Association of Sepsis-Related Mortality with Early Increase of TIMP-1/MMP-9 Ratio

Objective

Higher circulating levels of tissue inhibitor of matrix metalloproteinases (TIMP)-1 at the time of severe sepsis diagnosis have been reported in nonsurviving than in surviving patients. However, the following questions remain unanswered: 1) Does TIMP-1/MMP-9 ratio differ throughout the first week of intensive care between surviving and non-surviving patients? 2) Is there an association between TIMP-1/MMP-9 ratio and sepsis severity and mortality during such period? 3) Could TIMP-1/MMP-9 ratio during the first week be used as an early biomarker of sepsis outcome? 4) Is there an association between TIMP-1/MMP-9 ratio and coagulation state and circulating cytokine levels during the first week of intensive care in these patients? The present study sought to answer these questions.

Methods

Multicenter, observational and prospective study carried out in six Spanish Intensive Care Units (ICUs) of 295 patients with severe sepsis. Were measured circulating levels of TIMP-1, MMP-9, tumour necrosis factor (TNF)-alpha, interleukin (IL)-10 and plasminogen activator inhibitor (PAI)-1 at day 1, 4 and 8. End-point was 30-day mortality.

Results

We found higher TIMP-1/MMP-9 ratio during the first week in non-surviving (n = 98) than in surviving patients (n = 197) (p<0.01). Logistic regression analyses showed that TIMP-1/MMP-9 ratio at days 1, 4 and 8 was associated with mortality. Receiver operating characteristic (ROC) curves showed that TIMP-1/MMP-9 ratio at days 1, 4 and 8 could predict mortality. There was an association between TIMP-1/MMP-9 ratio and TNF-alpha, IL-10, PAI-1 and lactic acid levels, SOFA score and platelet count at days 1, 4 and 8.

Conclusions

The novel findings of our study were that non-surviving septic patients showed persistently higher TIMP-1/MMP-9 ratio than survivors ones during the first week, which was associated with severity, coagulation state, circulating cytokine levels and mortality; thus representing a new biomarker of sepsis outcome.     

Individualized Treatment of Genotype 1 Na?ve Patients: An Italian Multicenter Field Practice Experience

Background

Triple therapy including Telaprevir or Boceprevir still represents in many European countries the standard of care for patients with Hepatitis C Virus genotype 1 infection. The number of patients who received this treatment resulted generally lower than expected. We investigated, among naïve patients, number and characteristics of treatment candidates who were started on triple or dual therapy in comparison to those who were deferred.

Patients and Methods

621 naïve treatment candidates were prospectively evaluated at each center. Factors associated with decision to defer or treat with dual or triple therapy were investigated by univariate and multivariate analyses. Rates of Sustained Virological Response and safety profile were analysed.

Results

Of candidates to treatment, 33% did not received it. It was mostly due to high risk of Interferon-induced decompensation. Of 397 patients who were started on treatment, 266 (67%) received triple, 131 dual. Among patient receiving treatment, unfavorable IL28B, severe liver damage and higher albumin were independently associated with the physician decision to administer triple therapy. Sustained Virological Response after dual therapy was 66.4%, after triple 73.7% (p = 0.14). 142 patients received Telaprevir. The choice of Telaprevir-based therapy was associated with higher Body Mass Index and advanced liver disease. Sustained Virological Response rates were 71.1% after Telaprevir and 76.6% after Boceprevir.

Conclusions

Individualizing treatment with available regimens allows to maximize Sustained Virological Response and to reduce the number of patients who remain untreated. High proportion of patients with severe liver damage urgently need Interferon free treatment.     

Serum Levels of Caspase-Cleaved Cytokeratin-18 and Mortality Are Associated in Severe Septic Patients: Pilot Study

Objective

Apoptosis is increased in sepsis. Cytokeratin 18 (CK-18), a protein of the intermediate filament group present in most epithelial and parenchymal cells, is cleaved by the action of caspases and released into the blood as caspase-cleaved CK (CCCK)-18 during apoptosis. Circulating levels of CCCK-18 have scarcely been explored in septic patients. In one study with 101 severe septic patients, the authors reported higher serum CCCK-18 levels in non-survivors than in survivors; however, the sample size was too small to demonstrate an association between serum CCCK-18 levels and early mortality and whether they could be used as a biomarker to predict outcomes in septic patients. Thus, these were the objectives of this study with a large series of patients.

Methods

We performed a prospective, multicenter, observational study in six Spanish Intensive Care Units with 224 severe septic patients. Blood samples were collected at the time that severe sepsis was diagnosed to determine serum levels of CCCK-18, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-10. The end point was 30-day mortality.

Results

Non-surviving patients (n = 80) showed higher serum CCCK-18 levels (P<0.001) than survivors (n = 144). Multiple logistic regression analysis showed that serum CCCK-18 levels>391 u/L were associated with 30-day survival (Odds ratio = 2.687; 95% confidence interval = 1.449–4.983; P = 0.002), controlling for SOFA score, serum lactic acid levels and age. Kaplan-Meier survival analysis showed that the risk of death in septic patients with serum CCCK-18 levels >391 u/L was higher than in patients with lower values (Hazard Ratio = 3.1; 95% CI = 1.96–4.84; P<0.001). Serum CCCK-18 levels were positively associated with serum levels of IL-6 and lactic acid, and with SOFA and APACHE scores.

Conclusions

The major novel finding of our study, the largest cohort of septic patients providing data on circulating CCCK-18 levels, was that serum CCCK-18 levels are associated with mortality in severe septic patients.     

Arctic Lineage-Canine Distemper Virus as a Cause of Death in Apennine Wolves (Canis lupus) in Italy

Canine distemper virus (CDV) infection is a primary threat affecting a wide number of carnivore species, including wild animals. In January 2013, two carcasses of Apennine wolves (Canis lupus) were collected in Ortona dei Marsi (L''Aquila province, Italy) by the local Veterinary Services. CDV was immediately identified either by RT-PCR or immunohistochemistry in lung and central nervous tissue samples. At the same time, severe clinical signs consistent with CDV infection were identified and taped (Videos S1–S3) from three wolves rescued in the areas surrounding the National Parks of the Abruzzi region by the Veterinary Services. The samples collected from these symptomatic animals also turned out CDV positive by RT-PCR. So far, 30 carcasses of wolves were screened and CDV was detected in 20 of them. The sequencing of the haemagglutinin gene and subsequent phylogenetic analysis demonstrated that the identified virus belonged to the CDV Arctic lineage. Strains belonging to this lineage are known to circulate in Italy and in Eastern Europe amongst domestic dogs. To the best of our knowledge this is the first report of CDV Arctic lineage epidemics in the wild population in Europe.     

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals^1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo^3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex^5-9.This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.     

Loss of function of the ALS protein SigR1 leads to ER pathology associated with defective autophagy and lipid raft disturbances

Intracellular accumulations of altered, misfolded proteins in neuronal and other cells are pathological hallmarks shared by many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Mutations in several genes give rise to familial forms of ALS. Mutations in Sigma receptor 1 have been found to cause a juvenile form of ALS and frontotemporal lobar degeneration (FTLD). We recently described altered localization, abnormal modification and loss of function of SigR1 in sporadic ALS. In order to further elucidate the molecular mechanisms underlying SigR1-mediated alterations in sporadic and familial ALS, we extended our previous studies using neuronal SigR1 knockdown cell lines. We found that loss of SigR1 leads to abnormal ER morphology, mitochondrial abnormalities and impaired autophagic degradation. Consistent with these results, we found that endosomal trafficking of EGFR is impaired upon SigR1 knockdown. Furthermore, in SigR1-deficient cells the transport of vesicular stomatitis virus glycoprotein is inhibited, leading to the accumulation of this cargo protein in the Golgi apparatus. Moreover, depletion of SigR1 destabilized lipid rafts and associated calcium mobilization, confirming the crucial role of SigR1 in lipid raft and intracellular calcium homeostasis. Taken together, our results support the notion that loss of SigR1 function contributes to ALS pathology by causing abnormal ER morphology, lipid raft destabilization and defective endolysosomal pathways.ER stress causes alterations in protein quality control, autophagy, calcium imbalance and mitochondrial dysfunction.^{1, 2, 3} It is central to the pathogenesis of many neurodegenerative diseases.^{4, 5} Altered proteins are targeted by molecular chaperones for protein repair or refolding. However, failure of this first line of defense leads to abnormal aggregates of such proteins that then form ubiquitinated cellular inclusions and compromise UPS function.^{6, 7, 8, 9} Macroautophagy, the major lysosomal degradative pathway in cells, is responsible for degrading long-lived cytoplasmic constituents; it is the principal mechanism for turning over cellular organelles and protein aggregates too large to be degraded by the proteasome.^{10, 11, 12, 13} Macroautophagy, hereafter referred to as autophagy, is a multistep process, initiated primarily by sequestration of portions of the cytoplasm in double-membrane-bound vesicles to form an autophagosome. Autophagosomes along with their cargoes are then degraded upon fusing with late endosome- or lysosome-containing cathepsins, other acid hydrolases, and vacuolar [H+] ATPase.¹⁴ Even though autophagy is a selective and efficient mechanism for the degradation of misfolded and mutant proteins related to neurodegeneration, recent evidence indicates that the alterations in certain disease-related genes may actually impair autophagic activity at different levels, including accumulation of autophagic vacuoles,¹⁵ substrate recognition, lysosomal acidity and autophagosome membrane nucleation.^{16, 17}SigR1 interacts with a variety of ligands and is involved in a broad array of biological functions that have only been partially defined so far. They include regulation of neuronal survival, neuritogenesis, ion channel activity, IP3R-mediated Ca²⁺ signaling, memory and drug addiction. SigR1 is an ER chaperone that is located at the mitochondria–ER interface and is normally bound to another chaperone, BiP/GRP78.¹⁸ Upon IP₃ receptor stimulation or Ca²⁺ depletion within the ER, SigR1 dissociates from BiP and stabilizes IP₃ receptors, leading to prolonged Ca²⁺ signaling into mitochondria.¹⁸ Recently, a mutation in SigR1, E1O2Q, has been reported to cause a juvenile form of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD).¹⁹ SigR1 protein is decreased significantly in human sporadic ALS spinal cord.²⁰ In addition, functional relevance of SigR1 in ALS pathogenesis is demonstrated by SigR1 knockout mice that display motor deficits with a shorter latency period in rotarod experiments, and by another study showing that lack of SigR1 exacerbates ALS progression in SOD1 tg mice.^{21, 22} Furthermore, a recent study showed improvement in motor function and survival of motor neurons in SOD1 mice treated with a SigR1 agonist.²³ Recently, we reported altered localization and abnormal modification of SigR1 in sporadic ALS and showed that loss of SigR1 function leads to deformities in ER structure, formation of ER-derived autophagic vacuoles and induction of ER stress.²⁰ Loss of function of SigR1 also leads to aberrant calcium homeostasis and cell death.²⁰ Together, these data suggest a crucial role of SigR1 in neuronal function and survival.Autophagy has been known to be tightly linked to ER function and is decisive in neurodegeneration mediated by ER stress.^{24, 25, 26} Therefore, we hypothesized that loss of SigR1 function contributes to a vicious circle including ER stress, defective autophagy and altered calcium signaling that causes multifactorial ALS pathology. In the present study, we show that depletion of SigR1 leads to the accumulation of numerous autophagic vacuoles often filled with nondegraded autophagic substrates, and severe deformities in ER ultrastructure including loss of ER tethering. Biochemical analysis in SigR1-deficient cells revealed the accumulation of various autophagic substrates and defects in endosomal trafficking, suggesting an impairment of endolysosomal pathways. Finally, loss of SigR1 destabilized lipid rafts causing both impaired calcium mobilization and altered endosomal trafficking. Altogether, our results support the notion that loss of SigR1 contributes to ALS pathogenesis by causing abnormal ER morphology, lipid raft destabilization and defective autophagy.     

A monoclonal antibody to glucosylceramide inhibits the growth of Fonsecaea pedrosoi and enhances the antifungal action of mouse macrophages

Fungal glucosylceramides (GlcCer) are conserved lipid components in a large variety of pathogenic and non-pathogenic fungal species, but their biological functions are still unclear. Recent studies demonstrate that GlcCer are immunologically active components inducing the production of antifungal antibodies. In this work, a major GlcCer was purified and characterized from mycelial forms of Fonsecaea pedrosoi, the most frequent causative agent of chromoblastomycosis. As determined by fast atom bombardment mass spectrometry (FAB-MS) analysis, the purified molecule was identified as the conserved structure N-2'-hydroxyhexadecanoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine. A monoclonal antibody (Mab) against this structure was used in indirect immunofluorescence with the different morphological stages of F. pedrosoi. Only the surface of young dividing cells was recognized by the antibody. Treatment of F. pedrosoi conidia with the Mab against GlcCer resulted in a clear reduction in fungal growth. In addition, the Mab also enhanced phagocytosis and killing of F. pedrosoi by murine cells. These results suggest that, in F. pedrosoi, GlcCer seem to be cell wall components targeted by antifungal antibodies that directly inhibit fungal development and also enhance macrophage function, supporting the use of monoclonal antibodies to GlcCer as potential tools in antifungal immunotherapy.     

Origin of sucrose metabolism in higher plants: when,how and why?

Since the discovery of sucrose biosynthesis, considerable advances have been made in understanding its regulation and crucial role in the functional biology of plants. However, important aspects of this metabolism are still an enigma. Studies in cyanobacteria and the publication of the sequences of several complete genomes have recently significantly increased our knowledge of the structures of proteins involved in sucrose metabolism and given us new insights into their origin and further evolution.     

Protein interaction data curation: the International Molecular Exchange (IMEx) consortium

The International Molecular Exchange (IMEx) consortium is an international collaboration between major public interaction data providers to share literature-curation efforts and make a nonredundant set of protein interactions available in a single search interface on a common website (http://www.imexconsortium.org/). Common curation rules have been developed, and a central registry is used to manage the selection of articles to enter into the dataset. We discuss the advantages of such a service to the user, our quality-control measures and our data-distribution practices.