Elevated Resting and Postprandial Digestive Proteolytic Activity in Peripheral Blood of Individuals With Type-2 Diabetes Mellitus, With Uncontrolled Cleavage of Insulin Receptors.

Objective: To examine resting and postprandial peripheral protease activity in healthy controls and individuals with type 2 diabetes mellitus (T2DM) and pre-T2DM. Methods: Individuals with T2DM or pre-T2DM and healthy controls (mean age 55.8 years) were studied before and for a span of 300 minutes following a single high-calorie McDonald's breakfast. Metalloproteases-2/-9 (MMP-2/-9), elastase, and trypsin activities were assessed in whole blood before and following the meal using a novel high-precision electrophoretic platform. Also assessed were circulating levels of inflammatory biomarkers and insulin receptor density on peripheral blood mononuclear cells (PBMCs) in relationship to protease activity. Results: Premeal MMP-2/-9 and elastase activity levels in T2DM and in pre-T2DM participants were significantly elevated as compared to controls. The T2DM group showed a significant increase in elastase activity 15 minutes after the meal; elastase activity continued to increase to the 30-minute time point (p < 0.01). In control participants, MMP-2/-9, elastase, and trypsin were significantly increased at 15 minutes after the meal (p < 0.05) and returned to premeal values within a period of approximately 30 to 60 minutes post meal. PBMCs incubated for 1 hour with plasma from T2DM and pre-T2DM participants had significantly lower levels of insulin receptor density compared to those incubated with plasma from control participants (p < 0.001). Conclusions: The results of this study suggest that individuals with T2DM and pre-T2DM have higher resting systemic protease activity than nonsymptomatic controls. A single high-calorie/high-carbohydrate meal results in further elevations of protease activity in the systemic circulation of T2DM and pre-T2DM, as well as in healthy controls. The protease activity in turn can lead to a downregulation of insulin receptor density, potentially supporting a state of insulin resistance.

Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood.

BACKGROUND: Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a "liquid biopsy" may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. METHODS: We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 µL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification, PCR, and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. RESULTS: PCR and DNA sequencing results obtained by DEP from 25 µL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15-20 mL blood. CONCLUSIONS: Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.

Dielectrophoretic isolation and detection of cancer-related circulating cell-free DNA biomarkers from blood and plasma.

Conventional methods for the isolation of cancer-related circulating cell-free (ccf) DNA from patient blood (plasma) are time consuming and laborious. A DEP approach utilizing a microarray device now allows rapid isolation of ccf-DNA directly from a small volume of unprocessed blood. In this study, the DEP device is used to compare the ccf-DNA isolated directly from whole blood and plasma from 11 chronic lymphocytic leukemia (CLL) patients and one normal individual. Ccf-DNA from both blood and plasma samples was separated into DEP high-field regions, after which cells (blood), proteins, and other biomolecules were removed by a fluidic wash. The concentrated ccf-DNA was detected on-chip by fluorescence, and then eluted for PCR and DNA sequencing. The complete process from blood to PCR required less than 10 min; an additional 15 min was required to obtain plasma from whole blood. Ccf-DNA from the equivalent of 5 µL of CLL blood and 5 µL of plasma was amplified by PCR using Ig heavy-chain variable (IGHV) specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone. The PCR and DNA sequencing results obtained by DEP from all 11 CLL blood samples and from 8 of the 11 CLL plasma samples were exactly comparable to the DNA sequencing results obtained from genomic DNA isolated from CLL patient leukemic B cells (gold standard).

The Pseudomonas aeruginosa protease LasB directly activates IL-1ß.

BACKGROUND: Pulmonary damage by Pseudomonas aeruginosa during cystic fibrosis lung infection and ventilator-associated pneumonia is mediated both by pathogen virulence factors and host inflammation. Impaired immune function due to tissue damage and inflammation, coupled with pathogen multidrug resistance, complicates the management of these deep-seated infections. Pathological inflammation during infection is driven by interleukin-1ß (IL-1ß), but the molecular processes involved are not fully understood. METHODS: We examined IL-1ß activation in a pulmonary model infection of Pseudomonas aeruginosa and in vitro using genetics, specific inhibitors, recombinant proteins, and targeted reporters of protease activity and IL-1ß bioactivity. FINDINGS: Caspase-family inflammasome proteases canonically regulate maturation of this proinflammatory cytokine, but we report that plasticity in IL-1ß proteolytic activation allows for its direct maturation by the pseudomonal protease LasB. LasB promotes IL-1ß activation, neutrophilic inflammation, and destruction of lung architecture characteristic of severe P. aeruginosa pulmonary infection. INTERPRETATION: Preservation of lung function and effective immune clearance may be enhanced by selectively controlling inflammation. Discovery of this IL-1ß regulatory mechanism provides a distinct target for anti-inflammatory therapeutics, such as matrix metalloprotease inhibitors that inhibit LasB and limit inflammation and pathology during P. aeruginosa pulmonary infections. FUNDING: Full details are provided in the Acknowledgements section.