Patients and specimens
We have totally recruited 71 brucella patients who have referred to the either local health centers or hospitals of kerman province. Diagnosis of acute brucellosis was made based on a brief history of the disease,clinical examinations,results of serological tests,antibody titration in serums was performed by bacterial wright method, agglutination and 2ME with antigen and wright rate . The Elisa Juan behring device was used to read Elisa's results. Fifty (50) people of both sexes from the blood donors and students from kerman province of medical science who were normal without history of brucellosis or other types of bacterial infections control as participants in this study.
RNA isolation and preparation of cDNA
The expression of the CXCR3 mRNA was examined using quantitative real time PCR (qRT-PCR) for all of patients and controls. From the buficoat of blood samples total RNA was isolated employing Invitrogen Trizol according to the instructions of the manufacturer. Both quality and purity of the isolated RNA were examined by running on agarose gel and electrophoresis as well as measurement of optical density (A260/A280 ratio) by Nano Drop 1000 Spectrophotometer (Wilmington, DE, USA), respectively. The genomic DNA was removed from the RNA preparations, throughout DNase I, RNase-free kit from Thermo (Thermo Scientific, USA), according to instructions to manufacturers. To achieve this, ; 1 µg RNA, 1 μl 10X Reaction Buffer with MgCl2, 1 μl DNase, RNase-free, water nuclease-free up to 10 μl and was incubated at 37°C for 30 min and then 1 µL 50 mM ETDA was added and the incubation was prolonged at 65°C for 10 min. The processes of reverse transcription (RT) was undertaken using the Revert Aid First Strand complementary DNA (cDNA) Synthesis kit from Thermo (Thermo Scientific, USA). The RT reaction was made up 20μl and 11μl of solution that prepared in before staging for removal of genomic DNA, 1 μl Random Hexamer Primer, 4 μl 5x Reaction Buffer, 2 μl dNTP Mix (10 mM), 1 μl Ribo Lock RNase Inhibitor (20 U/μl), and 1 μl Revert Aid M-MuLV Reverse Transcriptase (200 U/μl) as a master mix. Adapted time scales and temperature profiles of RT reaction was as follow: incubation 5 min at 25 °C followed by 60 min at 42 °C. The reaction was terminated by heating at 70 °C for 5 min (13).
Isolation of PBMCs (peripheral blood mononuclear cells) by using LeucoSep tubes.
PBMCs were purified using LeucoSep tubes according to the instructions of the manufacturer (Greiner Bio-One). In brief, 3 ml of Ficoll-Paque was preloaded in a 14 ml LeucoSep tube by centrifugation for 30 s at 1,000 × g. The heparinized whole-blood samples were diluted with equal volumes of PBS, and 6 ml of the diluted blood was added to a LeucoSep tube. The cell separation tubes were centrifuged for 15 min at 800 × g without braking at room temperature. The cell suspension was collected, and the cells were washed twice in PBS (for 10 min at 640 and 470 × g, respectively, for the two successive washes) and resuspended in complete RPMI medium before counting (14, 31).
Cell yield/viability.
The both PBMCs yield and viability were determined using a NucleoCounter (ChemoMetec A/S, Allerød, Denmark). The NucleoCounter is able to detect nonviable cells using propidium iodide to stain cell nucleus and determines cellular viability of a blood sample employing total cell count and counting dead cells.
Quantitative-real-time-polymerase-chain-reaction (QRT-PCR)
Transcription levels of mRNA for the CXCR3 as the chemokine receptor for CXCL9, CXCL10 and CXCL11 was examined by QRT-PCR using previously described primers [table 1]. As stated earlier, further isolation PBMCs were twice rinsed with phosphate-buffered saline and their total RNA content was isolated by TRIzol reagent (Invitrogen, Life Technologies). The total RNA (2 μg) was then reversely transcribed by standard reagents (Invitrogen). The complementary DNA (cDNA) corresponding to 200 ng RNA was amplified in a PCR containing 0.1 mM of CXCR3 specific intron spanning primers (forward primer:5′-CTCTGCTGGACCCCCTATCA-3′; reverse primer:5′-GTCTCAGACCAGGATGAATC-3′, product: 255 bp) with an internal control for equal amounts of cDNA of a glyceraldehyde- 3-phosphate dehydrogenase (GAPDH)–specific intron spanning primer pair (5´-CCAGCCGAGCCACATCGCTC-3´; 5´-ATGAGCCCCAGCCTTCTCCAT-3´), which yielded a 360- bp amplified product. Amplification was performed using 25 to 40 cycles with denaturation at 94°C for 30s, primer annealing at 60°C for 30s, and extension at 72°C for 1 min. PCR products were subjected to electrophoresis on a 1% agarose gel and visualized by SYBR Safe staining (Invitrogen) (15).
Measurement of serum CXC chemokines by ELISA
Brucellosis patients CXCL9, CXCL10 and CXCL11 serum levels were detected by sandwich ELISA (R&D, Minneapolis or Peprotech respectively) using paired chemokine-specific mAbs, according to the manufacturer’s guidelines.
Following clinical examinations by the infectious disease specialists, intravenous blood samples were collected from patients and serum samples were then isolated from the patients and harvested samples have been kept at -80 °c for further chemokines (CXCL1 , CXCL9, CXCL10 and CXCL12) measurment.
Statistical Analysis:
Following of the collection the desired data, statistical evaluations on the mentioned variables with SPSS computer software (version18) of tukey test were used to analyze the findings.