Prospective observational study in two Dutch hospitals to assess the performance of inflammatory plasma markers to determine disease severity of viral respiratory tract infections in children.

INTRODUCTION: Respiratory viruses causing lower respiratory tract infections (LRTIs) are a major cause of hospital admissions in children. Since the course of these infections is unpredictable with potential fast deterioration into respiratory failure, infants are easily admitted to the hospital for observation. The aim of this study was to examine whether systemic inflammatory markers can be used to predict severity of disease in children with respiratory viral infections. METHODS: Blood and nasopharyngeal washings from children <3 years of age with viral LRTI attending a hospital were collected within 24 hours (acute) and after 4-6 weeks (recovery). Patients were assigned to a mild (observation only), moderate (supplemental oxygen and/or nasogastric feeding) or severe (mechanical ventilation) group. Linear regression analysis was used to design a prediction rule using plasma levels of C reactive protein (CRP), serum amyloid A (SAA), pentraxin 3 (PTX3), serum amyloid P component and properdin. This rule was tested in a validation cohort. RESULTS: One hundred and four children (52% male) were included. A combination of CRP, SAA, PTX3 and properdin was a better indicator of severe disease compared with any of the individual makers and age (69% sensitivity (95% CI 50 to 83), 90% specificity (95% CI 80 to 96)). Validation in 141 patients resulted in 71% sensitivity (95% CI 53 to 85), 87% specificity (95% CI 79 to 92), negative predictive value of 64% (95% CI 47 to 78) and positive predictive value of 90% (95% CI 82 to 95). The prediction rule was not able to identify patients with a mild course of disease. CONCLUSION: A combination of CRP, SAA, PTX3 and properdin was able to identify children with a severe course of viral LRTI disease, even in children under 2 months of age. To assess the true impact on clinical management, these results should be validated in a prospective randomised control study.

Sex matters: Systemic complement activity of female C57BL/6J and BALB/cJ mice is limited by serum terminal pathway components.

Experimental mouse models have been extensively used to elucidate the role of the complement system in different diseases and injuries. Contribution of gender has revealed an intriguing gender specific difference; female mice often show protection against most complement driven injuries such as ischemia/reperfusion injury, graft rejection and sepsis. Interestingly, early studies to the mouse complement system revealed that female mice have very low total complement activity (CH50), which is related to androgen regulation of hepatic complement synthesis. Here, our aim was to understand at which level the female specific differences in mouse complement resides. We have used recently developed complement assays to study the functional activities of female and male mice at the level of C3 and C9 activation, and furthermore assayed key complement factor levels in serum of age-matched female and male C57BL/6 mice. Our results show that the female mice have normal complement cascade functionality at the level of C3 activation, which was supported by determinations of early complement factors. However, all pathways are strongly reduced at the level of C9 activation, suggesting a terminal pathway specific difference. This was in line with C6 and C9 measurements, showing strongly decreased levels in females. Furthermore, similar gender differences were also found in BALB/cJ mice, but not in CD-1 mice. Our results clearly demonstrate that the complement system in females of frequently used mouse strains is restricted by the terminal pathway components and that the perceived female specific protection against experimental disease and injury might be in part explained by the inability promote inflammation through C5b-9.

Functional assessment of mouse complement pathway activities and quantification of C3b/C3c/iC3b in an experimental model of mouse renal ischaemia/reperfusion injury.

The complement system is an essential component of our innate immunity, both for the protection against infections and for proper handling of dying cells. However, the complement system can also contribute to tissue injury and inflammatory responses. In view of novel therapeutic possibilities, there is an increasing interest in measurement of the complement system activation in the systemic compartment, both in the clinical setting as well as in experimental models. Here we describe in parallel a sensitive and specific sandwich ELISA detecting mouse C3 activation fragments C3b/C3c/iC3b, as well as functional complement ELISAs detecting specific activities of the three complement pathways at the level of C3 and at the level of C9 activation. In a murine model of renal ischaemia/reperfusion injury (IRI) we found transient complement activation as shown by generation of C3b/C3c/iC3b fragments at 24 h following reperfusion, which returned to base-line at 3 and 7 days post reperfusion. When the pathway specific complement activities were measured at the level of C3 activation, we found no significant reduction in any of the pathways. However, the functional complement activity of all three pathways was significantly reduced when measured at the level of C9, with the strongest reduction being observed in the alternative pathway. For all three pathways there was a strong correlation between the amount of C3 fragments and the reduction in functional complement activity. Moreover, at 24 h both C3 fragments and the functional complement activities showed a correlation with the rise in serum creatinine. Together our results show that determination of the systemic pathway specific complement activity is feasible in experimental mouse models and that they are useful in understanding complement activation and inhibition in vivo.

Dis3-like 1: a novel exoribonuclease associated with the human exosome.

The exosome is an exoribonuclease complex involved in the degradation and maturation of a wide variety of RNAs. The nine-subunit core of the eukaryotic exosome is catalytically inactive and may have an architectural function and mediate substrate binding. In Saccharomyces cerevisiae, the associated Dis3 and Rrp6 provide the exoribonucleolytic activity. The human exosome-associated Rrp6 counterpart contributes to its activity, whereas the human Dis3 protein is not detectably associated with the exosome. Here, a proteomic analysis of immunoaffinity-purified human exosome complexes identified a novel exosome-associated exoribonuclease, human Dis3-like exonuclease 1 (hDis3L1), which was confirmed to associate with the exosome core by co-immunoprecipitation. In contrast to the nuclear localization of Dis3, hDis3L1 exclusively localized to the cytoplasm. The hDis3L1 isolated from transfected cells degraded RNA in an exoribonucleolytic manner, and its RNB domain seemed to mediate this activity. The siRNA-mediated knockdown of hDis3L1 in HeLa cells resulted in elevated levels of poly(A)-tailed 28S rRNA degradation intermediates, indicating the involvement of hDis3L1 in cytoplasmic RNA decay. Taken together, these data indicate that hDis3L1 is a novel exosome-associated exoribonuclease in the cytoplasm of human cells.

Biochemical studies of the mammalian exosome with intact cells.

A key component responsible for 3'- to 5'-RNA turnover in eukaryotic cells is the exosome, a multisubunit complex present in both the nucleus and cytoplasm of the cell. Here we describe several methods that can be applied to study the structure and function of the exosome in mammalian cell lines. The mammalian two-hybrid system has been successfully used to identify protein-protein interactions between exosome core components. Cell and glycerol gradient fractionation procedures are described that allow the identification and characterization of different exosome subsets. Finally, a protocol to study the function of the exosome in RNA turnover with RNA interference is presented.

C1D is a major autoantibody target in patients with the polymyositis-scleroderma overlap syndrome.

OBJECTIVE: To assess whether the recently discovered exosome-associated proteins MPP6, C1D, KIAA0052/hMtr4, hSki2, and hSki8 are targeted by autoantibodies, and to determine whether these autoantibodies are accompanied by antibodies directed to the established exosome-associated autoantigens PM-Scl-75 and PM-Scl-100. METHODS: Complementary DNAs encoding the recently identified human exosome-associated proteins were expressed as His-tagged fusion proteins in Escherichia coli cells. Sera obtained from patients with several different autoimmune diseases were analyzed for the presence of autoantibodies directed to these proteins, in an enzyme-linked immunosorbent assay (ELISA). The ELISA data obtained for C1D were confirmed by Western blot analysis, using recombinant C1D. RESULTS: All exosome-associated proteins were found to be targeted by autoantibodies, although the frequency with which such antibodies occurred in patient sera was relatively low, with the exception of anti-C1D antibodies. Autoantibodies recognizing C1D were detected in 7 of 30 patients (23%) with the polymyositis (PM)-scleroderma overlap syndrome; this frequency was similar to the frequencies for the established autoantigens PM-Scl-75c (27%) and PM-Scl-100 (23%). Importantly, several patients with the PM-scleroderma overlap syndrome had anti-C1D antibodies but no anti-PM-Scl antibodies. Anti-C1D autoantibodies were observed in only 2 of 204 patients with other diseases, including PM, dermatomyositis, and scleroderma. CONCLUSION: Our results demonstrate that the recently identified exosome-associated protein C1D is a major autoantigen in patients with the PM-scleroderma overlap syndrome and suggest that the use of recombinant C1D as an autoantibody target may aid in diagnosis of the PM-scleroderma overlap syndrome.

Human cell growth requires a functional cytoplasmic exosome, which is involved in various mRNA decay pathways.

The human exosome is a 3'-5' exoribonuclease complex that functions both in the nucleus and in the cytoplasm to either degrade or process RNA. Little is known yet about potential differences among core exosome complexes in these different cellular compartments and the roles of the individual subunits in maintaining a stable and functional complex. Glycerol gradient sedimentation analyses indicated that a significant subset of nuclear exosomes is present in much larger complexes (60-80S) than the cytoplasmic exosomes ( approximately 10S). Interestingly, siRNA-mediated knock-down experiments indicated that the cytoplasmic exosome is down-regulated much more efficiently than the nuclear exosome. In addition, we observed that knock-down of hRrp41p or hRrp4p but not PM/Scl-100 or PM/Scl-75 leads to codepletion of other subunits. Nevertheless, PM/Scl-100 and PM/Scl-75 are required to maintain normal levels of three different mRNA reporters: a wild-type beta-globin mRNA, a beta-globin mRNA containing an AU-rich (ARE) instability element, and a beta-globin mRNA bearing a premature termination codon (PTC). The increased levels of ARE- and the PTC-containing mRNAs upon down-regulation of the different exosome subunits, in particular PM/Scl-100, appeared to be due to decreased turnover rates. These results indicate that, although not required for exosome stability, PM/Scl-100 and PM/Scl-75 are involved in mRNA degradation, either as essential subunits of a functional exosome complex or as exosome-independent proteins.

C1D and hMtr4p associate with the human exosome subunit PM/Scl-100 and are involved in pre-rRNA processing.

The exosome is a complex of 3'-5' exoribonucleases and RNA-binding proteins, which is involved in processing or degradation of different classes of RNA. Previously, the characterization of purified exosome complexes from yeast and human cells suggested that C1D and KIAA0052/hMtr4p are associated with the exosome and thus might regulate its functional activities. Subcellular localization experiments demonstrated that C1D and KIAA0052/hMtr4p co-localize with exosome subunit PM/Scl-100 in the nucleoli of HEp-2 cells. Additionally, the nucleolar accumulation of C1D appeared to be dependent on PM/Scl-100. Protein-protein interaction studies showed that C1D binds to PM/Scl-100, whereas KIAA0052/hMtr4p was found to interact with MPP6, a previously identified exosome-associated protein. Moreover, we demonstrate that C1D, MPP6 and PM/Scl-100 form a stable trimeric complex in vitro. Knock-down of C1D, MPP6 and KIAA0052/hMtr4p by RNAi resulted in the accumulation of 3'-extended 5.8S rRNA precursors, showing that these proteins are required for rRNA processing. Interestingly, C1D appeared to contain RNA-binding activity with a potential preference for structured RNAs. Taken together, our results are consistent with a role for the exosome-associated proteins C1D, MPP6 and KIAA052/hMtr4p in the recruitment of the exosome to pre-rRNA to mediate the 3' end processing of the 5.8S rRNA.

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis.

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'-->5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD369 [see text] G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.

Cell and molecular biology of the exosome: how to make or break an RNA.

The identification and characterization of the exosome complex has shown that the exosome is a complex of 3' --> 5' exoribonucleases that plays a key role in the processing and degradation of a wide variety of RNA substrates. Advances in the understanding of exosome function have led to the identification of numerous cofactors that are required for a selective recruitment of the exosome to substrate RNAs, for their structural alterations to facilitate degradation, and to aid in their complete degradation/processing. Structural data obtained by two-hybrid interaction analyses and X-ray crystallography show that the core of the exosome adopts a doughnut-like structure and demonstrates that probably not all exosome subunits are active exoribonucleases. Despite all data obtained on the structure and function of the exosome during the last decade, there are still a lot of unanswered questions. What is the molecular mechanism by which cofactors select and target substrate RNAs to the exosome and modulate its function for correct processing or degradation? How can the exosome discriminate between processing or degradation of a specific substrate RNA? What is the precise structure of exosome subunits and how do they contribute to its function? Here we discuss studies that provide some insight to these questions and speculate on the mechanisms that control the exosome.

MPP6 is an exosome-associated RNA-binding protein involved in 5.8S rRNA maturation.

The exosome is a complex of 3'-->5' exoribonucleases which is involved in many RNA metabolic processes. To regulate these functions distinct proteins are believed to recruit the exosome to specific substrate RNAs. Here, we demonstrate that M-phase phosphoprotein 6 (MPP6), a protein reported previously to co-purify with the TAP-tagged human exosome, accumulates in the nucleoli of HEp-2 cells and associates with a subset of nuclear exosomes as evidenced by co-immunoprecipitation and biochemical fractionation experiments. In agreement with its nucleolar accumulation, siRNA-mediated knock-down experiments revealed that MPP6 is involved in the generation of the 3' end of the 5.8S rRNA. The accumulation of the same processing intermediates after reducing the levels of either MPP6 or exosome components strongly suggests that MPP6 is required for the recruitment of the exosome to the pre-rRNA. Interestingly, MPP6 appeared to display RNA-binding activity in vitro with a preference for pyrimidine-rich sequences, and to bind to the ITS2 element of pre-rRNAs. Our data indicate that MPP6 is a nucleolus-specific exosome co-factor required for its role in the maturation of 5.8S rRNA.

The Tyr113His polymorphism in exon 3 of the microsomal epoxide hydrolase gene is a risk factor for perinatal mortality.

BACKGROUND: A genetic predisposition to impaired detoxification of oxidative or chemical stress could play a role in the etiology of perinatal mortality. In this pilot study we investigated the risk of perinatal mortality in relation to genetic polymorphism in microsomal epoxide hydrolase (EPHX) and glutathione S-transferase P1 (GSTP1) in women who experienced perinatal mortality caused by placental pathology, congenital disorders and complications of premature delivery and their male partners. METHODS: Genomic DNA of couples (72 females and 46 males) with a history of perinatal mortality and control couples (71 females and 66 males) with no complications in their obstetric history were analyzed for the presence of the polymorphisms in exon 3 of EPHX (Tyr113His) and GSTP1 (Ile105Val). RESULTS: A similar distribution of the GSTP1 polymorphism was found in all subjects investigated. In women who experienced perinatal mortality, we demonstrated a higher prevalence of the EPHX His113/His113 genotype, which could result in a lower enzyme activity, compared with controls (25% vs. 9%; chi2 = 5.7 and p < 0.02), with an odds ratio (95% confidence interval) of 3.5 (1.1-12.7). CONCLUSION: Our results suggest that the maternal Tyr113His polymorphism in EPHX may be a risk factor for perinatal mortality. However, more research is needed to determine the implication of this finding.

The exosome, a molecular machine for controlled RNA degradation in both nucleus and cytoplasm.

One of the most important protein complexes involved in maintaining correct RNA levels in eukaryotic cells is the exosome, a complex consisting almost exclusively of exoribonucleolytic proteins. Since the identification of the exosome complex, seven years ago, much progress has been made in the characterization of its composition, structure and function in a variety of organisms. Although the exosome seems to accumulate in the nucleolus, it has been clearly established that it is also localized in cytoplasm and nucleoplasm. In accordance with its widespread intracellular distribution, the exosome has been implicated in a variety of RNA processing and degradation processes. Nevertheless, many questions still remain unanswered. What are the factors that regulate the activity of the exosome? How and where is the complex assembled? What are the differences in the composition of the nuclear and cytoplasmic exosome? What is the detailed structure of exosome subunits? What are the mechanisms by which the exosome is recruited to substrate RNAs? Here, we summarize the current knowledge on the composition and architecture of this complex, explain its role in both the production and degradation of various types of RNA molecules and discuss the implications of recent research developments that shed some light on the questions above and the mechanisms that are controlling the exosome.

N-Acetylcysteine improves the disturbed thiol redox balance after methionine loading.

Methionine loading seems to be accompanied by increased oxidative stress and damage. However, it is not known how this oxidative stress is generated. We performed the present crossover study to further elucidate the effects of methionine loading on oxidative stress in the blood of healthy volunteers, and to examine possible preventative effects of N -acetylcysteine (NAC) administration. A total of 18 healthy subjects were given two oral methionine loads of 100 mg/kg body weight, 4 weeks apart, one without NAC (Met group), and one in combination with supplementation with 2x900 mg doses of NAC (Met+NAC group). Blood samples were collected before and 2, 4, 8 and 24 h after methionine loading for measurements of thiol levels, protein carbonyls, lipid peroxidation, cellular fibronectin and ferric reducing ability of plasma (FRAP; i.e. antioxidant capacity). After methionine loading, whole-blood levels of free and oxidized cysteine and homocysteine were increased in both groups. Furthermore, the total plasma levels of homocysteine were higher, whereas those of cysteine were lower, after methionine loading in both groups. Lower levels of oxidized homocysteine and a higher free/oxidized ratio were found in the Met+NAC group compared with the Met group. Although the antioxidant capacity decreased after methionine loading, no major changes over time were found for protein carbonyls or cellular fibronectin in either group. Our results suggest that methionine loading may initiate the generation of reactive oxygen species by the (auto)-oxidation of homocysteine. In addition, supplementation with NAC seems to be able to partially prevent excessive increases in the levels of homocysteine in plasma and of oxidized homocysteine in whole blood, and might thereby contribute to the prevention of oxidative stress.