Patients
This study is a descriptive observational. Patients diagnosed with BS were recruited in spine surgery at two hospitals in Xinjiang, Urumqi from 2015 to 2019. The study was approved by the ethics committee of Xinjiang Medical University. The patients were categorized as acute phase (with symptoms less than 3 months), subacute phase (3–6 months) and chronic phase (more than 6 months). The clinical symptoms and laboratory results were collected. The paravertebral cartilage tissue (include close and distant tissue) and peripheral blood of 29 BS patients were collected. 15 healthy controls were enrolled. The consent of patients was received at the same time. The data used to support the findings of this study are available from the corresponding author upon request.
The inclusion criteria of BS patients: 1. Patients have a history of contact with Brucella infected livestock or livestock products, consumption of raw meat and unpasteurized dairy products. 2. The patients have a persistent pain in the lower back, sacroiliac, fever, sweating, fatigue, or joint pain for days or even weeks. 3. Brucella was detected after one-week blood culture. 4. Tube agglutination test is positive. 5. Magnetic Resonance Imaging (MRI) reports that the adjacent surface of vertebra is destroyed, and there are hyperplasia and sclerosis. 6. Exclude patients with other immune, neoplastic diseases and HIV infection.
Specimens
BS tissue specimen (n = 29) and corresponding peripheral blood samples (n = 29) was obtained. All samples were collected at the close and distant normal tissues, by which tissues in vitro were washed in PBS (Boster, CN), and fixed in 4% paraformaldehyde (Beijing Chemical Works, CN) for 24–48 h. Then the tissues were decalcified in 10% EDTA decalcification solution for 21–30 days. The samples were then embedded in paraffin and sectioned transversely at 4 μm continuously to make parallel sections. Peripheral blood (3 ml) was drawn from each subject and treated with Ficoll-Hypaque Solution (Tianjin TBD, CN) to get peripheral blood mononuclear cells (PBMCs).
Hematoxylin-eosin (H&E) staining
H&E staining was performed according to routine procedure, including hematoxylin (Solarbio, CN) staining for 3 min, eosin (Solarbio, CN) staining for 2 min, 1% hydrochloric acid differentiation, ethanol gradient dehydration, neutral gum seal, and microscopic observation of histopathological changes.
Immunohistochemistry (IHC) staining
In short, sections, 4 μm thick, were deparaffinised and hydrated. After antigen retrieval, they were treated with primary antibody (Dilution ratio: Brucella-Ab, 1:200; IFN-γ, 1:100; CXCR3, 1:200; CXCL9, 1:200; CXCL10, 1:200) (Concentration: Brucella-Ab, 5 μg/ml; IFN-γ, 10 μg/ml; CXCR3, 5 μg/ml; CXCL9, 5 μg/ml; CXCL10, 5 μg/ml) (Beijin Bioss, CN) at appropriate dilution at 4 °C overnight. Sections were incubated with secondary antibody (Zhongshan Jinqiao, CN) for 1.5 h. Visualisation was performed with 3, 3′-diaminobenzidine (DAB) as substrate, applied for 1.5 min. Sections were counterstained with Hematoxylin. Antigen expression was analyzed under × 20 medium power lens. Positive signals were required to meet two criteria: irregular patchy dark brown granules; more than five clusters. IHC positive expression area was processed by Image J. The average optical density (AOD) obtained was the final processing results.
Real time PCR (RT-PCR)
Total RNA was extracted from the PBMCs by Trizol reagent (Invitrogen, USA). RNA quantity (> 80 ng/μl) and purity (OD260/OD280 = 1.8–2.0) were determined by nucleic acid quantifier. RNA was treated with DNase I (Takara, Japan) before reverse transcription to eliminate contaminating genomic DNA. cDNA was synthesized by reverse transcription according to the PrimeScript™ RT reagent Kit (Takara, Japan). The total amount of RNA was 3.75 μl. For RT-PCR, we mixed a 2 μl cDNA, 1 μl forward primers and 1 μl reverse primers (the final concentration of the primers was 10 μM.), 12.5 μl TB Green Premix Ex Taq (Takara, Japan) and 8.5 μl DEPC water. Thermal cycle parameters were: 95 °C for 30 s for 1 cycle, then 95 °C for 5 s, and 60 °C for 30 s for 39 cycles. Primer sequences are shown in Table 1. GAPDH was used as the internal control. The relative expression was calculated by the comparative cycle threshold method (2-△△t).
Enzyme-linked immuno sorbent assay (ELISA)
Serum levels of IFN-γ, CXCL9, CXCL10 (eBioscience, Austria) and autoantibodies against CXCR3 (CellTrend GmbH Luckenwalde, Germany), were detected in accordance with ELISA kit instructions. Standard wells, blank control wells and sample wells were set on 96-well plates. The diluted standard was added to the standard well, the sample dilution was added to the blank control well, and the test serum was added to the sample well. Then they were placed in 37 °C incubator for 60 min. After washing, color development and termination, the absorbance (A) was detected at a wavelength of 450 nm. For anti-CXCR-3-antibodies, the CXCR-3-receptor has been pre-coated onto a microtiter plate. During the first incubation the anti-CXCR-3-antibodies of the samples are immobilised on the plate. The autoantibodies are detected with a POD labeled antihuman IgG antibody. In the following enzymatic substrate reaction the intensity of the colour correlates with the concentration and/ or avidity of anti-CXCR-3-antibodies. The concentration was determined according to the standard curve.
Statistical analysis
All data in this study were analyzed by SPSS 22.0 and GraphPad Prism 8.0. Quantitative data with normal distribution were expressed as mean ± standard deviation (SD) and t-test was used for comparison between groups. Quantitative data with a non-normal distribution are presented as medians (interquartile ranges), and the groups were compared using a Wilcoxon rank-sum test. The receiver operating characteristic (ROC) curve was established to evaluate the diagnostic value. The area under the curve (AUC) was calculated. The Youden index was used to determine the optimal cutoff value. A two-tailed p value of < 0.05 was considered to indicate significance.