Identification of Predictive Early Biomarkers for Sterile-SIRS after Cardiovascular Surgery

Systemic inflammatory response syndrome (SIRS) is a common complication after cardiovascular surgery that in severe cases can lead to multiple organ dysfunction syndrome and even death. We therefore set out to identify reliable early biomarkers for SIRS in a prospective small patient study for timely intervention. 21 Patients scheduled for planned cardiovascular surgery were recruited in the study, monitored for signs of SIRS and blood samples were taken to investigate biomarkers at pre-assigned time points: day of admission, start of surgery, end of surgery, days 1, 2, 3, 5 and 8 post surgery. Stored plasma and cryopreserved blood samples were analyzed for cytokine expression (IL1β, IL2, IL6, IL8, IL10, TNFα, IFNγ), other pro-inflammatory markers (sCD163, sTREM-1, ESM-1) and response to endotoxin. Acute phase proteins CRP, PCT and pro-inflammatory cytokines IL6 and IL8 were significantly increased (p<0.001) at the end of surgery in all patients but could not distinguish between groups. Normalization of samples revealed significant increases in IL1β changes (p<0.05) and decreased responses to endotoxin (p<0.01) in the SIRS group at the end of surgery. Soluble TREM-1 plasma concentrations were significantly increased in patients with SIRS (p<0.01). This small scale patient study could show that common sepsis markers PCT, CRP, IL6 and TNFα had low predictive value for early diagnosis of SIRS after cardiovascular surgery. A combination of normalized IL1β plasma levels, responses to endotoxin and soluble TREM-1 plasma concentrations at the end of surgery are predictive markers of SIRS development in this small scale study and could act as an indicator for starting early therapeutic interventions.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

Mutation of the binding site for Cbl (Tyr1045) in the EGF receptor (EGFR) results in impaired ubiquitination but does not affect EGFR internalization. However, the Y1045F mutation resulted in strongly decreased degradation of the EGFR, as well as efficient recycling of EGFR to the plasma membrane. Significantly, more wild-type EGFR than Y1045F EGFR was found localizing to multivesicular late endosomes. Ubiquitination of the EGFR was in HeLa cells inhibited both upon overexpressing the N-terminal part of Cbl and upon overexpressing a double mutant Grb2 incapable of interacting with Cbl and thereby being incapable of indirectly recruiting Cbl to the EGFR. Collectively, these data suggest that the ubiquitination resulting from direct binding of Cbl to pTyr1045 of the EGFR is critical for lysosomal sorting of the EGFR in contrast to ubiquitination resulting from Grb2-mediated binding of Cbl to the EGFR.     

Ras activation in response to phorbol ester proceeds independently of the EGFR via an unconventional nucleotide-exchange factor system in COS-7 cells

Ras is a major mediator of PE (phorbol ester) effects in mammalian cells. Various mechanisms for PE activation of Ras have been reported [Downward, Graves, Warne, Rayter and Cantrell (1990) Nature (London) 346, 719-723; Shu, Wu, Mosteller and Broek (2002) Mol. Cell. Biol. 22, 7758-7768; Roose, Mollenauer, Gupta, Stone and Weiss (2005) Mol. Cell. Biol. 25, 4426-4441; Grosse, Roelle, Herrlich, H?hn and Gudermann (2000) J. Biol. Chem. 275, 12251-12260], including pathways that target GAPs (GTPase-activating proteins) for inactivation and those that result in activation of GEFs (guanine nucleotide-exchange factors) Sos (son of sevenless homologue) or RasGRP (RAS guanyl releasing protein). However, a biochemical link between PE and GAP inactivation is missing and GEF stimulation is hard to reconcile with the observation that dominant-negative S17N-Ras does not compromise Ras-dependent ERK (extracellular-signal-regulated kinase) activation by PE. We have addressed this controversy and carried out an in-depth biochemical study of PE-induced Ras activation in COS-7 cells. Using a cell-permeabilization approach to monitor nucleotide exchange on Ras, we demonstrate that PE-induced Ras-GTP accumulation results from GEF stimulation. Nucleotide exchange stimulation by PE is prevented by PKC (protein kinase C) inhibition but not by EGFR [EGF (epidermal growth factor) receptor] blockade, despite the fact that EGFR inhibition aborts basal and PE-induced Shc (Src homology and collagen homology) phosphorylation and Shc-Grb2 (growth-factor-receptor-bound protein 2) association. In fact, EGFR inhibition ablates basal nucleotide exchange on Ras in growth-arrested COS-7 cells. These data disclose the existence of two separate GEF systems that operate independently from each other to accomplish PE-dependent formation of Ras-GTP and to maintain resting Ras-GTP levels respectively. We document that COS-7 cells do not express RasGRP and present evidence that the PE-responsive GEF system may involve PKC-dependent phosphorylation of Sos. More fundamentally, these observations shed new light on enigmatic issues such as the inefficacy of S17N-Ras in blocking PE action or the role of the EGFR in heterologous agonist activation of the Ras/ERK pathway.     

Beta Cell Count Instead of Beta Cell Mass to Assess and Localize Growth in Beta Cell Population following Pancreatic Duct Ligation in Mice

Background

Pancreatic-tail duct ligation (PDL) in adult rodents has been reported to induce beta cell generation and increase beta cell mass but increases in beta cell number have not been demonstrated. This study examines whether PDL increases beta cell number and whether this is caused by neogenesis of small clusters and/or their growth to larger aggregates.

Methodology

Total beta cell number and its distribution over small (<50 µm), medium, large (>100 µm) clusters was determined in pancreatic tails of 10-week-old mice, 2 weeks after PDL or sham.

Principal findings

PDL increased total beta cell mass but not total beta cell number. It induced neogenesis of small beta cell clusters (2.2-fold higher number) which contained a higher percent proliferating beta cells (1.9% Ki67+cells) than sham tails (<0.2%); their higher beta cell number represented <5% of total beta cell number and was associated with a similar increase in alpha cell number. It is unknown whether the regenerative process is causally related to the inflammatory infiltration in PDL-tails. Human pancreases with inflammatory infiltration also exhibited activation of proliferation in small beta cell clusters.

Conclusions/significance

The PDL model illustrates the advantage of direct beta cell counts over beta cell mass measurements when assessing and localizing beta cell regeneration in the pancreas. It demonstrates the ability of the adult mouse pancreas for neogenesis of small beta cell clusters with activated beta cell proliferation. Further studies should investigate conditions under which neoformed small beta cell clusters grow to larger aggregates and hence to higher total beta cell numbers.     

Targeting antibody responses to the membrane proximal external region of the envelope glycoprotein of human immunodeficiency virus

Although human immunodeficiency type 1 (HIV-1) infection induces strong antibody responses to the viral envelope glycoprotein (Env) only a few of these antibodies possess the capacity to neutralize a broad range of strains. The induction of such antibodies represents an important goal in the development of a preventive vaccine against the infection. Among the broadly neutralizing monoclonal antibodies discovered so far, three (2F5, Z13 and 4E10) target the short and hidden membrane proximal external region (MPER) of the gp41 transmembrane protein. Antibody responses to MPER are rarely observed in HIV-infected individuals or after immunization with Env immunogens. To initiate antibody responses to MPER in its membrane-embedded native conformation, we generated expression plasmids encoding the membrane-anchored ectodomain of gp41 with N-terminal deletions of various sizes. Following transfection of these plasmids, the MPER domains are displayed on the cell surface and incorporated into HIV virus like particles (VLP). Transfected cells displaying MPER mutants bound as efficiently to both 2F5 and 4E10 as cells transfected with a plasmid encoding full-length Env. Mice immunized with VLPs containing the MPER mutants produced MPER-specific antibodies, the levels of which could be increased by the trimerization of the displayed proteins as well as by a DNA prime-VLP boost immunization strategy. Although 2F5 competed for binding to MPER with antibodies in sera of some of the immunized mice, neutralizing activity could not be detected. Whether this is due to inefficient binding of the induced antibodies to MPER in the context of wild type Env or whether the overall MPER-specific antibody response induced by the MPER display mutants is too low to reveal neutralizing activity, remains to be determined.     

Transiently expressed ATG16L1 inhibits autophagosome biogenesis and aberrantly targets RAB11-positive recycling endosomes

The membrane source for autophagosome biogenesis is an unsolved mystery in the study of autophagy. ATG16L1 forms a complex with ATG12–ATG5 (the ATG16L1 complex). The ATG16L1 complex is recruited to autophagic membranes to convert MAP1LC3B-I to MAP1LC3B-II. The ATG16L1 complex dissociates from the phagophore before autophagosome membrane closure. Thus, ATG16L1 can be used as an early event marker for the study of autophagosome biogenesis. We found that among 3 proteins in the ATG16L1 complex, only ATG16L1 formed puncta-like structures when transiently overexpressed. ATG16L1⁺ puncta formed by transient expression could represent autophagic membrane structures. We thoroughly characterized the transiently expressed ATG16L1 in several mammalian cell lines. We found that transient expression of ATG16L1 not only inhibited autophagosome biogenesis, but also aberrantly targeted RAB11-positive recycling endosomes, resulting in recycling endosome aggregates. We conclude that transient expression of ATG16L1 is not a physiological model for the study of autophagy. Caution is warranted when reviewing findings derived from a transient expression model of ATG16L1.     

How well can post-traumatic stress disorder be predicted from pre-trauma risk factors? An exploratory study in the WHO World Mental Health Surveys

Post‐traumatic stress disorder (PTSD) should be one of the most preventable mental disorders, since many people exposed to traumatic experiences (TEs) could be targeted in first response settings in the immediate aftermath of exposure for preventive intervention. However, these interventions are costly and the proportion of TE‐exposed people who develop PTSD is small. To be cost‐effective, risk prediction rules are needed to target high‐risk people in the immediate aftermath of a TE. Although a number of studies have been carried out to examine prospective predictors of PTSD among people recently exposed to TEs, most were either small or focused on a narrow sample, making it unclear how well PTSD can be predicted in the total population of people exposed to TEs. The current report investigates this issue in a large sample based on the World Health Organization (WHO)'s World Mental Health Surveys. Retrospective reports were obtained on the predictors of PTSD associated with 47,466 TE exposures in representative community surveys carried out in 24 countries. Machine learning methods (random forests, penalized regression, super learner) were used to develop a model predicting PTSD from information about TE type, socio‐demographics, and prior histories of cumulative TE exposure and DSM‐IV disorders. DSM‐IV PTSD prevalence was 4.0% across the 47,466 TE exposures. 95.6% of these PTSD cases were associated with the 10.0% of exposures (i.e., 4,747) classified by machine learning algorithm as having highest predicted PTSD risk. The 47,466 exposures were divided into 20 ventiles (20 groups of equal size) ranked by predicted PTSD risk. PTSD occurred after 56.3% of the TEs in the highest‐risk ventile, 20.0% of the TEs in the second highest ventile, and 0.0‐1.3% of the TEs in the 18 remaining ventiles. These patterns of differential risk were quite stable across demographic‐geographic sub‐samples. These results demonstrate that a sensitive risk algorithm can be created using data collected in the immediate aftermath of TE exposure to target people at highest risk of PTSD. However, validation of the algorithm is needed in prospective samples, and additional work is warranted to refine the algorithm both in terms of determining a minimum required predictor set and developing a practical administration and scoring protocol that can be used in routine clinical practice.     

Methylated DNA/RNA in Body Fluids as Biomarkers for Lung Cancer

DNA/RNA methylation plays an important role in lung cancer initiation and progression. Liquid biopsy makes use of cells, nucleotides and proteins released from tumor cells into body fluids to help with cancer diagnosis and prognosis. Methylation of circulating tumor DNA (ctDNA) has gained increasing attention as biomarkers for lung cancer. Here we briefly introduce the biological basis and detection method of ctDNA methylation, and review various applications of methylated DNA in body fluids in lung cancer screening, diagnosis, prognosis, monitoring and treatment prediction. We also discuss the emerging role of RNA methylation as biomarkers for cancer.