J Clin Immunol DOI 10.1007/s10875-014-0118-0
ORIGINAL RESEARCH
Invariant Natural Killer T Cells are Reduced in Hereditary Hemochromatosis Patients M. L. Maia & C. S. Pereira & G. Melo & I. Pinheiro & M. A. Exley & G. Porto & M. F. Macedo
Received: 26 September 2014 / Accepted: 27 November 2014 # Springer Science+Business Media New York 2014
Abstract Purpose Invariant natural killer T (iNKT) cells are CD1d restricted-T cells that react to lipid antigens. iNKT cells were shown to be important in infection, autoimmunity and tumor surveillance. Alterations in the number and function of these cells were described in several pathological conditions including autoimmune and/or liver diseases. CD1d is critical for antigen presentation to iNKT cells, and its expression is increased in liver diseases. The liver is the major organ affected in Hereditary Hemochromatosis (HH), an autosomal recessive disorder caused by excessive iron absorption. Herein, we describe the study of iNKT cells of HH patients. Methods Twenty-eight HH patients and 24 control subjects from Santo António Hospital, Porto, were included in this study. Patient’s iron biochemical parameters (serum M. L. Maia : C. S. Pereira : I. Pinheiro : M. F. Macedo (*)
transferrin saturation and ferritin levels) and the liver function marker alanine transaminase (ALT) were determined at the time of study. Peripheral blood iNKT cells were analyzed by flow cytometry using an anti-CD3 antibody and the CD1d tetramer loaded with PBS57. Results We found a decrease in the percentage and number of circulating iNKT cells from HH patients when compared with control population independently of age. iNKT cell defects were more pronounced in untreated patients, relating with serum ferritin and transferrin saturation levels. No correlation was found with ALT, a marker of active liver dysfunction. Conclusions Altogether, our results demonstrate that HH patients have reduced numbers of iNKT cells and that these are influenced by iron overload. Keywords Invariant natural killer T cells . hereditary hemochromatosis . iron . lipid
UniLiPe, IBMC—Institute for Molecular and Cellular Biology, University Porto, Porto, Portugal e-mail: [email protected] G. Melo : G. Porto Clinical Hematology, CHP-HSA—Santo António General Hospital Porto, Porto, Portugal M. A. Exley University of Manchester, Manchester, UK M. A. Exley Brigham & Women’s Hospital, Harvard Medical School, Boston, MA, USA G. Porto BCRIB, IBMC—Institute for Molecular and Cellular Biology and ICBAS, Abel Salazar Institute for the Biomedical Sciences, University of Porto, Porto, Portugal M. F. Macedo Aveiro Health Sciences Program, University of Aveiro, Aveiro, Portugal
Introduction Invariant natural killer T (iNKT) cells are a distinct subset of T lymphocytes that react with lipid antigens presented by the MHC class I-like CD1d molecules. They are characterized by the expression of a semi-invariant T cell receptor (TCR) that in humans is composed of a Vα24Jα18 chain preferentially combined with the Vβ11 [1–3]. iNKT cell numbers are highly variable between individuals. The numbers can range from 0.001 % to occasionally over 3 % of human peripheral blood mononuclear cells (PBMCs). Human iNKT cells are more abundant in adipose tissue such as omentum in which iNKT cells represent 10 % of the T cells [4]. However, blood iNKT cells are the most studied in humans, since peripheral blood is the most easily available biological sample.
J Clin Immunol
iNKT cells have protective and regulatory functions in tumor surveillance, chronic infectious diseases, transplantation and autoimmunity [1]. A role in the pathogenesis of inflammatory bowel diseases and in asthma was also described [5]. In chronic infection disease caused by HIV and Mycobacterium tuberculosis, iNKT cells had phenotypes associated with immune activation [6]. In both primary biliary cirrhosis patients [7] and in HCV infected patients [8] peripheral blood iNKT cells are decreased [9]. In the liver of primary biliary cirrhosis patients, however, iNKT cells were increased compared with control subjects [7]. Consistent with iNKT cell role in autoimmunity, intrinsic iNKT cell defects were found to be associated with autoimmune diseases, such as type 1 diabetes [10, 11], multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis and Sjogren’s syndrome, cutaneous forms of lupus erythematosus, psoriasis, Crohn’s disease, and ulcerative colitis [12]. Currently, iNKT cells are being used as therapeutic targets in some clinical trials, mainly in oncological conditions, which further increases the need for understanding of their behavior in health and disease [2]. The normal function of iNKT cells relies on the efficient presentation of lipid antigens by CD1d molecules. The CD1d at the surface of antigen presenting cells (APCs) binds to the lipid antigen and this complex is presented to the iNKT cells. Alterations occurring in CD1d, APCs or in the metabolism of lipids can affect the activation of the iNKT cells, since they are required for the iNKT cell activation and proliferation. Human CD1d is expressed on the cell surface of APCs (monocytes, macrophages, B cells, and some myeloid dendritic cells), being responsible for lipid and glycolipid antigenpresentation to iNKT cells [13, 14]. CD1d can also be expressed, at low levels, in several non-leukocyte cells, including hepatocytes [15–17]. Hereditary Hemochromatosis (HH) is an autosomal recessive disease of iron metabolism, in which the major organ affected is the liver. This pathology is characterized by increased intestinal iron absorption, causing augmented plasma iron concentration and consequently an increase in transferrin saturation. Excessive iron in circulation may accumulate in the liver, as well as in other organs, as reflected by an increase in serum ferritin. From a pathogenic point of view, HH is classified as a liver disease with primary failure in the production of the liver iron hormone hepcidin, which is the key regulator of iron absorption and its systemic distribution [18, 19]. In the vast majority of cases, the C282Y HFE mutation in homozygosity is the cause of the hepcidin dysregulation [19, 20]. The signs and symptoms are both general and organ-specific, including diabetes, arthritis, hypogonadism, hyperpigmentation, cardiomyopathy and liver disease [18, 20]. Since iNKT cells frequency and phenotype are altered in several liver diseases [9], and given the strong liver
involvement in HH, we hypothesized that iNKT cells might be altered in this condition. Herein we analyzed the iNKT cells in blood samples from HH patients and correlated this immunological parameter with biochemical parameters of iron overload and liver function.
Methods Population Studied The study was approved by the Ethical Committee of Centro Hospitalar do Porto, Porto. Informed consent was obtained from participants according to the Helsinki declaration. HH Patients Twenty-eight HH patients, followed-up regularly at the Hemochromatosis Outpatient Clinic of Santo António Hospital, Porto, were included in this study. They were all Caucasians from the north of Portugal, with confirmed homozygosity for the C282Y HFE mutation and no confounding co-morbidities. Clinical evaluation of the group of patients was previously described [21–24]. Twenty were males and 8 were females, with a mean age of 58 years old (range 39–80). Biochemical parameters of iron metabolism, serum transferrin saturation (TfSat), serum ferritin levels (SF) and the liver function marker alanine transaminase (ALT) were routinely determined at the time of the study in the Clinical Chemistry Laboratory of Santo António Hospital, Porto, by standard techniques. Control Population Twenty-four apparently healthy subjects, volunteer blood donors at the Blood Bank of Santo António Hospital, Porto, were analyzed as controls to obtain the percentages of peripheral blood iNKT cells. This population included 13 males and 11 females, with a mean age of 45 years old (range 21–62). Mononuclear Cells Isolation from Human Peripheral Blood and Flow Cytometry Total lymphocyte counts were determined in an automatic blood cell counter (Adavia 120; Hematology Systems, Bayer®, Tarrytown, NY, USA). Peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque-1077® (Sigma) density centrifugation following the manufacturer’s instructions. Human PBMCs were stained with PerCP/ Cy5.5-labeled anti-human CD3 monoclonal (OKT3) antibody (from eBioscience) and with the PE-labeled human CD1d tetramer loaded with PBS57 (from the National Institute of Health Tetramer Core Facility, Emory University, Atlanta, GA, USA), diluted in PBS/2%FBS/0.01%NaN3 (flow cytometry solution) for 20 min, at 4 °C, in the dark. After staining, the cells were washed with flow cytometry
J Clin Immunol
solution and then fixed with PBS 1 % formaldehyde. Samples were run on a 3-laser BD FACS Canto™ II flow cytometer using BD FACSDiva™ software (BD Biosciences). All flow cytometry analyses were performed using FlowJo software (Tree Star). Statistical Analysis Correlations between variables were performed by simple linear regression analysis. Comparisons between groups were performed using the parametric Student’s T test or the nonparametric Kolmogorov-Smirnov test for sample distributions as appropriate. Because of data skewness, the logarithmic transformation was used for analyses of serum ferritin, iNKT cell percentages and iNKT cells numbers. All other continuous variables were not transformed. Because of the observed differences in age distributions between patients and controls, comparisons between these groups were always confirmed after correction for the effect of age. This was done by excluding subjects older than 62 years, which was the maximum value found in controls. Data were analyzed by the Statgraphics software (Version 7.0).
Results Study Population A description of the iron related parameters from HH patients analyzed in this study is given in Table 1 where HH patients are divided in two groups. One group is composed of 20 patients (patients 9–28 in Table 1) who had already undergone intensive treatment for iron depletion and are currently under maintenance treatment (regular phlebotomies every 3 months). These present lower ferritin values (ranging from 14 to 240 ng/mL). The second group includes 8 patients (patients 1–8 in Table 1) who were still in the beginning of intensive (weekly) phlebotomy treatment and have higher ferritin levels (ranging from 1160 to 5934 ng/ mL). The two groups differed significantly in terms of age (p=0.02), treated patients being on average older (61± 12 years) than the untreated (50±8 years). They also differ in the distribution of transferrin saturation and serum ferritin values. All untreated patients had serum ferritin values higher than 1000 ng/ml and transferrin saturation values higher than 65 %, except one patient who presented an acute infection at the time of the study (patient 1 in Table 1). Patients in maintenance treatment had all serum ferritin values below 240 ng/ml but they showed a high heterogeneity in transferrin saturation levels, ranging from 17 to 90 %. In total, seven patients showed an elevation of the ALT marker of active liver damage, most of these (6/7) corresponding to untreated patients.
Table 1 Sex, age, serum iron-related parameters and liver function marker of the Hereditary Hemochromatosis patients Subjects
Sex
Age
Trf Sat (%)
Ferritin (ng/ml)
ALT (U/L)
1* 2 3 4
M M M M
56 52 55 45
41 81 77 89
1925 1949 1431 3638
81 19 60 62
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
M M M M M M F M M F M F F F M F M M
39 48 42 61 50 48 51 67 60 55 68 77 52 60 39 80 44 71
81 92 65 81 52 61 43 23 43 84 65 19 56 33 84 45 22 17
1415 1646 1160 5934 89 160 205 39 34 75 172 23 94 50 215 119 39 39
47 34 222 74 25 24 10 19 16 16 25 16 15 14 78 29 25 24
23 24 25 26 27 28
F F M M M M
76 77 48 67 63 62
25 20 72 90 32 77
53 14 240 94 32 50
11 15 16 17 10 31
Trf Sat Transferrin saturation, ALT Alanine transaminase, M male, F female. *HH patient with an acute infection at the time of analyses
HH Patients Have Lower Numbers of iNKT Cells in the Peripheral Blood It has been previously reported that HH patients present lower numbers of peripheral blood CD8+ T cells [25, 26]. Accordingly, in the present patient cohort we found a significant decrease both in the percentage (p=0.002) and total number (p=0.012) of peripheral blood CD8+ T cells and no differences in the CD4+ T cell population (Table 2) and total lymphocyte numbers. iNKT cells were analyzed in blood samples from HH patients and control subjects by flow cytometry using antiCD3 antibody and the PBS57 loaded CD1d tetramer. Figure 1a illustrates the flow cytometry gating strategies. In the control group we found a percentage of iNKT cells over total T cells ranging from 0.012 to 0.53, whereas in HH patients it varied between undetectable to 0.313. For further
J Clin Immunol Table 2
T lymphocyte subsets in HH patients and control subjects Controls (n=24) HH Patients (n=28)
CD4+ T cells % 43±9 CD8+ T cells % 22±8 * iNKT cells % 0.1±0.1 CD4+ number (×106/mL cells) 0.96±0.3 CD8+ number (×106/mL cells) 0.5±0.3 * iNKT number (×106/mL cells) 0.001±0.002 *
49±11 15±6 * 0.05±0.09 1±0.5 0.3±0.2 * 0.0006±0.001 *
Values are given as the Mean±SD. * p
ORIGINAL RESEARCH
Invariant Natural Killer T Cells are Reduced in Hereditary Hemochromatosis Patients M. L. Maia & C. S. Pereira & G. Melo & I. Pinheiro & M. A. Exley & G. Porto & M. F. Macedo
Received: 26 September 2014 / Accepted: 27 November 2014 # Springer Science+Business Media New York 2014
Abstract Purpose Invariant natural killer T (iNKT) cells are CD1d restricted-T cells that react to lipid antigens. iNKT cells were shown to be important in infection, autoimmunity and tumor surveillance. Alterations in the number and function of these cells were described in several pathological conditions including autoimmune and/or liver diseases. CD1d is critical for antigen presentation to iNKT cells, and its expression is increased in liver diseases. The liver is the major organ affected in Hereditary Hemochromatosis (HH), an autosomal recessive disorder caused by excessive iron absorption. Herein, we describe the study of iNKT cells of HH patients. Methods Twenty-eight HH patients and 24 control subjects from Santo António Hospital, Porto, were included in this study. Patient’s iron biochemical parameters (serum M. L. Maia : C. S. Pereira : I. Pinheiro : M. F. Macedo (*)
transferrin saturation and ferritin levels) and the liver function marker alanine transaminase (ALT) were determined at the time of study. Peripheral blood iNKT cells were analyzed by flow cytometry using an anti-CD3 antibody and the CD1d tetramer loaded with PBS57. Results We found a decrease in the percentage and number of circulating iNKT cells from HH patients when compared with control population independently of age. iNKT cell defects were more pronounced in untreated patients, relating with serum ferritin and transferrin saturation levels. No correlation was found with ALT, a marker of active liver dysfunction. Conclusions Altogether, our results demonstrate that HH patients have reduced numbers of iNKT cells and that these are influenced by iron overload. Keywords Invariant natural killer T cells . hereditary hemochromatosis . iron . lipid
UniLiPe, IBMC—Institute for Molecular and Cellular Biology, University Porto, Porto, Portugal e-mail: [email protected] G. Melo : G. Porto Clinical Hematology, CHP-HSA—Santo António General Hospital Porto, Porto, Portugal M. A. Exley University of Manchester, Manchester, UK M. A. Exley Brigham & Women’s Hospital, Harvard Medical School, Boston, MA, USA G. Porto BCRIB, IBMC—Institute for Molecular and Cellular Biology and ICBAS, Abel Salazar Institute for the Biomedical Sciences, University of Porto, Porto, Portugal M. F. Macedo Aveiro Health Sciences Program, University of Aveiro, Aveiro, Portugal
Introduction Invariant natural killer T (iNKT) cells are a distinct subset of T lymphocytes that react with lipid antigens presented by the MHC class I-like CD1d molecules. They are characterized by the expression of a semi-invariant T cell receptor (TCR) that in humans is composed of a Vα24Jα18 chain preferentially combined with the Vβ11 [1–3]. iNKT cell numbers are highly variable between individuals. The numbers can range from 0.001 % to occasionally over 3 % of human peripheral blood mononuclear cells (PBMCs). Human iNKT cells are more abundant in adipose tissue such as omentum in which iNKT cells represent 10 % of the T cells [4]. However, blood iNKT cells are the most studied in humans, since peripheral blood is the most easily available biological sample.
J Clin Immunol
iNKT cells have protective and regulatory functions in tumor surveillance, chronic infectious diseases, transplantation and autoimmunity [1]. A role in the pathogenesis of inflammatory bowel diseases and in asthma was also described [5]. In chronic infection disease caused by HIV and Mycobacterium tuberculosis, iNKT cells had phenotypes associated with immune activation [6]. In both primary biliary cirrhosis patients [7] and in HCV infected patients [8] peripheral blood iNKT cells are decreased [9]. In the liver of primary biliary cirrhosis patients, however, iNKT cells were increased compared with control subjects [7]. Consistent with iNKT cell role in autoimmunity, intrinsic iNKT cell defects were found to be associated with autoimmune diseases, such as type 1 diabetes [10, 11], multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis and Sjogren’s syndrome, cutaneous forms of lupus erythematosus, psoriasis, Crohn’s disease, and ulcerative colitis [12]. Currently, iNKT cells are being used as therapeutic targets in some clinical trials, mainly in oncological conditions, which further increases the need for understanding of their behavior in health and disease [2]. The normal function of iNKT cells relies on the efficient presentation of lipid antigens by CD1d molecules. The CD1d at the surface of antigen presenting cells (APCs) binds to the lipid antigen and this complex is presented to the iNKT cells. Alterations occurring in CD1d, APCs or in the metabolism of lipids can affect the activation of the iNKT cells, since they are required for the iNKT cell activation and proliferation. Human CD1d is expressed on the cell surface of APCs (monocytes, macrophages, B cells, and some myeloid dendritic cells), being responsible for lipid and glycolipid antigenpresentation to iNKT cells [13, 14]. CD1d can also be expressed, at low levels, in several non-leukocyte cells, including hepatocytes [15–17]. Hereditary Hemochromatosis (HH) is an autosomal recessive disease of iron metabolism, in which the major organ affected is the liver. This pathology is characterized by increased intestinal iron absorption, causing augmented plasma iron concentration and consequently an increase in transferrin saturation. Excessive iron in circulation may accumulate in the liver, as well as in other organs, as reflected by an increase in serum ferritin. From a pathogenic point of view, HH is classified as a liver disease with primary failure in the production of the liver iron hormone hepcidin, which is the key regulator of iron absorption and its systemic distribution [18, 19]. In the vast majority of cases, the C282Y HFE mutation in homozygosity is the cause of the hepcidin dysregulation [19, 20]. The signs and symptoms are both general and organ-specific, including diabetes, arthritis, hypogonadism, hyperpigmentation, cardiomyopathy and liver disease [18, 20]. Since iNKT cells frequency and phenotype are altered in several liver diseases [9], and given the strong liver
involvement in HH, we hypothesized that iNKT cells might be altered in this condition. Herein we analyzed the iNKT cells in blood samples from HH patients and correlated this immunological parameter with biochemical parameters of iron overload and liver function.
Methods Population Studied The study was approved by the Ethical Committee of Centro Hospitalar do Porto, Porto. Informed consent was obtained from participants according to the Helsinki declaration. HH Patients Twenty-eight HH patients, followed-up regularly at the Hemochromatosis Outpatient Clinic of Santo António Hospital, Porto, were included in this study. They were all Caucasians from the north of Portugal, with confirmed homozygosity for the C282Y HFE mutation and no confounding co-morbidities. Clinical evaluation of the group of patients was previously described [21–24]. Twenty were males and 8 were females, with a mean age of 58 years old (range 39–80). Biochemical parameters of iron metabolism, serum transferrin saturation (TfSat), serum ferritin levels (SF) and the liver function marker alanine transaminase (ALT) were routinely determined at the time of the study in the Clinical Chemistry Laboratory of Santo António Hospital, Porto, by standard techniques. Control Population Twenty-four apparently healthy subjects, volunteer blood donors at the Blood Bank of Santo António Hospital, Porto, were analyzed as controls to obtain the percentages of peripheral blood iNKT cells. This population included 13 males and 11 females, with a mean age of 45 years old (range 21–62). Mononuclear Cells Isolation from Human Peripheral Blood and Flow Cytometry Total lymphocyte counts were determined in an automatic blood cell counter (Adavia 120; Hematology Systems, Bayer®, Tarrytown, NY, USA). Peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque-1077® (Sigma) density centrifugation following the manufacturer’s instructions. Human PBMCs were stained with PerCP/ Cy5.5-labeled anti-human CD3 monoclonal (OKT3) antibody (from eBioscience) and with the PE-labeled human CD1d tetramer loaded with PBS57 (from the National Institute of Health Tetramer Core Facility, Emory University, Atlanta, GA, USA), diluted in PBS/2%FBS/0.01%NaN3 (flow cytometry solution) for 20 min, at 4 °C, in the dark. After staining, the cells were washed with flow cytometry
J Clin Immunol
solution and then fixed with PBS 1 % formaldehyde. Samples were run on a 3-laser BD FACS Canto™ II flow cytometer using BD FACSDiva™ software (BD Biosciences). All flow cytometry analyses were performed using FlowJo software (Tree Star). Statistical Analysis Correlations between variables were performed by simple linear regression analysis. Comparisons between groups were performed using the parametric Student’s T test or the nonparametric Kolmogorov-Smirnov test for sample distributions as appropriate. Because of data skewness, the logarithmic transformation was used for analyses of serum ferritin, iNKT cell percentages and iNKT cells numbers. All other continuous variables were not transformed. Because of the observed differences in age distributions between patients and controls, comparisons between these groups were always confirmed after correction for the effect of age. This was done by excluding subjects older than 62 years, which was the maximum value found in controls. Data were analyzed by the Statgraphics software (Version 7.0).
Results Study Population A description of the iron related parameters from HH patients analyzed in this study is given in Table 1 where HH patients are divided in two groups. One group is composed of 20 patients (patients 9–28 in Table 1) who had already undergone intensive treatment for iron depletion and are currently under maintenance treatment (regular phlebotomies every 3 months). These present lower ferritin values (ranging from 14 to 240 ng/mL). The second group includes 8 patients (patients 1–8 in Table 1) who were still in the beginning of intensive (weekly) phlebotomy treatment and have higher ferritin levels (ranging from 1160 to 5934 ng/ mL). The two groups differed significantly in terms of age (p=0.02), treated patients being on average older (61± 12 years) than the untreated (50±8 years). They also differ in the distribution of transferrin saturation and serum ferritin values. All untreated patients had serum ferritin values higher than 1000 ng/ml and transferrin saturation values higher than 65 %, except one patient who presented an acute infection at the time of the study (patient 1 in Table 1). Patients in maintenance treatment had all serum ferritin values below 240 ng/ml but they showed a high heterogeneity in transferrin saturation levels, ranging from 17 to 90 %. In total, seven patients showed an elevation of the ALT marker of active liver damage, most of these (6/7) corresponding to untreated patients.
Table 1 Sex, age, serum iron-related parameters and liver function marker of the Hereditary Hemochromatosis patients Subjects
Sex
Age
Trf Sat (%)
Ferritin (ng/ml)
ALT (U/L)
1* 2 3 4
M M M M
56 52 55 45
41 81 77 89
1925 1949 1431 3638
81 19 60 62
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
M M M M M M F M M F M F F F M F M M
39 48 42 61 50 48 51 67 60 55 68 77 52 60 39 80 44 71
81 92 65 81 52 61 43 23 43 84 65 19 56 33 84 45 22 17
1415 1646 1160 5934 89 160 205 39 34 75 172 23 94 50 215 119 39 39
47 34 222 74 25 24 10 19 16 16 25 16 15 14 78 29 25 24
23 24 25 26 27 28
F F M M M M
76 77 48 67 63 62
25 20 72 90 32 77
53 14 240 94 32 50
11 15 16 17 10 31
Trf Sat Transferrin saturation, ALT Alanine transaminase, M male, F female. *HH patient with an acute infection at the time of analyses
HH Patients Have Lower Numbers of iNKT Cells in the Peripheral Blood It has been previously reported that HH patients present lower numbers of peripheral blood CD8+ T cells [25, 26]. Accordingly, in the present patient cohort we found a significant decrease both in the percentage (p=0.002) and total number (p=0.012) of peripheral blood CD8+ T cells and no differences in the CD4+ T cell population (Table 2) and total lymphocyte numbers. iNKT cells were analyzed in blood samples from HH patients and control subjects by flow cytometry using antiCD3 antibody and the PBS57 loaded CD1d tetramer. Figure 1a illustrates the flow cytometry gating strategies. In the control group we found a percentage of iNKT cells over total T cells ranging from 0.012 to 0.53, whereas in HH patients it varied between undetectable to 0.313. For further
J Clin Immunol Table 2
T lymphocyte subsets in HH patients and control subjects Controls (n=24) HH Patients (n=28)
CD4+ T cells % 43±9 CD8+ T cells % 22±8 * iNKT cells % 0.1±0.1 CD4+ number (×106/mL cells) 0.96±0.3 CD8+ number (×106/mL cells) 0.5±0.3 * iNKT number (×106/mL cells) 0.001±0.002 *
49±11 15±6 * 0.05±0.09 1±0.5 0.3±0.2 * 0.0006±0.001 *
Values are given as the Mean±SD. * p
