A Stable Bacterial Peroxidase with Novel Halogenating Activity and an Autocatalytically Linked Heme Prosthetic Group

Reconstructing the phylogenetic relationships of the main evolutionary lines of the mammalian peroxidases lactoperoxidase and myeloperoxidase revealed the presence of novel bacterial heme peroxidase subfamilies. Here, for the first time, an ancestral bacterial heme peroxidase is shown to possess a very high bromide oxidation activity (besides conventional peroxidase activity). The recombinant protein allowed monitoring of the autocatalytic peroxide-driven formation of covalent heme to protein bonds. Thereby, the high spin ferric rhombic heme spectrum became similar to lactoperoxidase, the standard reduction potential of the Fe(III)/Fe(II) couple shifted to more positive values (−145 ± 10 mV at pH 7), and the conformational and thermal stability of the protein increased significantly. We discuss structure-function relationships of this new peroxidase in relation to its mammalian counterparts and ask for its putative physiological role.     

The Design and Characterization of Receptor-selective APRIL Variants

A proliferation-inducing ligand (APRIL), a member of the TNF ligand superfamily with an important role in humoral immunity, is also implicated in several cancers as a prosurvival factor. APRIL binds two different TNF receptors, B cell maturation antigen (BCMA) and transmembrane activator and cylclophilin ligand interactor (TACI), and also interacts independently with heparan sulfate proteoglycans. Because APRIL shares binding of the TNF receptors with B cell activation factor, separating the precise signaling pathways activated by either ligand in a given context has proven quite difficult. In this study, we have used the protein design algorithm FoldX to successfully generate a BCMA-specific variant of APRIL, APRIL-R206E, and two TACI-selective variants, D132F and D132Y. These APRIL variants show selective activity toward their receptors in several in vitro assays. Moreover, we have used these ligands to show that BCMA and TACI have a distinct role in APRIL-induced B cell stimulation. We conclude that these ligands are useful tools for studying APRIL biology in the context of individual receptor activation.     

Current concepts of immune dysregulation in cystic fibrosis

Cystic fibrosis (CF) lung disease is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene and is characterized by a perpetuated feedback loop of bacterial infection and inflammation. Both intrinsic (CFTR-dependent) and extrinsic (CFTR-independent) mechanisms contribute to the inflammatory phenotype of CF lung disease. Innate immune cells, initially recruited to combat bacterial pathogens, are acting in a dysregulated and non-resolving fashion in CF airways and cause harm to the host by releasing proteases and oxidants. Targeting harmful immune pathways, while preserving protective ones, remains the challenge for the future. This review highlights current concepts of innate immune dysregulation in CF lung disease.This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.     

Soluble Ecto-5′-nucleotidase (5′-NT), Alkaline Phosphatase,and Adenosine Deaminase (ADA1) Activities in Neonatal Blood Favor Elevated Extracellular Adenosine

Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5′-nucleotidase (5′-NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine, whereas soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5′-NT and AP activity and lower adenosine-metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant because supplementation with AMP inhibited whereas selective pharmacologic inhibition of 5′-NT enhanced Toll-like receptor-mediated TNF-α production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine-metabolizing enzymes may benefit this vulnerable population.     

Spatial and temporal expression patterns of chitinase genes in developing zebrafish embryos

Chitinases and chitinase like proteins play an important role in mammalian immunity and functions in early zebrafish development have been suggested. Here we report identification of six zebrafish chitinases and chitinase like proteins (called CHIA.1–6) belonging to the glycoside hydrolase family 18, and determine their spatial and temporal expression at 10 stages of zebrafish development.CHIA.4 is highly maternally expressed and it is expressed 100 fold above any other CHIA gene at zygote through to blastula stage. Later, after the maternal to zygotic transition, CHIA.4 expression decreases to the same level as CHIA.5 and CHIA.6. Subsequently, CHIA.1, CHIA.2, CHIA.3 and CHIA.4, CHIA.5, CHIA.6 each follow distinct paths in terms of expression levels.Until 4 days post fertilization the spatial expression patterns of all six CHIA genes overlap extensively, with expression detected predominantly in vascular, ocular and intestinal tissues. At 5 days post fertilization CHIA.1, CHIA.2 and CHIA.3 are expressed almost exclusively in the stomach, whereas CHIA.4, CHIA.5 and CHIA.6 are also prominently expressed in the liver. These different expression patterns may contribute to the establishment of a basis on which functional analysis in older larvae may be founded.     

Orthogonal test design for optimization of the extraction of polysaccharides from Phascolosoma esulenta and evaluation of its immunity activity

Yield of polysaccharides from Phascolosoma esulenta obtained by phosphate buffer extraction through an orthogonal experiment (L₉(3)⁴) were investigated to get the best extraction conditions. The results showed that extraction temperature, ratio of phosphate buffer to raw material, extraction time, and ratio of trypsinase to raw material were the main four variables that influenced the yields of extracts. The highest yield was obtained when extraction temperature, ratio of phosphate buffer to raw material, extraction time and ratio of trypsinase to raw material were 40 °C, 2, 5.5 h and 1.6, respectively. The immunity-stimulating method showed that polysaccharides from P. esulenta could significantly raise liver, spleen and thymus index of mice and enhance Con A-stimulated mouse spleen cells proliferation. These results indicate that polysaccharides from P. esulenta had significantly higher immunity-stimulating activities.     

Granuloma formation to Capillaria hepatica eggs. I. Descriptive definition

The course of the cellular response in the liver to nonembryonated Capillaria hepatica (Bancroft, 1893) eggs given by intravenous injection into the portal circulation of unsensitized and sensitized mice was studied qualitatively and quantitatively. A gradual infiltration of predominantly mononuclear cells occurred around the eggs in the liver, leading to the formation of distinct granulomatous lesions characterized by macrophages and lymphocytes. This was followed by an infiltration of eosinophils. Previous intraperitoneal sensitization led to an earlier and an enhanced reaction to an intravenous challenge with eggs. Specificity of the cellular response was suggested by the lack of an enhanced reaction to presensitization with eggs of a closely related species, Trichuris muris. These studies indicate that granuloma formation to C. hepatica eggs has an immunological basis and furthermore the cell composition of the granuloma would suggest that a cell-mediated component may be involved as part of the specific response.     

Schistosoma mansoni: The attrition of a challenge infection in mice immunized with highly irradiated live cercariae

Mice immunized percutaneously with 400 Schistosoma mansoni cercariae given 20 kR of ⁶⁰Co irradiation were shown to develop an immunity in which nearly 80% of the parasites that would be expected to survive in control mice were killed. The major attrition of parasites was shown to occur within the first 4 days after challenge. Marked differences in the number of parasites which were recovered from the skin of immune mice and the failure of the majority of parasites to reach the lungs of immune mice indicated that the major site of attrition was in the skin. A further trickle of parasite deaths was evident beyond Day 5, but after Day 14 no further attrition of parasites appeared to occur. Mice immunized in the abdominal skin demonstrated similar levels of immunity whether challenged in the abdominal skin or in the ear. Immunization intramuscularly with irradiated schistosomula induced a much lower level of resistance and the marked parasite attrition in the skin at Day 2 was absent. Immunization with only 50 irradiated cercariae was shown to induce a level of skin immunity equivalent to that seen with 400 irradiated cercariae. The majority of cercariae given 20 kR of ⁶⁰Co irradiation remained in the skin; approximately 2% only reached the lungs. These studies demonstrate that percutaneous immunization of mice with highly irradiated cercariae induced a strong immunity which was largely effective in the skin. This immunity differed from that developed by chronically infected mice where the major attrition of parasites occurs after the lung phase of migration. The results also suggest that the penetration or persistence in the skin of live attenuated schistosomula may play a crucial role in the induction of a high level of skin immunity.