A total of 28 AE patients admitted at the First Affiliated Hospital of Xinjiang Medical University from February 2016 to October 2019 were included in the study cohort. Patients were diagnosed with AE following international recommendations. The exclusion criteria included: (1) Patients with cystic echinococcosis. (2) Patients with viral hepatitis, autoimmune diseases, and tumors. We grouped 28 AE patients following the WHO Informal Working Group on Echinococcosis PNM (P = parasitic mass in the liver, N = involvement of neighboring organs, and M = metastasis) classification and staging of alveolar echinococcosis. 28 healthy volunteers from the same demographic origin who received annual health examinations were included as the control group. Detailed baseline information for all subjects is summarized in Table S1.The handling of human subjects followed the principles of the Helsinki Declaration and was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University. We obtained informed consent from all participants or their legal custody before commencing the study.
Grouping patients based on lesion ‘metabolic activity’
We performed the [18F] Fluorodeoxyglucose positron emission tomography/computed tomography ([18F] FDG PET-CT) scan of the patients immediately after admission or before the operation. The PET/CT acquisitions were conducted 3 hours after 18F-FDG injection . The images were analyzed by two experienced nuclear physicians blinded to the clinical, biological, and pathological characteristics of the patients. Subsequently, following the maximum standardized uptake value (SUVMAX) of [18F]FDG PET-CT, patients were grouped into High Metabolically Active AE (HMAE SUVMAX>4.0) and Low Metabolically Active AE (LMAE SUVMAX≤4.0) categories. The demographic characteristics of subgroups (HMAE vs. LMAE) did not exhibit any significant difference(Table S1).
Blood samples were collected from the subjects on the admission day. Serum was extracted from the samples through centrifugation at 2,500 × g for 10 minutes and stored at -80°C awaiting further analysis. Besides, a similar procedure was used to process samples from the control subjects.
Sections of the liver tissue were obtained from the 28 AE patients previously underwent surgical resection. One specimen was obtained from a region close to the parasitic lesion including the metacestode (close liver tissue [CLT], about 0.5 cm from lesion). Another specimen was obtained from the macroscopically normal liver distant from the lesion (distant liver tissue [DLT], at least 2 cm distant from lesion) as previously described .
The quantities of sPDGF-BB were estimated via enzyme-linked immunosorbent assay (ELISA) tests (Quantikine; R&D Systems, Minneapolis, MN, USA). The concentrations of sPDGF-BB were adjusted to the platelet count levels which were obtained from a complete blood count.
Liver tissue samples were prepared for immunohistochemical analysis. We analyzed the PDGF-BB staining results using IPWIN (version 184.108.40.206 Image-Pro Plus, California, USA). IHC staining was conducted as described previously by Chan et al. Stained sections were visualized by two senior pathologists blinded to each other’s results. Briefly, we incubated tissue sections with primary antibodies for mouse PDGF-BB (Abcam, 1:200 Polyclonal) overnight at 4°C. Subsequent incubation with corresponding secondary antibodies was performed at room temperature for 1 hour in the dark. Immunoreactivity was observed using a horseradish peroxidase-streptavidin detection system (Abcam).
Statistical analyses were performed using the SPSS statistical software package version 20.0 (SPSS, Inc, Chicago, IL, USA) and GraphPad Prism (version 7.0d for MacOS X, USA, GraphPad Software, San Diego, California, USA). Non-parametric data were analyzed via the Mann-Whitney U test, and categorical data were subjected to the Chi-square test. A Spearman correlation coefficient ≥ 0.4 was considered a relevant correlation and a p-value less than 0.05 was considered significant. The immunohistochemical results of PDGF-BB and CD31 were normalized using the following formula.
We mapped the range of values of a set of data X to an interval , where Y is the mapped value, Xmax is the maximum value, and Xmin is the minimum score in this data set. Data that did not conform to the normal distribution such as ALT, AST, and hospitalization days were transformed by Log.