the liver is the largest internal organ in the body and performs many important functions , so it is not surprising that diseases of the liver are a major cause of morbidity and mortality throughout the world . among the most common liver disorders hcv infection is considered a major worldwide public health problem with a global prevalence of 3% ( lower in europe ~1.03% and americas ~1.7% and highest in africa ~5.3% ) . in egypt , about 10- 13% of the population is infected with hcv , meanwhile , hcv antibody prevalence among blood donors ranged from 6% to 38% with a mean of 15% . hcv , the most common cause of viral hepatitis , may present with skin manifestations in the early stages of the disease . skin manifestations could be the first signal of hcv , even in the absence of hepatic symptoms . the skin manifestations of hcv include lichen planus ( lp ) , necrolytic acral erythema ( nae ) , porphyria cutanea tarda , mixed cryoglobulinemia , polyarteritis nodosa , psoriasis , erythema multiforme , pyoderma gangrenosum , and dermatomyositis . cell death can be separated into two distinct forms : necrotic death and programmed death or apoptosis . necrosis results from massive cell injury and is often accompanied by inflammation . on the other hand , apoptosis is a programmed death , occurring in response to either external or internal stimuli . increasing evidence suggests that most mammalian cells exist in a state of unstable equilibrium , poised either to proliferate or die depending on the balance between influencing factors . thus , most if not all of the cell death machinery may be present at all times . whether it actually gets used depends on the physiological status of one or more check points that regulate the decision whether to divide , differentiate , and initiate dna repair , or eliminate the cells by apoptosis . apoptosis is an essential strategy of dynamic balance in the living system to maintain homeostasis . it is generally believed that the ratio of proapoptotic to antiapoptotic bcl-2 family proteins is the critical determinant of cell fate . the bcl-2 family of proteins contains both proapoptotic ( bax ) and antiapoptotic ( bcl-2 , bcl - xl ) proteins , which have special significance since they can determine if the cell commits to apoptosis or aborts the process . the tumor suppressor protein p53 is a nuclear phosphoprotein that , in its natural form ( wild - type ) , can bind to dna and prevent cells from entering the s ( synthesis ) phase of the cell cycle until repair of dna damage , or alternatively eliminate the cells by sending them down an irreversible apoptotic pathway . this tumor suppressor protein p53 has a critical role in regulation of the bcl-2 family of proteins . under normal conditions however , significantly higher p53 expression has been reported in sun - exposed and photo - aged skin as a result of sun - induced dna damage . death receptor  is a transmembrane receptor that is a member of tnf / nerve growth factor ( ngf ) receptor superfamily . caspase-3 is considered to be the most important of the executioner caspases and is activated by any of the initiator caspases ( caspase-8 , caspase-9 , or caspase-10 ) . to the best of our knowledge , no previous research has investigated the effect of advanced liver disease due to hcv infection , with its biochemical , metabolic , and hormonal alterations on the skin , regarding changes in apoptosis and its regulatory proteins . the present work aims to investigate the effect of advanced hcv liver disease on epidermal thickness , changes in the expression of various proapoptotic ( bax , fas , p53 , caspase-3 ) , and antiapoptotic ( bcl-2 , bcl - xl ) regulatory markers as well as the tunel technique for identification of apoptotic cells in skin samples of liver patients in comparison with normal healthy control subjects . the present study was conducted on 20 patients ( 13 males and 7 females ) , with advanced liver disease ( hcv ) attending the out - patient clinic of dermatology , std 's , and andrology department and both the out - patient clinic and the in - patient section of internal medicine department , al - minya university hospital . age of patients ranged from 22 to 70 years with a mean and sd of 47.711.63 . after informed consent , 4-mm punch biopsies were obtained from apparently normal skin of the thigh of 20 hcv patients with advanced liver disease . the study was approved by the committee for postgraduate studies and research of al - minya university . control samples were obtained from the thighs of five healthy subjects ( three males and two females ) , performing dermatosurgical procedures for another condition , and serving as a normal control group after taking an informed consent . the age of the control subjects ranged from 30 to 55 years , with a mean age of 40.49.4 . biopsies were fixed in buffered 10% formalin , embedded in paraffin , sectioned into 5-m thick sections , and stained with hematoxylin and eosin ( h&e ) for histometric analysis . paraffin sections were also utilized for immunohistochemical examination of apoptosis regulatory markers ( bax , fas , p53 , caspase-3 , bcl-2 , bcl - xl ) as well as the tunel technique for identification of apoptotic cells . a computer - assisted program ( analysis five by olympus soft imaging solutions gmbh , johann  krane - weg 39 , d-48149 mnster , germany ) was employed to measure epidermal thickness in h&e stained sections of biopsy specimens . the mean distance between the outermost surfaces of the epidermis , excluding stratum corneum , and the dermo - epidermal junction through the entire length of three - examined sections was determined at multiple points . for evaluation of bax and fas expression in the keratinocytes , : e3381 , ready - to - use rabbit antibody , spring bioscience , fermont , ca 94538 , usa ) , ( fas : code no . : m3554 , mouse monoclonal antihuman antibody , dako , carpinteria , 00ca , usa : at a dilution of 1:30 with the hrp method ) ] . the ready - to - use lsab2 peroxidase , dab detection system ( code no . : k0673 , dako , carpinteria , ca , usa ) was used to demonstrate antibodies expression according to the manufacturer 's instructions . for evaluation of p53 , caspase-3 , bcl-2 , and bcl - xl expression in the keratinocytes , : rmpd 016 , ready - to - use rabbit monoclonal antibody , dbs , ca , usa ) , ( caspase-3 : code no . : ms1770r7 , ready - to - use antibody , thermoscientific , ca , usa ) , ( bcl-2 : code no . : pdm 016 , ready - to - use monoclonal mouse antibody , dbs , ca , usa ) , ( bcl - xl : code no . : # ms-1334-po , mouse monoclonal antihuman antibody , thermoscientific , ca , usa : at a dilution of 1:200 with the hrp method ) ] . the ready - to - use detection system , universal hrp immunostaining kit ( code no . : kp - dbs , ca , usa ) was used to demonstrate antibodies expression according to the manufacturer 's instructions . the level of apoptosis regulatory markers expression in keratinocytes was evaluated , by two blinded histopathologists , in accordance with the scoring system devised by liang et al . this system results in a score ranging from 0 to 3 for both the degree of positivity ( percentage of positively stained epidermal cells : 0 : < 1% , 1 : 1 - 10% , 2 : 10 - 50% , 3:>50% ) and the degree of intensity of staining ( from faint - brown [ score 1 ] to deep - brown [ score 3 ] ) . the sum of the two scores was taken as the level of expression . detection of apoptotic cells by the tunel technique was performed using a universal apoptosis detection kit which contains equilibrium buffer , biotin-11-dutp , tdt enzyme , streptavidin - hrp , and dab ( code : kga7031 , keygen biotech 3f , 15 block no.439 changhong road , nanjing , china ) to detect apoptotic keratinocytes . apoptotic index was then calculated : data were coded , entered , and analyzed using an spss software package for statistical science ( spss for windows , version 16.0.1 , spss inc . statistical analysis included descriptive analysis as mean value and standard deviation ( sd ) , independent - samples t - test and correlation coefficient ( r ) for the results . significance was expressed in terms of p - value and the level of significance was 0.05 . a computer - assisted program ( analysis five by olympus soft imaging solutions gmbh , johann  krane - weg 39 , d-48149 mnster , germany ) was employed to measure epidermal thickness in h&e stained sections of biopsy specimens . the mean distance between the outermost surfaces of the epidermis , excluding stratum corneum , and the dermo - epidermal junction through the entire length of three - examined sections was determined at multiple points . for evaluation of bax and fas expression in the keratinocytes , sections were stained by the primary antibodies [ ( bax : code no . : e3381 , ready - to - use rabbit antibody , spring bioscience , fermont , ca 94538 , usa ) , ( fas : code no . : m3554 , mouse monoclonal antihuman antibody , dako , carpinteria , 00ca , usa : at a dilution of 1:30 with the hrp method ) ] . the ready - to - use lsab2 peroxidase , dab detection system ( code no . : k0673 , dako , carpinteria , ca , usa ) was used to demonstrate antibodies expression according to the manufacturer 's instructions . for evaluation of p53 , caspase-3 , bcl-2 , and bcl - xl expression in the keratinocytes , : rmpd 016 , ready - to - use rabbit monoclonal antibody , dbs , ca , usa ) , ( caspase-3 : code no . : ms1770r7 , ready - to - use antibody , thermoscientific , ca , usa ) , ( bcl-2 : code no . : pdm 016 , ready - to - use monoclonal mouse antibody , dbs , ca , usa ) , ( bcl - xl : code no . : # ms-1334-po , mouse monoclonal antihuman antibody , thermoscientific , ca , usa : at a dilution of 1:200 with the hrp method ) ] . the ready - to - use detection system , universal hrp immunostaining kit ( code no . : kp - dbs , ca , usa ) was used to demonstrate antibodies expression according to the manufacturer 's instructions . the level of apoptosis regulatory markers expression in keratinocytes was evaluated , by two blinded histopathologists , in accordance with the scoring system devised by liang et al . this system results in a score ranging from 0 to 3 for both the degree of positivity ( percentage of positively stained epidermal cells : 0 : < 1% , 1 : 1 - 10% , 2 : 10 - 50% , 3:>50% ) and the degree of intensity of staining ( from faint - brown [ score 1 ] to deep - brown [ score 3 ] ) . detection of apoptotic cells by the tunel technique was performed using a universal apoptosis detection kit which contains equilibrium buffer , biotin-11-dutp , tdt enzyme , streptavidin - hrp , and dab ( code : kga7031 , keygen biotech 3f , 15 block no.439 changhong road , nanjing , china ) to detect apoptotic keratinocytes . data were coded , entered , and analyzed using an spss software package for statistical science ( spss for windows , version 16.0.1 , spss inc . statistical analysis included descriptive analysis as mean value and standard deviation ( sd ) , independent - samples t - test and correlation coefficient ( r ) for the results . significance was expressed in terms of p - value and the level of significance was 0.05 . the epidermal thickness of liver disease patients ranged from 39.6 to 55.4 m with a mean of 46.2 m4.4 . on the other hand , the epidermal thickness of the control group ranged from 57.4 to 75 m with a mean of 647 m . the epidermis of liver disease patients showed significantly lower thickness than the control group ( p = 0.000 ) [ table 1 , figure 1 ] . mean value of epidermal thickness , score of pro - apoptotic ( bax , fas , p53 and caspase-3 ) and anti - apoptotic markers ( bcl-2 and bcl - xl ) and apoptotic index in hcv liver patients and controls the epidermis of advanced hcv liver disease patients ( a ) is lower in thickness when compared to controls ( b ) ( h and e , 100 ) cytoplasmic staining was observed in all bax positive specimens of both patients and controls . in liver disease patients , the stain was observed in both basal and squamous cell layers with a score ranging from 1.6 to 3.8 with a mean of 30.6 . in controls , staining was mainly confined to the basal cell layer with a range from 0.2 to 2.5 with a mean of 2.30.1 . the former was significantly higher than the control group ( p=0.03 ) [ table 1 , figures 2 and 3 ] . expression of pro - apoptotic ( bax , fas , p53 , caspase-3 ) and anti - apoptotic ( bcl-2 , bcl - xl ) markers , and apoptotic index in hcv liver patients and controls expression of pro - apoptotic markers ( bax , fas , p53 , caspase-3 ) in epidermal keratinocytes of advanced hcv liver disease patients ( a , c , e , g ) and in controls ( b , d , f , h ) respectively showing the following : 
 significant increase of cytoplasmic expression of bax staining in basal and squamous cell layers in patients ( a ) when compared to that confined to the basal cell layer of controls ( b ) ( immunohistochemical , 400).significant increase of membranous expression of fas staining in basal and lower 2/3 of squamous cell layers in patients ( c ) when compared to that confined to the basal cell layer of controls ( d ) ( immunohistochemical , 400).significant increase of nuclear expression of p53 staining in basal and lower 2/3 of squamous cell layers in patients ( e ) when compared to that confined to the basal cell layer of controls ( f ) ( immunohistochemical , 200).significant increase of cytoplasmic expression of caspase 3 staining in basal and squamous cell layers in patients ( g ) when compared to minimal staining confined to the basal cell layer of controls ( h ) ( immunohistochemical , 400 ) 
  significant increase of cytoplasmic expression of bax staining in basal and squamous cell layers in patients ( a ) when compared to that confined to the basal cell layer of controls ( b ) ( immunohistochemical , 400 ) . significant increase of membranous expression of fas staining in basal and lower 2/3 of squamous cell layers in patients ( c ) when compared to that confined to the basal cell layer of controls ( d ) ( immunohistochemical , 400 ) . significant increase of nuclear expression of p53 staining in basal and lower 2/3 of squamous cell layers in patients ( e ) when compared to that confined to the basal cell layer of controls ( f ) ( immunohistochemical , 200 ) . significant increase of cytoplasmic expression of caspase 3 staining in basal and squamous cell layers in patients ( g ) when compared to minimal staining confined to the basal cell layer of controls ( h ) ( immunohistochemical , 400 ) membranous staining was observed in all fas - positive specimens . in liver patients , the stain was observed in the basal and lower 2/3 of squamous cell layers with a score ranging from 1.7 to 3.8 , with a mean of 2.70.6 . in the control group , the stain was mainly confined to the basal cell layer and it ranged from 1.5 to 2 with a mean of 1.70.2 . fas expression in liver patients was significantly higher than control group ( p=0.003 ) [ table 1 , figures 2 and 3 ] . the stain was observed in the basal and lower 2/3 of squamous cell layers with score ranging from 0.6 to 1.9 with a mean of 1.30.5 . in control group , the positive staining was mainly seen in the basal cell layer and it showed a score ranging from 0.7 to 1.1 with a mean of 0.80.2 . p53 expression in liver patients was significantly higher than control group ( p=0.03 ) [ table 1 , figures 2 and 3 ] . p53 was positively correlated with bax ( p=0.000 ) , caspase-3 ( p=0.04 ) and apoptotic index ( p=0.000 ) . while , it was inversely correlated with epidermal thickness ( p=0.002 ) . caspase-3 expression cytoplasmic staining was observed in all caspase-3 positive specimens of both patients and controls . in liver disease patients the positively stained cells were observed in the basal and squamous cell layers with a score ranging from 2.3 to 4.3 with a mean of 3.10.6 . in the control group , minimal staining was noticed in the basal cell layer with a score ranging from 1.8 to 2.4 with a mean of 2.10.2 . the marker was significantly higher in patients than controls ( p=0.003 ) [ table 1 , figures 2 and 3 ] . caspase-3 was positively correlated with bax ( p=0.05 ) and p53 ( p=0.04 ) , while it was inversely correlated with epidermal thickness ( p=0.002 ) . cytoplasmic staining was observed in all bcl-2 positive specimens of both patients and controls . in the liver patients group , the positive cells were observed in basal cell layer with a score ranging from 0.5 to 3.2 with a mean of 2.30.8 . in the control group , the stain was mainly confined to basal layer showing a score ranging from 2.4 to 2.6 with a mean of 2.560.1 . there was no statistically significant difference between the two groups ( p=0.5 ) [ table 1 , figures 2 and 4 ] . bcl-2 expression was positively correlated with epidermal thickness ( p=0.03 ) . while , it was inversely correlated with caspase-3 expression ( p=0.001 ) . expression of anti - apoptotic markers ( bcl-2 , bcl - xl ) in epidermal keratinocytes of advanced hcv liver disease patients ( a , c ) and in controls ( b , d ) : 
 insiginificant lower cytoplasmic expression of bcl-2 staining , mainly confined to the basal cell layer , in patients ( a ) when compared to controls ( b).insiginificant lower perinuclear cytoplasmic expression of bcl - xl staining , mainly confined to the basal cell layer , in patients ( c ) when compared to that confined to the basal and supra basal cell layer of controls ( d ) ( immunohistochemical , 400 ) . insiginificant lower cytoplasmic expression of bcl-2 staining , mainly confined to the basal cell layer , in patients ( a ) when compared to controls ( b ) . insiginificant lower perinuclear cytoplasmic expression of bcl - xl staining , mainly confined to the basal cell layer , in patients ( c ) when compared to that confined to the basal and supra basal cell layer of controls ( d ) ( immunohistochemical , 400 ) . perinuclear cytoplasmic staining was observed in all bcl - xl positive specimens of patients and controls . in liver disease patients , the positively stained cells were observed in the basal cell layer with a range from 1 to 2.5 with a mean of 1.90.5 . in control subjects , they were also observed mainly in the basal cell layer ; however , in some specimens positive suprabasal cells were also noticed . no statistically significant difference was found between the two groups ( p=0.9 ) [ table 1 , figures 2 and 4 ] . apoptotic cells were demonstrated in basal and squamous cell layers in hcv liver patients with an apoptotic index ranging from 0 to 2 with a mean of 1.30.7 . control subjects showed few apoptotic cells , if present , with an apoptotic index ranging from 0 - 1 with a mean of 0.20.4 . the apoptotic index in liver disease patients was significantly higher than the control group ( p=0.002 ) [ table 1 , figures 2 and 5 ] . apoptotic index was positively correlated with bax ( p=0.001 ) and p53 expression ( p=0.000 ) , while it was inversely correlated with epidermal thickness ( p=0.001 ) . many apoptotic cells present in basal and squamous cell layer in advanced hcv liver disease patients ( a ) , compared to few apoptotic cells seen in controls ( b ) ( tunel , 400 ) the epidermal thickness of liver disease patients ranged from 39.6 to 55.4 m with a mean of 46.2 m4.4 . on the other hand , the epidermal thickness of the control group ranged from 57.4 to 75 m with a mean of 647 m . the epidermis of liver disease patients showed significantly lower thickness than the control group ( p = 0.000 ) [ table 1 , figure 1 ] . mean value of epidermal thickness , score of pro - apoptotic ( bax , fas , p53 and caspase-3 ) and anti - apoptotic markers ( bcl-2 and bcl - xl ) and apoptotic index in hcv liver patients and controls the epidermis of advanced hcv liver disease patients ( a ) is lower in thickness when compared to controls ( b ) ( h and e , 100 ) cytoplasmic staining was observed in all bax positive specimens of both patients and controls . in liver disease patients , the stain was observed in both basal and squamous cell layers with a score ranging from 1.6 to 3.8 with a mean of 30.6 . in controls , staining was mainly confined to the basal cell layer with a range from 0.2 to 2.5 with a mean of 2.30.1 . the former was significantly higher than the control group ( p=0.03 ) [ table 1 , figures 2 and 3 ] . expression of pro - apoptotic ( bax , fas , p53 , caspase-3 ) and anti - apoptotic ( bcl-2 , bcl - xl ) markers , and apoptotic index in hcv liver patients and controls expression of pro - apoptotic markers ( bax , fas , p53 , caspase-3 ) in epidermal keratinocytes of advanced hcv liver disease patients ( a , c , e , g ) and in controls ( b , d , f , h ) respectively showing the following : 
 significant increase of cytoplasmic expression of bax staining in basal and squamous cell layers in patients ( a ) when compared to that confined to the basal cell layer of controls ( b ) ( immunohistochemical , 400).significant increase of membranous expression of fas staining in basal and lower 2/3 of squamous cell layers in patients ( c ) when compared to that confined to the basal cell layer of controls ( d ) ( immunohistochemical , 400).significant increase of nuclear expression of p53 staining in basal and lower 2/3 of squamous cell layers in patients ( e ) when compared to that confined to the basal cell layer of controls ( f ) ( immunohistochemical , 200).significant increase of cytoplasmic expression of caspase 3 staining in basal and squamous cell layers in patients ( g ) when compared to minimal staining confined to the basal cell layer of controls ( h ) ( immunohistochemical , 400 ) 
  significant increase of cytoplasmic expression of bax staining in basal and squamous cell layers in patients ( a ) when compared to that confined to the basal cell layer of controls ( b ) ( immunohistochemical , 400 ) . significant increase of membranous expression of fas staining in basal and lower 2/3 of squamous cell layers in patients ( c ) when compared to that confined to the basal cell layer of controls ( d ) ( immunohistochemical , 400 ) . significant increase of nuclear expression of p53 staining in basal and lower 2/3 of squamous cell layers in patients ( e ) when compared to that confined to the basal cell layer of controls ( f ) ( immunohistochemical , 200 ) . significant increase of cytoplasmic expression of caspase 3 staining in basal and squamous cell layers in patients ( g ) when compared to minimal staining confined to the basal cell layer of controls ( h ) ( immunohistochemical , 400 ) membranous staining was observed in all fas - positive specimens . in liver patients , the stain was observed in the basal and lower 2/3 of squamous cell layers with a score ranging from 1.7 to 3.8 , with a mean of 2.70.6 . in the control group , the stain was mainly confined to the basal cell layer and it ranged from 1.5 to 2 with a mean of 1.70.2 . fas expression in liver patients was significantly higher than control group ( p=0.003 ) [ table 1 , figures 2 and 3 ] . the stain was observed in the basal and lower 2/3 of squamous cell layers with score ranging from 0.6 to 1.9 with a mean of 1.30.5 . in control group , the positive staining was mainly seen in the basal cell layer and it showed a score ranging from 0.7 to 1.1 with a mean of 0.80.2 . p53 expression in liver patients was significantly higher than control group ( p=0.03 ) [ table 1 , figures 2 and 3 ] . p53 was positively correlated with bax ( p=0.000 ) , caspase-3 ( p=0.04 ) and apoptotic index ( p=0.000 ) . while , it was inversely correlated with epidermal thickness ( p=0.002 ) . caspase-3 expression cytoplasmic staining was observed in all caspase-3 positive specimens of both patients and controls . in liver disease patients the positively stained cells were observed in the basal and squamous cell layers with a score ranging from 2.3 to 4.3 with a mean of 3.10.6 . in the control group , minimal staining was noticed in the basal cell layer with a score ranging from 1.8 to 2.4 with a mean of 2.10.2 . the marker was significantly higher in patients than controls ( p=0.003 ) [ table 1 , figures 2 and 3 ] . caspase-3 was positively correlated with bax ( p=0.05 ) and p53 ( p=0.04 ) , while it was inversely correlated with epidermal thickness ( p=0.002 ) . cytoplasmic staining was observed in all bcl-2 positive specimens of both patients and controls . in the liver patients group , the positive cells were observed in basal cell layer with a score ranging from 0.5 to 3.2 with a mean of 2.30.8 . in the control group , the stain was mainly confined to basal layer showing a score ranging from 2.4 to 2.6 with a mean of 2.560.1 . there was no statistically significant difference between the two groups ( p=0.5 ) [ table 1 , figures 2 and 4 ] . bcl-2 expression was positively correlated with epidermal thickness ( p=0.03 ) . while , it was inversely correlated with caspase-3 expression ( p=0.001 ) . expression of anti - apoptotic markers ( bcl-2 , bcl - xl ) in epidermal keratinocytes of advanced hcv liver disease patients ( a , c ) and in controls ( b , d ) : 
 insiginificant lower cytoplasmic expression of bcl-2 staining , mainly confined to the basal cell layer , in patients ( a ) when compared to controls ( b).insiginificant lower perinuclear cytoplasmic expression of bcl - xl staining , mainly confined to the basal cell layer , in patients ( c ) when compared to that confined to the basal and supra basal cell layer of controls ( d ) ( immunohistochemical , 400 ) . insiginificant lower cytoplasmic expression of bcl-2 staining , mainly confined to the basal cell layer , in patients ( a ) when compared to controls ( b ) . insiginificant lower perinuclear cytoplasmic expression of bcl - xl staining , mainly confined to the basal cell layer , in patients ( c ) when compared to that confined to the basal and supra basal cell layer of controls ( d ) ( immunohistochemical , 400 ) . perinuclear cytoplasmic staining was observed in all bcl - xl positive specimens of patients and controls . in liver disease patients , the positively stained cells were observed in the basal cell layer with a range from 1 to 2.5 with a mean of 1.90.5 . in control subjects , they were also observed mainly in the basal cell layer ; however , in some specimens positive suprabasal cells were also noticed . no statistically significant difference was found between the two groups ( p=0.9 ) [ table 1 , figures 2 and 4 ] . apoptotic cells were demonstrated in basal and squamous cell layers in hcv liver patients with an apoptotic index ranging from 0 to 2 with a mean of 1.30.7 . control subjects showed few apoptotic cells , if present , with an apoptotic index ranging from 0 - 1 with a mean of 0.20.4 . the apoptotic index in liver disease patients was significantly higher than the control group ( p=0.002 ) [ table 1 , figures 2 and 5 ] . apoptotic index was positively correlated with bax ( p=0.001 ) and p53 expression ( p=0.000 ) , while it was inversely correlated with epidermal thickness ( p=0.001 ) . many apoptotic cells present in basal and squamous cell layer in advanced hcv liver disease patients ( a ) , compared to few apoptotic cells seen in controls ( b ) ( tunel , 400 ) cytoplasmic staining was observed in all bax positive specimens of both patients and controls . in liver disease patients , the stain was observed in both basal and squamous cell layers with a score ranging from 1.6 to 3.8 with a mean of 30.6 . in controls , staining was mainly confined to the basal cell layer with a range from 0.2 to 2.5 with a mean of 2.30.1 . the former was significantly higher than the control group ( p=0.03 ) [ table 1 , figures 2 and 3 ] . expression of pro - apoptotic ( bax , fas , p53 , caspase-3 ) and anti - apoptotic ( bcl-2 , bcl - xl ) markers , and apoptotic index in hcv liver patients and controls expression of pro - apoptotic markers ( bax , fas , p53 , caspase-3 ) in epidermal keratinocytes of advanced hcv liver disease patients ( a , c , e , g ) and in controls ( b , d , f , h ) respectively showing the following : 
 significant increase of cytoplasmic expression of bax staining in basal and squamous cell layers in patients ( a ) when compared to that confined to the basal cell layer of controls ( b ) ( immunohistochemical , 400).significant increase of membranous expression of fas staining in basal and lower 2/3 of squamous cell layers in patients ( c ) when compared to that confined to the basal cell layer of controls ( d ) ( immunohistochemical , 400).significant increase of nuclear expression of p53 staining in basal and lower 2/3 of squamous cell layers in patients ( e ) when compared to that confined to the basal cell layer of controls ( f ) ( immunohistochemical , 200).significant increase of cytoplasmic expression of caspase 3 staining in basal and squamous cell layers in patients ( g ) when compared to minimal staining confined to the basal cell layer of controls ( h ) ( immunohistochemical , 400 ) 
  significant increase of cytoplasmic expression of bax staining in basal and squamous cell layers in patients ( a ) when compared to that confined to the basal cell layer of controls ( b ) ( immunohistochemical , 400 ) . significant increase of membranous expression of fas staining in basal and lower 2/3 of squamous cell layers in patients ( c ) when compared to that confined to the basal cell layer of controls ( d ) ( immunohistochemical , 400 ) . significant increase of nuclear expression of p53 staining in basal and lower 2/3 of squamous cell layers in patients ( e ) when compared to that confined to the basal cell layer of controls ( f ) ( immunohistochemical , 200 ) . significant increase of cytoplasmic expression of caspase 3 staining in basal and squamous cell layers in patients ( g ) when compared to minimal staining confined to the basal cell layer of controls ( h ) ( immunohistochemical , 400 ) membranous staining was observed in all fas - positive specimens . in liver patients , the stain was observed in the basal and lower 2/3 of squamous cell layers with a score ranging from 1.7 to 3.8 , with a mean of 2.70.6 . in the control group , the stain was mainly confined to the basal cell layer and it ranged from 1.5 to 2 with a mean of 1.70.2 . fas expression in liver patients was significantly higher than control group ( p=0.003 ) [ table 1 , figures 2 and 3 ] . the stain was observed in the basal and lower 2/3 of squamous cell layers with score ranging from 0.6 to 1.9 with a mean of 1.30.5 . in control group , the positive staining was mainly seen in the basal cell layer and it showed a score ranging from 0.7 to 1.1 with a mean of 0.80.2 . p53 expression in liver patients was significantly higher than control group ( p=0.03 ) [ table 1 , figures 2 and 3 ] . p53 was positively correlated with bax ( p=0.000 ) , caspase-3 ( p=0.04 ) and apoptotic index ( p=0.000 ) . while , it was inversely correlated with epidermal thickness ( p=0.002 ) . caspase-3 expression cytoplasmic staining was observed in all caspase-3 positive specimens of both patients and controls . in liver disease patients the positively stained cells were observed in the basal and squamous cell layers with a score ranging from 2.3 to 4.3 with a mean of 3.10.6 . in the control group , minimal staining was noticed in the basal cell layer with a score ranging from 1.8 to 2.4 with a mean of 2.10.2 . the marker was significantly higher in patients than controls ( p=0.003 ) [ table 1 , figures 2 and 3 ] . caspase-3 was positively correlated with bax ( p=0.05 ) and p53 ( p=0.04 ) , while it was inversely correlated with epidermal thickness ( p=0.002 ) . cytoplasmic staining was observed in all bcl-2 positive specimens of both patients and controls . in the liver patients group , the positive cells were observed in basal cell layer with a score ranging from 0.5 to 3.2 with a mean of 2.30.8 . in the control group , the stain was mainly confined to basal layer showing a score ranging from 2.4 to 2.6 with a mean of 2.560.1 . there was no statistically significant difference between the two groups ( p=0.5 ) [ table 1 , figures 2 and 4 ] . bcl-2 expression was positively correlated with epidermal thickness ( p=0.03 ) . while , it was inversely correlated with caspase-3 expression ( p=0.001 ) . expression of anti - apoptotic markers ( bcl-2 , bcl - xl ) in epidermal keratinocytes of advanced hcv liver disease patients ( a , c ) and in controls ( b , d ) : 
 insiginificant lower cytoplasmic expression of bcl-2 staining , mainly confined to the basal cell layer , in patients ( a ) when compared to controls ( b).insiginificant lower perinuclear cytoplasmic expression of bcl - xl staining , mainly confined to the basal cell layer , in patients ( c ) when compared to that confined to the basal and supra basal cell layer of controls ( d ) ( immunohistochemical , 400 ) . insiginificant lower cytoplasmic expression of bcl-2 staining , mainly confined to the basal cell layer , in patients ( a ) when compared to controls ( b ) . insiginificant lower perinuclear cytoplasmic expression of bcl - xl staining , mainly confined to the basal cell layer , in patients ( c ) when compared to that confined to the basal and supra basal cell layer of controls ( d ) ( immunohistochemical , 400 ) . perinuclear cytoplasmic staining was observed in all bcl - xl positive specimens of patients and controls . in liver disease patients , the positively stained cells were observed in the basal cell layer with a range from 1 to 2.5 with a mean of 1.90.5 . in control subjects , they were also observed mainly in the basal cell layer ; however , in some specimens positive suprabasal cells were also noticed . no statistically significant difference was found between the two groups ( p=0.9 ) [ table 1 , figures 2 and 4 ] . apoptotic cells were demonstrated in basal and squamous cell layers in hcv liver patients with an apoptotic index ranging from 0 to 2 with a mean of 1.30.7 . control subjects showed few apoptotic cells , if present , with an apoptotic index ranging from 0 - 1 with a mean of 0.20.4 . the apoptotic index in liver disease patients was significantly higher than the control group ( p=0.002 ) [ table 1 , figures 2 and 5 ] . apoptotic index was positively correlated with bax ( p=0.001 ) and p53 expression ( p=0.000 ) , while it was inversely correlated with epidermal thickness ( p=0.001 ) . many apoptotic cells present in basal and squamous cell layer in advanced hcv liver disease patients ( a ) , compared to few apoptotic cells seen in controls ( b ) ( tunel , 400 ) programmed cell death ( apoptosis ) is an essential strategy of dynamic balance in the living system . apoptosis is the major mechanism by which homeostasis of a number of physiological systems in the body is regulated . although apoptosis is an intrinsic process present in all cells , it can be regulated by extrinsic factors including growth factors , cell surface receptors , cellular stress , and hormones . apoptosis is morphologically and biochemically characterized by cell shrinkage , dense chromatin condensation , cellular budding , fragmentation , rapid phagocytosis by nearby cells , and dna fragmentation . in the skin , there is considerable evidence that apoptosis plays an important role in the pathogenesis of a wide variety of skin diseases . death of hepatocytes and other hepatic cell types is a characteristic feature of liver diseases as cholestasis , viral hepatitis , ischemia / reperfusion , liver preservation for transplantation , and drug / toxicant - induced injury . meanwhile , skin diseases characterized by increased apoptosis ( lp ) or necrosis ( nae ) of keratinocytes were previously reported in hcv liver disease patients . overactivation of the apoptotic process can lead to significant hepatocellular damage , while inhibition of apoptosis can promote the proliferation and transformation of cells . for example , apoptosis induction in infected liver cells , by hbv or hcv , is a defense mechanism to limit viral replication and promote their elimination . liver cell failure develops when the functional capacity of the liver can no longer maintain normal physiological conditions . this may be manifested on the skin , and it may affect other body systems in the form of hepatic encephalopathy , cardiovascular changes , portal hypertension , hepatopulmonary syndrome , and hepatorenal syndrome . in a similar model to liver cell failure , renal failure which is a systemic disease with cutaneous manifestations . the degree of renal failure in hepatorenal syndrome is a reflection of the degree of hepatocellular failure . interestingly , a recent study reported over expression of epidermal p53 and bcl-2 with increased epidermal thickness in chronic renal failure patients on maintenance hemodialysis : suggesting that an alteration in the proliferation/ apoptosis balance is most likely present in the skin of such patients . usually , apoptosis represents a counterbalance to proliferation , and decreased apoptosis is generally thought to be associated with epidermal hyperproliferation . in the present work , significant overexpression of proapoptotic markers ( bax , fas , p53 , and caspase-3 ) were detected in patients ( p=0.03 , 0.03 , 0.003 , and 0.003 , respectively ) : that could explain the increased apoptotic index in hcv liver patients ( 0.002 ) . whether these changes were due to the metabolic and biochemical alterations present in such patients or to the direct effect of the virus remain to be elucidated . especially when taking into consideration that apoptosis induction in infected liver cells , by hbv or hcv , is considered by some authors as a defense mechanism to limit viral replication and promote their elimination moreover , the epidermal thickness of skin biopsies from patients was inversely correlated with the increased proapoptotic markers ( bax : p=0.01 , fas : p=0.04 , p53 : p=0.002 , and caspase-3 : p=0.002 ) and apoptotic index ( p=0.001 ) . these findings suggest that an alteration in the proliferation / apoptosis balance is present in the skin of such patients . it is still unclear whether skin diseases associated with hcv infection and characterized histopathologically by apoptosis ( lp ) or necrosis ( nae ) are due to an exaggerated response to such alteration or to another mechanism . on the other hand , no statistically significant difference in expression of antiapoptotic markers ( bcl-2 and bcl - xl ) in epidermal keratinocytes of hcv patients and the control group ( p=0.5 and p=0.9 respectively ) were detected . the result of hcv liver disease patients was different from that reported in chronic renal failure where the expression of bcl-2 protein ( antiapoptotic ) was found to be higher in skin samples obtained from apparently normal skin of chronic renal failure patients on maintenance hemodialysis than in skin samples obtained from control cases . the difference between the mean values of the two groups was highly significant ( p=0.0003 ) . meanwhile , in the present study , the epidermis of hcv liver disease patients showed significantly lower thickness than the control group ( p=0.000 ) when measured by computer image analysis ( histometry ) . on the contrary , in renal failure patients there were significant increase of the epidermal thickness in patients than controls ( p<0.0001 ) . however , overexpression of p53 protein was the only common finding in both chronic renal failure and hcv liver disease patients , which could be attributed to significant dna damage . the results of the present study suggest an alteration in keratinocyte proliferation / apoptosis balance in advanced hcv liver patients , which are associated with an increase in the expression of skin pro - apoptotic markers ( bax , fas , p53 , and caspase-3 ) with consequent increase of the apoptotic index and decreased epidermal thickness . whether this alteration in the skin proliferation/ apoptosis balance is attributed to the metabolic and biochemical changes present in liver cell failure or to the direct effect of the virus future research on a larger group of patients is mandatory , and should address various apoptotic regulatory proteins in the skin as well as other organs , in hcv patients and other liver diseases , to explain how the metabolic effects of liver diseases may affect skin apoptosis .