Chong Shen

Capture and isolation of flowing cells and particulates from body fluids has enormous implications in diagnosis, monitoring, and drug testing, yet monovalent adhesion molecules used for this purpose result in inefficient cell capture and difficulty in retrieving the captured cells. Inspired by marine creatures that present long tentacles containing multiple adhesive domains to effectively capture flowing food particulates, we developed a platform approach to capture and isolate cells using a 3D DNA network comprising repeating adhesive aptamer domains that extend over tens of micrometers into the solution. The DNA network was synthesized from a microfluidic surface by rolling circle amplification where critical parameters, including DNA graft density, length, and sequence, could readily be tailored. Using an aptamer that binds to protein tyrosine kinase-7 (PTK7) that is overexpressed on many human cancer cells, we demonstrate that the 3D DNA network significantly enhances the capture efficiency of lymphoblast CCRF-CEM cells over monovalent aptamers and antibodies, yet maintains a high purity of the captured cells. When incorporated in a herringbone microfluidic device, the 3D DNA network not only possessed significantly higher capture efficiency than monovalent aptamers and antibodies, but also outperformed previously reported cell-capture microfluidic devices at high flow rates. This work suggests that 3D DNA networks may have broad implications for detection and isolation of cells and other bioparticles.

BACKGROUND: Adalimumab, a fully human monoclonal antibody to tumour necrosis factor, was recently introduced for therapy of Crohn's disease. AIM: Since induction of apoptosis of inflammatory cells is thought to be an important mechanism of action of the antitumour necrosis factor monoclonal antibody infliximab, we studied the induction of apoptosis of activated peripheral blood monocytes by adalimumab. METHOD: Apoptosis was analysed at the levels of the cell membrane, mitochondria and DNA by flow cytometry. RESULTS: We found that both adalimumab and infliximab induced apoptosis in cultured monocytes, while etanercept did not. Apoptosis induction was caspase-dependent and detectable already after 2 h. The production of interleukin-10 and interleukin-12 by monocytes was down-regulated significantly by adalimumab and infliximab but not by etanercept, while levels of soluble tumour necrosis factor in monocyte cultures were down-regulated by all three reagents. CONCLUSIONS: These data show that both adalimumab and infliximab affect monocyte cytokine production and induce apoptosis of activated monocytes. Our findings will have to be further correlated to therapeutic efficacy of these antitumour necrosis factor reagents.

While some probiotic strains might have adjuvant effects in the therapy for inflammatory bowel diseases (IBD), these effects remain controversial and cannot be generalized. In this study, a dltD mutant of the model probiotic Lactobacillus rhamnosus GG (LGG), having a drastic modification in its lipoteichoic acid (LTA) molecules, was analysed for its effects in an experimental colitis model. Dextran sulphate sodium (DSS) was used to induce either moderate to severe or mild chronic colitis in mice. Mice received either phosphate-buffered saline (PBS), LGG wild-type or the dltD mutant via the drinking water. Macroscopic parameters, histological abnormalities, cytokine and Toll-like receptor (TLR) expression were analysed to assess disease activity. LGG wild-type did not show efficacy in the different experimental colitis set-ups. This wild-type strain even seemed to exacerbate the severity of colitic parameters in the moderate to severe colitis model compared to untreated mice. In contrast, mice treated with the dltD mutant showed an improvement of some colitic parameters compared to LGG wild-type-treated mice in both experimental models. In addition, treatment with the dltD mutant correlated with a significant down-regulation of Toll-like receptor-2 expression and of downstream proinflammatory cytokine expression in the colitic mice. These results show that molecular cell surface characteristics of probiotics are crucial when probiotics are considered for use as supporting therapy in IBD.

BACKGROUND: Adalimumab is a fully human monoclonal antibody to tumor necrosis factor (TNF), which was recently introduced as a therapy for Crohn's disease and rheumatoid arthritis. Besides neutralization, induction of apoptosis of monocytes/macrophages and T cells is thought to be an important mechanism of action of the anti-tumor necrosis factor monoclonal antibody infliximab, at least in Crohn's disease therapy. AIM: To study caspase activation and the induction of apoptosis by adalimumab and the effect of a caspase inhibitor in vivo. METHODS: For in vitro studies, THP-1 cells (human monocytic cell line) were incubated with adalimumab, infliximab, or human immunoglobulin G, and annexin V + propidium iodide, Apo2.7, and 7-amino actinomycin-D were used to study apoptosis on the cell membrane, mitochodrial, and DNA level, respectively. Active caspase-3 was detected by intracellular staining. For in vivo studies, a chimeric human-mouse model was used, in which THP-1 cells were injected intraperitoneally in SCID-Beige mice followed by treatment with adalimumab, infliximab, or human immunoglobulin G. Effects of a pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyketone on apoptosis induction were evaluated. RESULTS: In vitro analysis revealed that apoptosis could be induced in THP-1 cells by both adalimumab and infliximab. Activation of caspase-3 after incubation with adalimumab was demonstrated by intracellular staining. In addition, in the chimeric mouse model, a higher percentage of residual THP-1 cells were apoptotic, and lower cell numbers were recovered in the adalimumab- or infliximab-treated mouse. Apoptosis induction by adalimumab could be abrogated through in vivo pretreatment of mice with the pan-caspase inhibitor. CONCLUSIONS: Adalimumab, besides neutralizing tumor necrosis factor, also induces apoptosis of transmembrane tumor necrosis factor-positive THP-1 cells by activating intracellular caspases. This activity is likely to be important for the clinical effect of this biodrug.

It is generally believed that probiotic bacteria need to survive gastrointestinal transit to exert a health-promoting effect. In this study, a genuine luxS mutant and a luxS mutant containing unknown suppressor mutations of the probiotic strain Lactobacillus rhamnosus GG were compared to the wild type for survival and persistence in the murine gastrointestinal tract. The LuxS enzyme, catalyzing the production of the autoinducer-2 signaling molecule, also forms an integral part of the activated methyl cycle and the metabolism of methionine and cysteine. The genuine luxS mutant CMPG5412 showed drastically reduced persistence in mice, which was related to less survival in simulated gastric juice, indicating that LuxS metabolism is crucial for the gastric stress resistance of L. rhamnosus GG. The suppressor mutations in the other luxS mutant, CMPG5413, appear to compensate for the metabolic defects of the luxS mutation and to restore the resistance to gastric juice but cause a defect in adherence, biofilm formation, and exopolysaccharide production. The shorter residence time of this suppressor mutant in the murine gastrointestinal tract indicates a role for biofilm formation and exopolysaccharides in the persistence capacity of L. rhamnosus GG.