Guidelines for specialized nutritional and metabolic support in the critically-ill patient. Update. Consensus SEMICYUC-SENPE: Septic patient

Nutritional metabolic management, together with other treatment and support measures used, is one of the mainstays of the treatment of septic patients. Nutritional support should be started early, after initial life support measures, to avoid the consequences of malnutrition, to provide adequate nutritional intake and to prevent the development of secondary complications such as superinfection or multiorgan failure. As in other critically-ill patients, when the enteral route cannot be used to ensure calorie-protein requirements, the association of parenteral nutrition has been shown to be safe in this subgroup of patients. Studies evaluating the effect of specific pharmaconutrients in septic patients are scarce and are insufficient to allow recommendations to be made. To date, enteral diets with a mixture of substrates with distinct pharmaconutrient properties do not seem to be superior to standard diets in altering the course of sepsis, although equally there is no evidence that these diets are harmful. There is insufficient evidence to recommend the use of glutamine in septic patients receiving parenteral nutrition. However, given the good results and absence of glutamine-related adverse effects in the various studies performed in the general population of critically-ill patients, these patients could benefit from the use of this substance. Routine use of omega-3 fatty acids cannot be recommended until further evidence has been gathered, although the use of lipid emulsions with a high omega-6 fatty acid content should be avoided. Septic patients should receive an adequate supply of essential trace elements and vitamins. Further studies are required before the use of high-dose selenium can be recommended.

Timing of adequate antibiotic therapy is a greater determinant of outcome than are TNF and IL-10 polymorphisms in patients with sepsis.

INTRODUCTION Genetic variations may influence clinical outcomes in patients with sepsis. The present study was conducted to evaluate the impact on mortality of three polymorphisms after adjusting for confounding variables, and to assess the factors involved in progression of the inflammatory response in septic patients. METHOD The inception cohort study included all Caucasian adults admitted to the hospital with sepsis. Sepsis severity, microbiological information and clinical variables were recorded. Three polymorphisms were identified in all patients by PCR: the tumour necrosis factor (TNF)-alpha 308 promoter polymorphism; the polymorphism in the first intron of the TNF-beta gene; and the IL-10-1082 promoter polymorphism. Patients included in the study were followed up for 90 days after hospital admission. RESULTS A group of 224 patients was enrolled in the present study. We did not find a significant association among any of the three polymorphisms and mortality or worsening inflammatory response. By multivariate logistic regression analysis, only two factors were independently associated with mortality, namely Acute Physiology and Chronic Health Evaluation (APACHE) II score and delayed initiation of adequate antibiotic therapy. In septic shock patients (n = 114), the delay in initiation of adequate antibiotic therapy was the only independent predictor of mortality. Risk factors for impairment in inflammatory response were APACHE II score, positive blood culture and delayed initiation of adequate antibiotic therapy. CONCLUSION This study emphasizes that prompt and adequate antibiotic therapy is the cornerstone of therapy in sepsis. The three polymorphisms evaluated in the present study appear not to influence the outcome of patients admitted to the hospital with sepsis.

Overlooked post-translational modifications of proteins in Plasmodium falciparum: N- and O-glycosylation - A Review

Human malignant malaria is caused by Plasmodium falciparum and accounts for almost 900,000 deaths per year, the majority of which are children and pregnant women in developing countries. There has been significant effort to understand the biology of P. falciparum and its interactions with the host. However, these studies are hindered because several aspects of parasite biology remain controversial, such as N- and O-glycosylation. This review describes work that has been done to elucidate protein glycosylation in P. falciparum and it focuses on describing biochemical evidence for N- and O-glycosylation. Although there has been significant work in this field, these aspects of parasite biochemistry need to be explored further.

Amoebae as a tool to isolate new bacterial species, to discover new virulence factors and to study the host-pathogen interactions.

Amoebae are unicellular protozoan present worldwide in several environments mainly feeding on bacteria. Some of them, the amoebae-resistant bacteria (ARBs), have evolved mechanisms to survive and replicate inside amoebal species. These mainly include legionella, mycobacteria and Chlamydia-related bacteria. Amoebae can provide a replicative niche, can act as reservoir for bacteria whereas the cystic form can protect the internalized bacteria. Moreover, the amoebae represent a Trojan horse for ARBs to infect animals. The long interaction between amoebae and bacteria has likely selected for bacterial virulence traits leading to the adaptation towards an intracellular lifestyle, and some ARBs have acquired the ability to infect mammals. This review intends to highlight the important uses of amoebae in several fields in microbiology by describing the main tools developed using amoebal cells. First, amoebae such as Acanthamoeba are used to isolate and discover new intracellular bacterial species by two main techniques: the amoebal co-culture and the amoebal enrichment. In the second part, taking Waddlia chondrophila as example, we summarize some important recent applications of amoebae to discover new bacterial virulence factors, in particular thanks to the amoebal plaque assay. Finally, the genetically tractable Dictyostelium discoideum is used as a model organism to study host-pathogen interactions, in particular with the development of several approaches to manipulate its genome that allowed the creation of a wide range of mutated strains largely shared within the Dictyostelium community.

Differential phosphorylation of some proteins of the neuronal cytoskeleton during brain development.

The cytoskeleton is important for neuronal morphogenesis. During the postnatal development of cat brain, the molecular composition of the neuronal cytoskeleton changes with maturation. Several of its proteins change in their rate of expression, in their degree of phosphorylation, in their subcellular distribution, or in their biochemical properties. It is proposed that phosphorylation is an essential mechanism to regulate the plasticity of the early, juvenile-type cytoskeleton. Among such proteins are several microtubule-associated proteins (MAPs), such as MAP5a, MAP2c or the juvenile tau proteins. Phosphorylation may also act on neurofilaments, postulated to be involved in the adult-type stabilization of axons. These observations imply that phosphorylation may affect cytoskeleton function in axons and dendrites at various developmental stages. Yet, the mechanisms of phosphorylation and its regulation cascades are largely unknown. In view of the topic of this issue on CD15, the potential role of matrix molecules being involved in the modulation of phosphorylation activity and of cytoskeletal properties is addressed.

The ability of haemolysins expressed by atypical enteropathogenic Escherichia coli to bind to extracellular matrix components

Typical and atypical enteropathogenic Escherichia coli (EPEC) are considered important bacterial causes of diarrhoea. Considering the repertoire of virulence genes, atypical EPEC (aEPEC) is a heterogeneous group, harbouring genes that are found in other diarrheagenic E. coli pathotypes, such as those encoding haemolysins. Haemolysins are cytolytic toxins that lyse host cells disrupting the function of the plasma membrane. In addition, these cytolysins mediate a connection to vascular tissue and/or blood components, such as plasma and cellular fibronectin. Therefore, we investigated the haemolytic activity of 72 aEPEC isolates and determined the correlation of this phenotype with the presence of genes encoding enterohaemolysins (Ehly) and cytolysin A (ClyA). In addition, the correlation between the expression of haemolysins and the ability of these secreted proteins to adhere to extracellular matrix (ECM) components was also assessed in this study. Our findings demonstrate that a subset of aEPEC presents haemolytic activity due to the expression of Ehlys and/or ClyA and that this activity is closely related to the ability of these isolates to bind to ECM components.

Modulation of phosphorylation of neuronal cytoskeletal proteins by neuronal depolarization.

1. The neuronal cytoskeletal protein tau and the carboxy tails of cytoskeletal proteins neurofilament-M (NF-M) and neurofilament-H (NF-H) are phosphorylated on serine residues by the cyclin-dependent kinase cdk-5. 2. In aggregating neuronal-glial cultures we show that veratridine-mediated cation influx causes dephosphorylation of tau, NF-M and NF-H. Dephosphorylation was blocked specifically by cyclosporine A but not by okadiac acid at concentrations up to 200 nM. 3. These results suggest that veratridine-triggered cation influx causes activation of PP-2B (calcineurin) leading to dephosphorylation of these cytoskeletal proteins.

Characterization and binding affinities of SmLANP: a new Schistosoma mansoni member of the ANP32 family of regulatory proteins.

Members of the leucine-rich repeat protein family are involved in diverse functions including protein phosphatase 2-inhibition, cell cycle regulation, gene regulation and signalling pathways. A novel Schistosoma mansoni gene, called SmLANP, presenting homology to various genes coding for proteins that belong to the super family of leucine-rich repeat proteins, was characterized here. SmLANP was 1184bp in length as determined from cDNA and genomic sequences and encoded a 296 amino acid open reading frame that spanning from 6 to 894bp. The predicted amino acid sequence had a calculated molecular weight of 32kDa. Analysis of the predicted sequence indicated the presence of 3 leucine-rich domains (LRR) located in the N-terminal region and an aspartic acid rich region in the C-terminal end. SmLANP transcript is expressed in all stages of the S. mansoni life cycle analyzed, exhibiting the highest expression level in males. The SmLANP protein was expressed in a GST expression system and antibodies raised in mice against the recombinant protein. By immunolocalization assay, using adult worms, it was shown that the protein is mainly present in the cell nucleus through the whole body and strongly expressed along the tegument cell body nuclei of adult worms. As members of this family are usually involved in protein-protein interaction, a yeast two hybrid assay was conducted to identify putative binding partners for SmLANP. Thirty-six possible partners were identified, and a protein ATP synthase subunit alpha was confirmed by pull down assays, as a binding partner of the SmLANP protein.

Granulocyte colony-stimulating factor in the treatment of high-risk febrile neutropenia: a multicenter randomized trial.

BACKGROUND Granulocyte colony-stimulating factors (G-CSFs) have been shown to help prevent febrile neutropenia in certain subgroups of cancer patients undergoing chemotherapy, but their role in treating febrile neutropenia is controversial. The purpose of our study was to evaluate-in a prospective multicenter randomized clinical trial-the efficacy of adding G-CSF to broad-spectrum antibiotic treatment of patients with solid tumors and high-risk febrile neutropenia. METHODS A total of 210 patients with solid tumors treated with conventional-dose chemotherapy who presented with fever and grade IV neutropenia were considered to be eligible for the trial. They met at least one of the following high-risk criteria: profound neutropenia (absolute neutrophil count <100/mm(3)), short latency from previous chemotherapy cycle (<10 days), sepsis or clinically documented infection at presentation, severe comorbidity, performance status of 3-4 (Eastern Cooperative Oncology Group scale), or prior inpatient status. Eligible patients were randomly assigned to receive the antibiotics ceftazidime and amikacin, with or without G-CSF (5 microg/kg per day). The primary study end point was the duration of hospitalization. All P values were two-sided. RESULTS Patients randomly assigned to receive G-CSF had a significantly shorter duration of grade IV neutropenia (median, 2 days versus 3 days; P = 0.0004), antibiotic therapy (median, 5 days versus 6 days; P = 0.013), and hospital stay (median, 5 days versus 7 days; P = 0.015) than patients in the control arm. The incidence of serious medical complications not present at the initial clinical evaluation was 10% in the G-CSF group and 17% in the control group (P = 0.12), including five deaths in each study arm. The median cost of hospital stay and the median overall cost per patient admission were reduced by 17% (P = 0.01) and by 11% (P = 0.07), respectively, in the G-CSF arm compared with the control arm. CONCLUSIONS Adding G-CSF to antibiotic therapy shortens the duration of neutropenia, reduces the duration of antibiotic therapy and hospitalization, and decreases hospital costs in patients with high-risk febrile neutropenia.

Efficiency diagnostic and advantages of procalcitonin and C-reactive protein in the early diagnosis of sepsis.

The goal of our study is to assess the diagnostic profi tability of procalcitonin (PCT) in septic shock and another biomarker as C-reactive protein (CRP). Results: Fifty-four septic patients were assessed, 66% were males; mean age, 63 years. Eighty-eight percent was diagnosed as septic shock and 11% severe sepsis. Seventy-six percent were medical patients. Positive blood cultures in 42.5%. Sepsis origin: respiratory 46%, neurological 5%, digestive 37% and urinary 3%. Average SOFA score was 10.4. Conclusions: PCT and CRP have the same efficiency in early sepsis diagnosis. The PCT and CRP effi ciency diagnostic together is signifi cant but small. We suggest using both with the doubt of sepsis.

Dectin-1 and DC-SIGN polymorphisms associated with invasive pulmonary Aspergillosis infection.

The recognition of pathogen-derived structures by C-type lectins and the chemotactic activity mediated by the CCL2/CCR2 axis are critical steps in determining the host immune response to fungi. The present study was designed to investigate whether the presence of single nucleotide polymorphisms (SNPs) within DC-SIGN, Dectin-1, Dectin-2, CCL2 and CCR2 genes influence the risk of developing Invasive Pulmonary Aspergillosis (IPA). Twenty-seven SNPs were selected using a hybrid functional/tagging approach and genotyped in 182 haematological patients, fifty-seven of them diagnosed with proven or probable IPA according to the 2008 EORTC/MSG criteria. Association analysis revealed that carriers of the Dectin-1(rs3901533 T/T) and Dectin-1(rs7309123 G/G) genotypes and DC-SIGN(rs4804800 G), DC-SIGN(rs11465384 T), DC-SIGN(7248637 A) and DC-SIGN(7252229 C) alleles had a significantly increased risk of IPA infection (OR = 5.59 95%CI 1.37-22.77; OR = 4.91 95%CI 1.52-15.89; OR = 2.75 95%CI 1.27-5.95; OR = 2.70 95%CI 1.24-5.90; OR = 2.39 95%CI 1.09-5.22 and OR = 2.05 95%CI 1.00-4.22, respectively). There was also a significantly increased frequency of galactomannan positivity among patients carrying the Dectin-1(rs3901533_T) allele and Dectin-1(rs7309123_G/G) genotype. In addition, healthy individuals with this latter genotype showed a significantly decreased level of Dectin-1 mRNA expression compared to C-allele carriers, suggesting a role of the Dectin-1(rs7309123) polymorphism in determining the levels of Dectin-1 and, consequently, the level of susceptibility to IPA infection. SNP-SNP interaction (epistasis) analysis revealed significant interactions models including SNPs in Dectin-1, Dectin-2, CCL2 and CCR2 genes, with synergistic genetic effects. Although these results need to be further validated in larger cohorts, they suggest that Dectin-1, DC-SIGN, Dectin-2, CCL2 and CCR2 genetic variants influence the risk of IPA infection and might be useful in developing a risk-adapted prophylaxis.

NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies.

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

Molecular and immunological characterization of gluten proteins isolated from oat cultivars that differ in toxicity for celiac disease.

A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease (CD). Traditionally, treatment with a GFD has excluded wheat, barley and rye, while the presence of oats is a subject of debate. The most-recent research indicates that some cultivars of oats can be a safe part of a GFD. In order to elucidate the toxicity of the prolamins from oat varieties with low, medium, and high CD toxicity, the avenin genes of these varieties were cloned and sequenced, and their expression quantified throughout the grain development. At the protein level, we have accomplished an exhaustive characterization and quantification of avenins by RP-HPLC and an analysis of immunogenicity of peptides present in prolamins of different oat cultivars. Avenin sequences were classified into three different groups, which have homology with S-rich prolamins of Triticeae. Avenin proteins presented a lower proline content than that of wheat gliadin; this may contribute to the low toxicity shown by oat avenins. The expression of avenin genes throughout the development stages has shown a pattern similar to that of prolamins of wheat and barley. RP-HPLC chromatograms showed protein peaks in the alcohol-soluble and reduced-soluble fractions. Therefore, oat grains had both monomeric and polymeric avenins, termed in this paper gliadin- and glutenin-like avenins. We found a direct correlation between the immunogenicity of the different oat varieties and the presence of the specific peptides with a higher/lower potential immunotoxicity. The specific peptides from the oat variety with the highest toxicity have shown a higher potential immunotoxicity. These results suggest that there is wide range of variation of potential immunotoxicity of oat cultivars that could be due to differences in the degree of immunogenicity in their sequences.

Differential expression and modification of neurofilament triplet proteins during cat cerebellar development.

Neurofilament (NF) proteins consist of three subunits of different molecular weights defined as NF-H, NF-M, and NF-L. They are typical structures of the neuronal cytoskeleton. Their immunocytochemical distribution during postnatal development of cat cerebellum was studied with several monoclonal and polyclonal antibodies against phosphorylated or unmodified sites. Expression and distribution of the triplet neurofilament proteins changed with maturation. Afferent mossy and climbing fibers in the medullary layer contained NF-M and NF-L already at birth, whereas NF-H appeared later. Within the first three postnatal weeks, all three subunits appeared in mossy and climbing fibers in the internal granular and molecular layers and in the axons of Purkinje cells. Axons of local circuit neurons such as basket cells expressed these proteins at the end of the first month, whereas parallel fibers expressed them last, at the beginning of the third postnatal month. Differential localization was especially observed for NF-H. Depending on phosphorylation, NF-H proteins were found in different axon types in climbing, mossy, and basket fibers or additionally in parallel fibers. A nonphosphorylated NF-H subunit was exclusively located in some Purkinje cells at early developmental stages and in some smaller interneurons later. A novel finding is the presence of a phosphorylation site in the NF-H subunit that is localized in dendrites of Purkinje cells but not in axons. Expression and phosphorylation of the NF-H subunit, especially, is cell-type specific and possibly involved in the adult-type stabilization of the axonal and dendritic cytoskeleton.

The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray

Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.