Proportions of DCs were determined in suspensions of cells from MLNs and lamina propria by five-color quantitative flow cytometry in a FACSAria cytometer using FACSDiva software (Becton Dickinson). Analyses see more were carried out using FlowJo software (TreeStar Inc., Ashland, OR). Cell suspensions were incubated with a combination of the following mAbs to define DCs: PE-OX62 (OX62) (Serotec), PE-cyanin 5-CD45RA (OX33) (BD Pharmingen), APC-RT1B (HIS19) (eBioscience), and Alexa Fluor 700-CD3 (1F4) (Serotec). After surface staining, cells were fixed, permeabilized, and stained with FITC-TNF-α (eBioscience). TNF-α production was
determined after culturing DCs in DMEM (BioWhittaker) or stimulated with lipolysaccharide (LPS) (055:B5; 25 μg/mL; Sigma-Aldrich). selleck products MLNs and blood samples were inoculated into thioglycollate medium (Scharlab, Barcelona, Spain) and incubated at 37°C for 48 hours. Microorganisms were identified by a manual biochemical test or an automated system (Microscan; Baxter, Irvine, CA).
GBT and bacteriemia were defined by the presence of viable organisms in the MLNs or blood culture, respectively (i.e., a positive bacteriological culture). Total intestinal aerobic count was defined as the sum of all the aerobic bacteria present in the ileal content sample expressed as log10 colony-forming units (CFU)/g of stool.6 MLNs were homogenized in PBS by sonication (UP100H Ultrasonic Processor; Hielscher, Teltow, Germany). Genomic DNA from homogenized MLNs was isolated using the QIAmp Tissue Kit (Qiagen, Hilden, Germany), as described elsewhere.7, 16 Bacterial DNA (Bact-DNA) was identified by running a broad-range polymerase chain reaction, followed by nucleotide sequencing of a conserved region of the 16SrRNA gene. Results are shown as mean ± standard deviation. Quantitative variables were compared using the unpaired Student t test with Bonferroni’s correction for multiple comparisons.
The level of statistical significance was set at P < 0.05. Statistical analyses were performed using SPSS 15.0 for MCE公司 Windows (SPSS, Inc., Chicago, IL). Sixty-six rats were entered into the protocol of cirrhosis with ascites induction, of which 41 survived (62%). On average, rats developed ascites 15 weeks (range, 12-20) after the initial CCl4 dose. The average amount of ascites was greater in cirrhotic rats with than in those without GBT (19.6 ± 10.8 versus 12.1 ± 8.4 mL; P < 0.05). Twenty-two of the forty-one (54%) rats with cirrhosis showed GBT to the MLNs. Bact-DNA fragments were present in the MLNs of all animals with GBT and in 14 of the 19 (74%) animals without GBT. None of the 14 healthy control rats showed GBT or Bact-DNA in MLNs. Enteric aerobic bacterial loads were significantly higher and serum total protein and albumin levels lower in cirrhotic rats with GBT than in those lacking GBT (Table 1). None of the studied animals had bacteremia.