All of the samples of human patients (A to I) were obtained from an outbreak of eosinophilic meningitis in 1999. Those samples were collected at the research laboratory of Infectious Diseases Department, Kaohsiung Veterans General Hospital (KVGH) and the patient’s information was delinked. Institutional review board (IRB) approval to use the samples was obtained from the Commission on Medical Ethics of the Kaohsiung Veterans General Hospital (VGHKS98-CT8-07). All participants were informed about the study procedures and gave their written informed consent initially when the outbreak occurred. Animal studies were carried out in strict accordance with the recommendations from Taiwan’s Animal Protection Act. The protocol was approved by the Animal Committee of the Kaohsiung Veterans General Hospital.
A case of eosinophilic meningitis was clinically defined as an acute onset of headache, eosinophil pleocytosis in the blood/CSF, accompanied by at least of one of the following: fever, ataxia, visual disturbances, photophobia, nuchal rigidity, neck pain, hyperesthesias, or paresthesias. All patients who had eaten raw snails within 3 weeks of the outbreak’s onset were included in the study. We recorded demographic information, the date of snails were eaten and the amount ingested, clinical symptoms, and prior parasitic infections. Each patient underwent a physical, neurologic, and ophthalmic examination. The headache intensity was rated on a 4-point scale ranging from none to severe (0 = none, 1+ = mild, 2+ = moderate, 3+ = severe). The hyperintense basal ganglia lesion on T1-weighted MRI was also graded as a 4-point scale ranging from none to severe by 2 independent radiologists. Spinal taps were performed on all patients. The patients were observed daily during their hospital course. CSF was examined weekly. Peripheral blood was obtained weekly for the first 2 months, every other week for the next 2 months, and monthly thereafter for as long as 6 months. Antibodies to A. cantonensis were detected in serum and CSF by a microenzyme-linked immunosorbent assay (ELISA) using young-adult worm antigen, molecular weight 204 kDa purified by monoclonal antibody.
Infection of Balb/C mice with third stage larvae of A. cantonensis
Twenty-four Balb/C mice, aged 6–7 weeks, were purchased from the National Laboratory Animal Breeding Research Centre. They were raised and maintained in an air-conditioned animal facility (25 ± 2°C and 50 ± 10% relative humidity). Third-stage larvae of A. cantonensis were harvested from infected Biomphalaria glabrata after treatment with artificial gastric juice (pepsin, 2 g; concentrated HCl, 7 mL; distilled water, 1 L). Mice were orally infected with 50 A. cantonensis L3 via an orogastric tube after slight ether anesthesia and then mice were euthanized every week for 3 consecutive weeks after infection.
Collection of serum and CSF specimens
Blood samples from experimental mice were collected by a heart puncture under ketamine anesthesia. Serum specimens separated from blood samples after centrifugation at 3500 × g (Hermle, Z326K, Germany) for 5 min at 4°C were stored at - 70°C until they were measured.
The skull of the mice was opened after complete bleeding. Careful surgery was conducted in order to avoid blood contamination of the CSF. The brain was removed and washed with 50 μL 0.15 M phosphate buffered saline (PBS). Concurrently, the cerebral ventricles and cranial cavity were washed with 350 μL PBS. The CSF was, thus, harvested with PBS from above, which was then centrifuged in an eppendorf tube at 3000 × g (Hermle, Z326K, Germany) for 10 min at 4°C to eliminate cells. The supernatant was stored at -70°C until further use.
Measurement of permeability of the blood–brain barrier by Evans blue method
Evans blue was used to assess the permeability of the blood–brain barrier to macromolecules. When the blood–brain barrier had been compromised, albumin-bound Evans blue entered the CNS. In brief, a volume of 200 μL of 2% (w/v) solution of Evans blue in PBS was injected into the tail vein of a mouse. One hour later the brain of the mouse was removed after anesthesia with ketamine, which was ground with 1.0 mL PBS in a glass-tissue grinder with a Teflon pestle. The extract was then centrifuged at 18,000 × g (Hermle, Z326K, Germany) for 10 min at room temperature. The optical density (OD) of the supernatant was read at 595 nm wavelength using a colorimeter (Thermo scientific multiskan FC, USA). Then the OD values of mice brain supernatant after 1 to 3 weeks infections were compared with the control.
Measurement of 14-3-3β protein concentrations in CSF/serum by Western blot analysis
CSF/serum aliquots of 100 μL from 9 patients and mice were mixed with 7 volumes of cold methanol, kept at -20°C for 2 h, and then centrifuged at 20,800 g for 30 min. The pellet was dissolved in 40 μL of sample buffer (3% SDS, 3% β-mercaptoethanol, 2 mM EDTA, 10% glycerol, and 62.5 mM Tris, pH, 6.8) and boiled for 5 min. For each sample, 10 μL (the equivalent of 25 μL of CSF), 5 mL, and 1.25 mL of sample buffer/well were loaded onto a 13% polyacrylamide gel and transferred to polyvinyliden difluoride membranes (Immobilon P; Millipore). Membranes were incubated with anti 14-3-3β polyclonal rabbit IgG (Santa Cruz Biotechnology) at a 1:500 dilution and revealed with anti-rabbit horseradish peroxidase IgG (Amersham) at a 1:3000 dilution. The blots were developed using an enhanced chemiluminescent system (Amersham). Densitometric values for each sample were obtained with a computer-assisted laser scanner (GS-710 Calibrated Imaging Densitometry; BioRad), after correction for background. The total amount of 14-3-3 protein as quantified from each diluted and undiluted CSF sample was expressed in arbitrary units. The human control group (n = 9), matched for age and gender, consisted of patients with headache or altered consciousness who underwent lumbar puncture to exclude meningitis. CSF samples were centrifuged and the supernatants were frozen at -80°C until assayed.
Generation of recombinant 14-3-3β
Recombinant human 14-3-3β protein was purified from E. coli for antibodies generation as previously described. The human 14-3-3β cDNA was amplified from a human fetal brain cDNA library (Stratagene, La Jolla, CA) using the polymerase chain reaction (PCR). The PCR primers used to clone the human 14-3-3β cDNA were designed based on the 14-3-3β sequence in the Gen-Bank database (accession number, NM_003404.3; forward primer, 5′- cgcggatccatgacaatggataaaagtgagctg -3′; reverse primer, 5′- ggcgaattcttagttctctccctccccagc-3′). After DNA sequencing analysis, the PCR-amplified 14-3-3β cDNA was subcloned into the EcoR I and BamH I sites of the pET28a vector (Novagen, Madison, WI) and transformed into BL-21 cells (DE3, pLysS; Novagen). After induction, the 6x-histidine-tagged 14-3-3β protein was purified on an NTA-agarose affinity column (Qiagen, Hilden, Germany) and desalted on a G25 Sephadex column (Amersham Pharmacia, Little Chalfont, United Kingdom). The recombinant protein was passed through Detoxi-Gel (Pierce Biotechnology, Rockford, IL) to minimize contamination by endotoxin. The 14-3-3β antibodies were raised by periodic injection of recombinant 14-3-3β protein into rabbits. The serum was collected from immunized rabbits and analyzed using Western blot analysis.
Production of anti-human 14-3-3β polyclonal antisera
Purified recombinant 14-3-3β protein in PBS (500 μg in 500 μl) is mixed with complete Freunds adjuvant in a three-way stopcock until mixture becomes emulsified. The mixture is then transferred to a 3 ml 24-gauge syringe and was injected subcutaneously into adult New Zealand white rabbits (2 to 5 kg body weight) under restrain. Two weeks after the primary immunization, the rabbits were then boosted with recombinant 14-3-3β protein mixed with incomplete Freunds adjuvant at 2-week intervals for a total of nine boosts.
Blood serum preparation
Blood was collected from the ear marginal veins of restrained rabbits before immunization (pre-immune serum) and after subsequent boosts for a total of six batches. About 50 ml of blood was collected during each interval. After removing blood clots, the serum samples were placed overnight in 4°C and centrifuged 10 minutes at 5,000 g to remove red blood cell pellets and other cell debris. The samples were stored at -70°C prior to purification.
Serum samples containing 14-3-3β antibodies were purified using Protein A column (Pharmacia Biotech) and quantified with Coomassie Plus Bradford assay kit (Pierce, #23236). Antibody efficacy was checked with Western blot analysis using recombinant 14-3-3β protein at concentration of 1, 0.1 and 0.01ug/ml.
14-3-3β enzyme-linked immunosorbent assay (ELISA)
The in-house 14-3-3β ELISA consisted of rabbit anti-14-3-3β polyclonal antibody as the capture antibody and mouse anti-14-3-3β IgG conjugated with horse-radish peroxidase (Santa Cruz Biotechnologies Inc.; sc-1657 HRP (H-8), 200 μg/ml) as the detection antibody, with the purified recombinant 14-3-3β protein as standard. Briefly, 96-well plate were coated with 50 μl per well of diluted capture antibody (2.5 μg/ml) overnight at 4°C. After washing with buffer containing 0.05% Tween 20 in PBS, wells were blocked with buffer containing 0.05% Tween 20, 0.1% BSA, 5% sucrose in PBS at room temperature for 30 min. The CSF/serum samples (50 μl) or standards were applied to wells and incubated at room temperature for 1 hour. After wash, the wells were incubated with detection antibody (mouse anti-14-3-3β, 1 μg/ml) at room temperature for 1 hour. Finally, 100 μl of TMB substrate solutions were added and reacted at room temperature for 20 min. After adding 50 μl of 2 N H2SO4, the optical density in each well was measured at 450 nm using an ELISA reader.
The relation between the amount of 14-3-3β protein in CSF, laboratory abnormalities, and clinical severity and MRI findings were analyzed with Spearman’s correlation coefficient test. Wilcoxon signed-rank test was used to compare the change of 14-3-3β protein in every week of lumbar puncture. Mann Whitney U test was used to compare the changes of 14-3-3β protein or Evans blue in third week relative to the controls. A P value <0.05 was considered statistically significant.