Plasma surfactant protein-D as a diagnostic biomarker for acute respiratory distress syndrome: validation in US and Korean cohorts

Background

Acute respiratory distress syndrome (ARDS) is potentially underrecognized by clinicians. Early recognition and subsequent optimal treatment of patients with ARDS may be facilitated by usage of biomarkers. Surfactant protein D (SP-D), a marker of alveolar epithelial injury, has been proposed as a potentially useful biomarker for diagnosis of ARDS in a few studies. We tried to validate the performance of plasma SP-D levels for diagnosis of ARDS.

Methods

We conducted a retrospective analysis using data from three (two in USA and one in Korea) prospective biobank cohorts involving 407 critically ill patients admitted to medical intensive care unit (ICU). A propensity score matched analysis (patients with versus without ARDS, matched 1:1) was carried out using significant variables from multiple logistic regression. The diagnostic accuracy of plasma SP-D as a diagnostic marker of ARDS was assessed by receiver operating characteristic curve analysis.

Results

Out of the 407 subjects included in this study, 39 (10%) patients fulfilled ARDS criteria. Patients with ARDS had higher SP-D levels in plasma (p?<?0.01) and higher hospital-mortality (p?<?0.001) than those without ARDS. Thirty eight subjects with ARDS (cases) were successfully matched for propensity for ARDS with 38 subjects without ARDS (controls). Plasma levels of SP-D were higher in cases with ARDS compared to their matched controls without ARDS [median 20.8 ng/mL (interquartile range, 12.7–38.4) versus 7.9 (4.1–17.0); p?=?0.001]. The area under the receiver operating characteristic curve for SP-D for the diagnosis of ARDS was 0.71 (95% confidence intervals, 0.60–0.83). A cut-off point of 12.7 ng/mL for SP-D yielded sensitivity of 74% and specificity of 63%.

Conclusions

High levels of SP-D within 48 h after ICU admission might serve as a diagnostic marker for ARDS in patients hospitalized in medical ICU. Further prospective trials are required to validate the diagnostic role of SP-D in ARDS, and if its usefulness is greater in direct than in indirect ARDS, as well as across different strata of severity of ARDS.

     

Nanomechanical In Situ Monitoring of Proteolysis of Peptide by Cathepsin B

Characterization and control of proteolysis of peptides by specific cellular protease is a priori requisite for effective drug discovery. Here, we report the nanomechanical, in situ monitoring of proteolysis of peptide chain attributed to protease (Cathepsin B) by using a resonant nanomechanical microcantilever immersed in a liquid. Specifically, the detection is based on measurement of resonant frequency shift arising from proteolysis of peptides (leading to decrease of cantilever''s overall mass, and consequently, increases in the resonance). It is shown that resonant microcantilever enables the quantification of proteolysis efficacy with respect to protease concentration. Remarkably, the nanomechanical, in situ monitoring of proteolysis allows us to gain insight into the kinetics of proteolysis of peptides, which is well depicted by Langmuir kinetic model. This implies that nanomechanical biosensor enables the characterization of specific cellular protease such as its kinetics.     

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi‐lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2‐DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin–avidin affinity column were separated by 2‐DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole‐time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin‐related proteins, F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF‐induced morphological change of MSCs.     

Intra-arterial delivery of triolein emulsion increases vascular permeability in skeletal muscles of rabbits

Background  

To test the hypothesis that triolein emulsion will increase vascular permeability of skeletal muscle.     

Proteolytic activity of Co(III) complex of 1-oxa-4,7,10-triazacyclododecane: a new catalytic center for peptide-cleavage agents

Catalytic drugs based on target-selective artificial proteases have been proposed as a new paradigm in drug design. Peptide-cleavage agents selective for pathogenic proteins of Alzheimer’s disease, type 2 diabetes mellitus or Parkinson’s disease have been prepared using the Co(III) aqua complex (Co(III)cyclen) of 1,4,7,10-tetraazacyclododecane as the catalytic center. In the present study, the Co(III) aqua complex (Co(III)oxacyclen) of 1-oxa-4,7,10-triazacyclododecane was examined in search of an improved catalytic center for peptide-cleavage agents. An X-ray crystallographic study of [Co(oxacyclen)(CO₃)](ClO₄), titration of Co(III)oxacyclen, and kinetic studies on the cleavage of albumin, γ-globulin, lysozyme, and myoglobin by Co(III)oxacyclen were carried out. Considerably higher proteolytic activity was observed for Co(III)oxacyclen in comparison with Co(III)cyclen, indicating that better target-selective artificial metalloproteases would be obtained using Co(III)oxacyclen as the catalytic center. The improved proteolytic activity was attributed to either steric effects or the increased Lewis acidity of the Co(III) center. The kinetic data also predicted that side effects due to the cleavage of nontarget proteins by a catalytic drug based on Co(III)oxacyclen would be insignificant.     

Phosphorylation of Phospholipase C‐δ1 Regulates its Enzymatic Activity

Phosphorylation of phospholipase C‐δ₁ (PLC‐δ₁) in vitro and in vivo was investigated. Of the serine/threonine kinases tested, protein kinase C (PKC) phosphorylated the serine residue(s) of bacterially expressed PLC‐δ₁ most potently. It was also demonstrated that PLC‐δ₁ directly bound PKC‐α via its pleckstrin homology (PH) domain. Using deletion mutants of PLC‐δ₁ and synthetic peptides, Ser35 in the PH domain was defined as the PKC mediated in vitro phosphorylation site of PLC‐δ₁. In vitro phosphorylation of PLC‐δ₁ by PKC stimulated [³H]PtdIns(4,5)P₂ hydrolyzing activity and [³H]Ins(1,4,5)P₃‐binding of the PLC‐δ₁. On the other hand, endogenous PLC‐δ₁ was constitutively phosphorylated and phosphoamino acid analysis revealed that major phosphorylation sites were threonine residues in quiescent cells. The phosphorylation level and the species of phosphoamino acid were not changed by various stimuli such as PMA, EGF, NGF, and forskolin. Using matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry, we determined that Thr209 of PLC‐δ₁ is one of the constitutively phosphorylated sites in quiescent cells. The PLC activity was potentiated when constitutively phosphorylated PLC‐δ₁ was dephosphorylated by endogenous phosphatase(s) in vitro. Additionally, coexpression with PKC‐α reduced serine phosphorylation of PLC‐δ₁ detected by an anti‐phosphoserine antibody and PLC‐δ₁‐dependent basal production of inositol phosphates in NIH‐3T3 cells, suggesting PKC‐α activates phosphatase or inactivates another kinase involved in PLC‐δ₁ serine phosphorylation to modulate the PLC‐δ₁ activity in vivo. Taken together, these results suggest that PLC‐δ₁ has multiple phosphorylation sites and phosphorylation status of PLC‐δ₁ regulates its activity positively or negatively depends on the phosphorylation sites. J. Cell. Biochem. 108: 638–650, 2009. © 2009 Wiley‐Liss, Inc.     

Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy

Renal fibrosis is the common histological feature of advanced glomerular and tubulointerstitial disease leading to end-stage renal disease (ESRD). However, specific antifibrotic therapies to slow down the evolution to ESRD are still absent. Because persistent inflammation is a key event in the development of fibrosis, we hypothesized that the proinflammatory kinin B1 receptor (B1R) could be such a new target. Here we show that, in the unilateral ureteral obstruction model of renal fibrosis, the B1R is overexpressed and that delayed treatment with an orally active nonpeptide B1R antagonist blocks macrophage infiltration, leading to a reversal of the level of renal fibrosis. In vivo bone marrow transplantation studies as well as in vitro studies on renal cells show that part of this antifibrotic mechanism of B1R blockade involves a direct effect on resident renal cells by inhibiting chemokine CCL2 and CCL7 expression. These findings suggest that blocking the B1R is a promising antifibrotic therapy.