Peritoneal Level of CD206 Associates With Mortality and an Inflammatory Macrophage Phenotype in Patients With Decompensated Cirrhosis and Spontaneous Bacterial Peritonitis.

BACKGROUND & AIMS: Peritoneal macrophages (PMs) regulate inflammation and control bacterial infections in patients with decompensated cirrhosis. We aimed to characterize PMs and associate their activation with outcomes of patients with spontaneous bacterial peritonitis (SBP). METHODS: We isolated PMs from ascites samples of 66 patients with decompensated cirrhosis (19 with SBP) and analyzed them by flow cytometry, quantitative real-time polymerase chain reaction, functional analysis, and RNA microarrays. We used ascites samples of a separate cohort of 111 patients with decompensated cirrhosis (67 with SBP) and quantified the soluble form of the mannose receptor (CD206) and tumor necrosis factor by enzyme-linked immunosorbent assay (test cohort). We performed logistic regression analysis to identify factors associated with 90-day mortality. We validated our findings using data from 71 patients with cirrhosis and SBP. Data from 14 patients undergoing peritoneal dialysis for end-stage renal disease but without cirrhosis were included as controls. RESULTS: We used surface levels of CD206 to identify subsets of large PMs (LPM) and small PMs (SPM), which differed in granularity and maturation markers, in ascites samples from patients with cirrhosis. LPMs vs SPMs from patients with cirrhosis had different transcriptomes; we identified more than 4000 genes that were differentially regulated in LPMs vs SPMs, including those that regulate the cycle, metabolism, self-renewal, and immune cell signaling. LPMs had an inflammatory phenotype, were less susceptible to tolerance induction, and released more tumor necrosis factor than SPMs. LPMs from patients with cirrhosis produced more inflammatory cytokines than LPMs from controls. Activation of PMs by Toll-like receptor agonists and live bacteria altered levels of CD206 on the surface of LPMs and release of soluble CD206. Analysis of serial ascites fluid from patients with SBP revealed loss of LPMs in the early phase of SBP, but levels increased after treatment. In the test and validation cohorts, patients with SBP and higher concentrations of soluble CD206 in ascites fluid (>0.53 mg/L) were less likely to survive for 90 days than those with lower levels. CONCLUSIONS: Surface level of CD206 can be used to identify mature, resident, inflammatory PMs in patients with cirrhosis. Soluble CD206 is released from activated LPMs and increased concentrations in patients with cirrhosis and SBP indicate reduced odds of surviving for 90 days.

Nonlinear Multimodal Imaging Characteristics of Early Septic Liver Injury in a Mouse Model of Peritonitis.

Sepsis constitutes a life-threatening organ failure caused by a deregulated host response to infection. Identifying early biomolecular indicators of organ dysfunction may improve clinical decision-making and outcome of patients. Herein we utilized label-free nonlinear multimodal imaging, combining coherent anti-Stokes Raman scattering (CARS), two-photon excited autofluorescence (TPEF), and second-harmonic generation (SHG) to investigate the consequences of early septic liver injury in a murine model of polymicrobial abdominal infection. Liver tissue sections from mice with and without abdominal sepsis were analyzed using multimodal nonlinear microscopy, immunofluorescence, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Twenty-four hours after the induction of sepsis, hepatic mRNA of inflammatory cytokines and acute phase proteins was upregulated, and liver-infiltrating myeloid cells could be visualized alongside hepatocellular cytoplasmic translocation of high mobility group box 1. According to the statistical analysis based on texture feature extraction followed by the combination of dimension reduction and linear discriminant analysis, CARS (AUC = 0.93) and TPEF (AUC = 0.83) showed an excellent discrimination between liver sections from septic mice and sham-treated mice in contrast to SHG (AUC = 0.49). Spatial analysis revealed no major differences in the distribution of sepsis-associated changes between periportal and pericentral zones. These data suggest early alterations in hepatic lipid distribution and metabolism during liver injury and confirm nonlinear multimodal imaging as a promising complementary method for the real-time, label-free study of septic liver damage.

Simultaneous isolation and detection of single breast cancer cells using surface-enhanced Raman spectroscopy.

Nowadays, cancer is one of the most dangerous and deadly disease all around the world. Cancer that is diagnosed at early stages is more likely to be treated successfully. Treatment of progressed cancer is very difficult, and generally surviving rates are much lower. Therefore, much research has been focused on developing non-invasive methods for detection of cancer and monitoring of its progress. Within this contribution, we present a novel strategy for selective isolation and detection of breast cancer cell lines (MCF-7 and BT-20) based on surface enhanced Raman scattering (SERS). A simplified protocol based on cell-aptamer interaction has been developed in which core-shell (Au@Fe3O4) nanoparticles (CSNs) were functionalized with a mucin 1 (MUC1) specific aptamer (Apt1) to capture cells through the interaction between Apt1 and overexpressed protein (MUC1) on the surface of the tumor cells. Meanwhile, a SERS nano-tag, synthesized by the conjugation of Apt1 to the surface of BSA coated and with 4-mercaptopyridine (4-Mpy) functionalized gold nanoparticles, was used to detect the isolated cells. As a conclusion, the proposed strategy can be extended to isolate and detect cells more precisely based on the detection of different kinds of biomarkers on the surface of cancer cells, simultaneously.

Dicistronic expression of human proinsulin-protein A fusion in tobacco chloroplast.

Different expression systems such as bacteria and mammalian cells have been used to produce pharmaceutical proteins. In recent years, the use of plants as bioreactors offers efficient and economical systems in recombinant protein production. Furthermore, because of the large number of plastid copies in plants, chloroplast engineering functions as an effective method to increase recombinant protein expression. Because the commercially available insulin for treatment does not contain C-peptide, which is of great importance for type 1 diabetic patients, the current study introduces the human proinsulin gene fused with protein A into the tobacco chloroplast genome using the biolistic method. To achieve homoplasmy, three rounds of selection and regeneration of transforming cells were performed on the medium that contained spectinomycin antibiotic and hormones. The PCR analysis indicated the presence of the proinsulin gene in transplastomic plants. The reverse-transcription PCR analysis confirmed the expression of the proinsulin-protein A fusion at the transcription level. Immunoblot assays of leaf-derived protein extracts confirmed that the target gene expression is up to 0.2% of the total soluble protein. Our study showed that protein A fusion is not as efficient as other reported fusions. The transplastomic plants were also confirmed for homoplasmy using Southern blot analysis.