HCV Infection: A Risk Factor for Parkinson's Disease
The study population was derived from a community-based integrated screening programme in Keelung (KCIS), the northernmost area in Taiwan. A total of 63 163 subjects aged 40 or older were enrolled between 2000 and 2004. The ascertainment of PD was either through active community-based survey or hospital-based clinical series cases. The medical record consisted of information on medical visits, such as ICD codes, date of visit and prescriptions. The three major diagnostic codes were kept for financial reimbursement for all treatments, therapies and prescriptions. The major diagnostic code 332.0 was used to identify patients with PD and excluded parkinsonism caused by other reasons, such as vascular disease-related parkinsonism, drug-induced parkinsonism, multiple system atrophy and parkinsonism secondary to brain insults. Finally, 887 PD cases were found.
In the KCIS programme, HBsAg was detected using radioimmunoassay kits (Abbott Laboratories, Chicago, IL, USA), and anti-HCV was detected using a third-generation enzyme immunoassay (Abbott Laboratories). Venous blood samples were taken after the subjects had fasted at least 12 h for the measurement of plasma glucose, triacylglycerol, total cholesterol, high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol. The definition of metabolic syndrome was based on the modified National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria, that is when ≥3 of the following criteria were satisfied: (i) central obesity (waist circumference ≥90 cm for men and ≥80 cm for women), (ii) hypertriacylglycerolemia (≥150 mg/dL), (iii) an abnormally low HDL-cholesterol concentration (<40 mg/dL for men and <50 mg/dL for women), (iv) raised blood pressure (≥130 mmHg systolic or ≥85 mmHg diastolic) or (v) a raised fasting glucose concentration (≥100 mg/dL).
To prepare the HCV or hepatitis B virus (HBV) viral particles, 1 mL of serum from the chronic hepatitis B or C patients, and 0.5 mL of 30% polyethylene glycol 6000 in 1.5 m NaCl was added and left overnight at 4 °C. The sample was then centrifuged for 30 min at 3300 g, and the pellet was resuspended in culture medium for the coculture experiments. The viral titre of the medium was quantified by real-time PCR using an ABI 5700 sequence detection system (PE Applied Biosystems, Warrington, UK).
Neuron–glia cocultures from E-14 Wistar embryonic rat midbrain were obtained. Midbrain neuro-glia coculture has been used to study neuroinflammation in vitro, such as the potential neuroprotective effect of anti-inflammatory compounds. For the details of the test refer to Hung's paper. The cells were seeded in Dulbecco's Modified Eagle's Medium (DMEM) with 10% foetal bovine serum at 5 × 10 in 24-well plates previously coated with poly-D-lysine. The culture was kept in a humidified chamber at 37 °C in a 5% CO2 atmosphere. Twenty-four hours after plating, the culture was changed to Minimum Essential Medium (MEM) with 2% FBS and 2% horse serum. After 1 week, the primary midbrain neuron–glia cocultures were treated with 100 nm MPP in the presence or absence of testing compounds for 48 h. Dopaminergic neurons were characterized by immunostaining with a rabbit antityrosine hydroxylase (anti-TH) antibody (1:5000; Calbiochem, Darmstadt, Germany). A biotinylated goat anti-rabbit IgG was used for staining revealed by the ABC method (Vector Laboratories, Burlingame, CA, USA) and developed using 0.04% (w/v) diaminobenzidine to produce a brown reaction product. The number of TH (+) cell was counted under a microscope.
Commercially available cytokine antibody arrays from R&D Systems (Minneapolis, MN, USA, Proteome Profiler Rat Cytokine Array Panel A; ARY008) were used to measure differences in the relative amounts of cytokines released from midbrain in the presence and absence of virus. The array consists of a nitrocellulose membrane dotted with antibodies that recognize 29 distinct cytokines, chemokines and growth factors. Following a 48 h incubation of midbrain cultures with or without HBV or HCV, the culture media were collected for analysis. A detection antibody cocktail consisting of 29 biotinylated antibodies, each targeted to a specific cytokine, was added to the treated media samples. The media were then incubated overnight at 4 °C with the cytokine antibody arrays. Following this, the media was washed from the array and a Streptavidin–HRP solution was added to the membrane for 30 min at room temperature. The cytokine array was again washed, and a chemiluminescent HRP substrate (EMD Millipore, Darmstadt, Germany) was added to the membrane. The membranes were then exposed to X-ray film for 1–5 min, and the relative pixel density of each spot on the array was quantified using Image J (National Institutes of Health, Bethesda, MD, USA) software.
The relationships between hepatitis virus infection status, demographic factors, smoking habits, metabolic syndrome and PD were expressed as odds ratios (OR) and 95% confidence intervals (CI) using a univariate logistic regression model. Interactions between all pairs of factors were tested in the model. Afterwards, the adjusted ORs of hepatitis virus, smoking and metabolic syndrome were estimated after controlling for age, gender, education level and also possible interactions between the two variables. To compute adjusted odds ratios for the association between HCV and PD, smoking and metabolic syndrome together with age, gender and education levels were retained in the final model.
Materials and Methods
Study Population and Community-based Model for Ascertained PD
The study population was derived from a community-based integrated screening programme in Keelung (KCIS), the northernmost area in Taiwan. A total of 63 163 subjects aged 40 or older were enrolled between 2000 and 2004. The ascertainment of PD was either through active community-based survey or hospital-based clinical series cases. The medical record consisted of information on medical visits, such as ICD codes, date of visit and prescriptions. The three major diagnostic codes were kept for financial reimbursement for all treatments, therapies and prescriptions. The major diagnostic code 332.0 was used to identify patients with PD and excluded parkinsonism caused by other reasons, such as vascular disease-related parkinsonism, drug-induced parkinsonism, multiple system atrophy and parkinsonism secondary to brain insults. Finally, 887 PD cases were found.
Serum Viral Markers and Biochemical Variables
In the KCIS programme, HBsAg was detected using radioimmunoassay kits (Abbott Laboratories, Chicago, IL, USA), and anti-HCV was detected using a third-generation enzyme immunoassay (Abbott Laboratories). Venous blood samples were taken after the subjects had fasted at least 12 h for the measurement of plasma glucose, triacylglycerol, total cholesterol, high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol. The definition of metabolic syndrome was based on the modified National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria, that is when ≥3 of the following criteria were satisfied: (i) central obesity (waist circumference ≥90 cm for men and ≥80 cm for women), (ii) hypertriacylglycerolemia (≥150 mg/dL), (iii) an abnormally low HDL-cholesterol concentration (<40 mg/dL for men and <50 mg/dL for women), (iv) raised blood pressure (≥130 mmHg systolic or ≥85 mmHg diastolic) or (v) a raised fasting glucose concentration (≥100 mg/dL).
Harvest of HCV or HBV Viral Particles From Serum
To prepare the HCV or hepatitis B virus (HBV) viral particles, 1 mL of serum from the chronic hepatitis B or C patients, and 0.5 mL of 30% polyethylene glycol 6000 in 1.5 m NaCl was added and left overnight at 4 °C. The sample was then centrifuged for 30 min at 3300 g, and the pellet was resuspended in culture medium for the coculture experiments. The viral titre of the medium was quantified by real-time PCR using an ABI 5700 sequence detection system (PE Applied Biosystems, Warrington, UK).
Midbrain Neuron–Glia Coculture
Neuron–glia cocultures from E-14 Wistar embryonic rat midbrain were obtained. Midbrain neuro-glia coculture has been used to study neuroinflammation in vitro, such as the potential neuroprotective effect of anti-inflammatory compounds. For the details of the test refer to Hung's paper. The cells were seeded in Dulbecco's Modified Eagle's Medium (DMEM) with 10% foetal bovine serum at 5 × 10 in 24-well plates previously coated with poly-D-lysine. The culture was kept in a humidified chamber at 37 °C in a 5% CO2 atmosphere. Twenty-four hours after plating, the culture was changed to Minimum Essential Medium (MEM) with 2% FBS and 2% horse serum. After 1 week, the primary midbrain neuron–glia cocultures were treated with 100 nm MPP in the presence or absence of testing compounds for 48 h. Dopaminergic neurons were characterized by immunostaining with a rabbit antityrosine hydroxylase (anti-TH) antibody (1:5000; Calbiochem, Darmstadt, Germany). A biotinylated goat anti-rabbit IgG was used for staining revealed by the ABC method (Vector Laboratories, Burlingame, CA, USA) and developed using 0.04% (w/v) diaminobenzidine to produce a brown reaction product. The number of TH (+) cell was counted under a microscope.
Cytokine/Chemokine Arrays
Commercially available cytokine antibody arrays from R&D Systems (Minneapolis, MN, USA, Proteome Profiler Rat Cytokine Array Panel A; ARY008) were used to measure differences in the relative amounts of cytokines released from midbrain in the presence and absence of virus. The array consists of a nitrocellulose membrane dotted with antibodies that recognize 29 distinct cytokines, chemokines and growth factors. Following a 48 h incubation of midbrain cultures with or without HBV or HCV, the culture media were collected for analysis. A detection antibody cocktail consisting of 29 biotinylated antibodies, each targeted to a specific cytokine, was added to the treated media samples. The media were then incubated overnight at 4 °C with the cytokine antibody arrays. Following this, the media was washed from the array and a Streptavidin–HRP solution was added to the membrane for 30 min at room temperature. The cytokine array was again washed, and a chemiluminescent HRP substrate (EMD Millipore, Darmstadt, Germany) was added to the membrane. The membranes were then exposed to X-ray film for 1–5 min, and the relative pixel density of each spot on the array was quantified using Image J (National Institutes of Health, Bethesda, MD, USA) software.
Statistical Analysis
The relationships between hepatitis virus infection status, demographic factors, smoking habits, metabolic syndrome and PD were expressed as odds ratios (OR) and 95% confidence intervals (CI) using a univariate logistic regression model. Interactions between all pairs of factors were tested in the model. Afterwards, the adjusted ORs of hepatitis virus, smoking and metabolic syndrome were estimated after controlling for age, gender, education level and also possible interactions between the two variables. To compute adjusted odds ratios for the association between HCV and PD, smoking and metabolic syndrome together with age, gender and education levels were retained in the final model.
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