - treated chc patients , who : b eliminated hcv rna from both sera and pbmcs ( 0/0 ) , c only from sera ( 0/1 ) , or d neither from sera nor from pbmcs ( 1/1 ) .
3differences in gaba a 1 and 3 subunits expression in pbmcs evaluated by western blot analysis .
the results are shown after normalization to the -actin expression . a relative expression level of gaba a 1 protein in pbmcs from healthy donors and from post - treatment chc patients .
b relative expression level of gaba a 3 protein expression in pbmcs from healthy donors and three groups of anti - hcv - treated chc patients ( 0/0 , 0/1 , 1/1 ) expression of 1 and 3 subunits of the gaba a receptor in pbmcs as revealed by western blot analysis .
representative images of western blot analysis come from groups as follows : a healthy donors ( hd ) and ( b  d ) anti - hcv - treated chc patients , who : b eliminated hcv rna from both sera and pbmcs ( 0/0 ) , c only from sera ( 0/1 ) , or d neither from sera nor from pbmcs ( 1/1 ) .
the analysis of -actin was performed as a control of gene expression differences in gaba a 1 and 3 subunits expression in pbmcs evaluated by western blot analysis .
the results are shown after normalization to the -actin expression . a relative expression level of gaba a 1 protein in pbmcs from healthy donors and from post - treatment chc patients .
b relative expression level of gaba a 3 protein expression in pbmcs from healthy donors and three groups of anti - hcv - treated chc patients ( 0/0 , 0/1 , 1/1 ) an opposite relation was found for the expression of 3 subunit . in pbmcs from anti - hcv - treated chc patients , the 3 protein showed decreased levels in comparison to the group of healthy donors . as presented in fig.3b ,
significant differences ( p  =  0.0038 ) in the 3 expression among healthy donors and the three subgroups of chc patients were observed as a result of the 3 expression analysis .
the lowest expression level of 3 protein , compared to the healthy donors , was observed in pbmcs from patients , who , despite the antiviral treatment , have retained hcv rna in both sera and pbmcs ( p  =  0.00047 ) .
several studies have revealed the contribution of a variety of host factors to the development of hcv rna persistence in chronically infected patients , despite having used antiviral treatment [ 13 , 16 , 17 ] .
our study was designed to evaluate whether the expression of chosen subunits of gaba a receptors in pbmcs bears any relation to hcv infection and/or the path of hcv rna elimination after anti - viral treatment .
studies that were carried out on other viruses like herpes simplex virus ( hsv ) and human immunodeficiency virus ( hiv ) indicated that the expression of gaba a receptor on the target cells could be modulated upon the viral infection [ 18 , 19 ] .
although gaba a receptors tend to exist as a pentameric structure consisting of six major gaba a receptor subunits , we decided to screen the expression of two of them : 1 and 3 .
this choice was grounded on alam et al.s study   that confirmed the significant content of the 1 subunit in pbmcs and fractionated t - cell populations .
the evaluation of 3 expression , despite the lower abundance of this subunit in pbmcs , seemed interesting due to the confirmed impact on a proliferative activity of other cells [ 20 , 21 ] .
if governing the hcv infection indeed plays any role in the gaba a receptor activity , the modulation of their expression would be an expected phenomenon . to address this issue , we analyzed the 1 and 3 gaba a expression in pbmcs using nested pcr and quantitative immunoblotting .
our results showed , for the first time , that the expression of the 1 and 3 subunits of the gaba a receptor is common not only for healthy donors ( 15 ) , but also for anti - hcv - treated patients .
moreover , the expression of these subunits at the protein level displayed differences between healthy donors and chc patients .
a marginally significant elevation of 1 gaba a in pbmcs was demonstrated for anti - hcv - treated patients when compared with healthy donors .
it seems interesting that the expression of 1 gaba a receptor subunit is detectable in the majority of pbmcs subtypes , such as : t - cells , b - cells , and monocytes , in other words , in cells that are also able to maintain hcv during chronic infection .
taking into account that the increased expression of these receptors in t - cells downregulates the effector t - cell response [ 23 , 24 ] and that the defective function of hcv - specific t - cells contribute to the chronicity of infection [ 2527 ] , we can hypothesize that gaba a activation may contribute to the impaired response to the hcv infection .
in contrast , the majority of pbmcs from the anti - hcv - treated patients represented significantly lower expression of the 3 subunit of the gaba a receptor than the healthy donors pbmcs .
thus , also in case of the 3 subunit , expression measured at the protein level does not reflect precisely the gene expression level .
this type of discrepancy , probably connected with the post - transcriptional regulation , was observed for gaba a receptor expression previously .
the lowest level of gaba a 3 receptor expression was observed in these pbmcs , where hcv rna presence in cells was accompanied by the hcv rna positivity of sera . as was demonstrated , the transfection of hepatoma cells with 3-specific cdna resulted in a significant decrease of cell proliferation .
similarly , an increased gaba a 3 receptor expression was found to act as an inhibitory signal for hepatic cell proliferation , whereas the downregulation of the gaba a 3 receptor expression was observed in malignant hepatocyte cell lines .
although less is known about the role of 3 subunit expression of the gaba a receptor in pbmcs , we can hypothesize that , like in the case of hepatic cells , the decreased expression of gaba a 3 receptor in pbmcs may alter the proliferative activity of these cells .
since hcv - infected cells present enhanced proliferation in comparison to non - infected cells [ 30 , 31 ] , this process appears to play an important role in hcv rna replication . on the other hand
, it was previously demonstrated that the drug - dependent inhibition of gaba a receptors expression does not alter hcv load , which may suggest that , rather , hcv infection is responsible for inducing such changes in gaba a expression that favor hcv propagation in target cells . in conclusion , the current study provides evidence for the 1 and 3 expression in pbmcs from hcv - infected patients . decreased gaba a 3 expression , which is observed in hcv rna - positive pbmcs ,
future studies should elucidate whether the alteration of gaba a expression , which is observed during chc infection , has an impact on the development of hepatocellular carcinoma .
 the modulation of the gamma - aminobutyric acid type a ( gaba a ) receptors activity was observed in several chronic hepatitis failures , including hepatitis c. the expression of gaba a receptor subunits 1 and 3 was detected in peripheral blood mononuclear cells ( pbmcs ) originated from healthy donors . 
 the aim of the study was to evaluate if gaba a 1 and 3 expression can also be observed in pbmcs from chronic hepatitis c ( chc ) patients and to evaluate a possible association between their expression and the course of hepatitis c virus ( hcv ) infection . gaba a 1- and 3-specific mrnas presence and a protein expression in pbmcs from healthy donors and chc patients were screened by reverse transcription polymerase chain reaction ( rt - pcr ) and western blot , respectively . in patients , 
 hcv rna was determined in sera and pbmcs . 
 it was shown that gaba a 1 and 3 expression was significantly different in pbmcs from chc patients and healthy donors . in comparison to healthy donors , 
 chc patients were found to present an increase in the expression of gaba a 1 subunit and a decrease in the expression of 3 subunit in their pbmcs . 
 the modulation of 1 and 3 gaba a receptors subunits expression in pbmcs may be associated with ongoing or past hcv infection . 