A differential concentration-dependent effect of IVIg on neutrophil functions: relevance for anti-microbial and anti-inflammatory mechanisms

Background

Polymorphonuclear neutrophils (PMN) play a key role in host defences against invading microorganisms but can also potentiate detrimental inflammatory reactions in case of excessive or misdirected responses. Intravenous immunoglobulins (IVIg) are used to treat patients with immune deficiencies and, at higher doses, in autoimmune, allergic and systemic inflammatory disorders.

Methodology/Principal Findings

We used flow cytometry to examine the effects of IVIg on PMN functions and survival, using whole-blood conditions in order to avoid artifacts due to isolation procedures. IVIg at low concentrations induced PMN activation, as reflected by decreased L-selectin and increased CD11b expression at the PMN surface, oxidative burst enhancement, and prolonged cell survival. In contrast, IVIg at higher concentrations inhibited LPS-induced CD11b degranulation and oxidative burst priming, and counteracted LPS-induced PMN lifespan prolongation.

Conclusions/Significance

IVIg appears to have differential, concentration-dependent effects on PMN, possibly supporting the use of IVIg as either an anti-microbial or an anti-inflammatory agent.     

Heritability of morningness-eveningness and self-report sleep measures in a family-based sample of 521 hutterites

Individual variation in the phase and amplitude of human circadian rhythms is well known, but the impact of heritable factors on such variation is less clear. We estimated the narrow-sense heritability for selected circadian and sleep timing, quality, and duration measures among related members of the Hutterites, an endogamous, religious community (n=521 participants). “Morningness-eveningness” (M/E), a stable trait reflecting circadian phase, was evaluated using the Composite Scale (CS). Subjective sleep measures were assessed using the Sleep Timing Questionnaire. Initial analyses reconfirmed the impact of age on M/E. Previously reported correlations between M/E scores and the sleep measures were also noted, demonstrating the construct validity of the questionnaires among the participants. Following corrections for age, gender, and colony of residence, significant narrow-sense heritability was noted for M/E (23%). The heritability for subjective sleep measures (related to timing, duration, and quality) were statistically significant for all but one variable, and varied between 12.4% and 29.4%. Thus, significant heritable influences on human circadian phase and subjective sleep indices can be detected through family-based studies. In view of the impact of circadian malfunction on human health, it may be worthwhile to map genetic factors impacting circadian and sleep variation.     

Relative contributions of measles virus hemagglutinin- and fusion protein-specific serum antibodies to virus neutralization

The relative contribution of measles virus hemagglutinin (H)- or fusion protein (F)-specific antibodies to virus neutralization (VN) has not been demonstrated. We have depleted these specific antibodies from sera collected from young adults, who had been vaccinated during childhood, by prolonged incubation with viable transfected human melanoma cells expressing H or F. Simultaneous depletion of antibodies of both specificities completely abrogated VN activity. Depletion of F-specific antibodies only had a minimal effect, whereas removal of H-specific antibodies resulted in almost complete reduction of VN activity. These results demonstrate that measles virus neutralizing antibodies are mainly directed to the H protein.     

Effects of hyperbaric oxygen on Leishmania amazonensis promastigotes and amastigotes

In the present study, we evaluated the effects of hyperbaric oxygen (HBO) exposure in both Leishmania amazonensis life stages (promastigotes and amastigotes) and on macrophage cultures infected with the parasite. HBO treatment protocols, which can be tolerated by humans and animals, induced irreversible metabolic damage and affected parasite morphology, growth and ability to transform. The observation that the antioxidant N-acetylcysteine (NAC) prevents some of these deleterious effects indicated an involvement of oxidative stress during parasite HBO exposure. In addition, HBO exposed L. amazonensis-infected macrophage cultures showed reduction of the percentage of infected cells and of the number of intracellular parasites per cell. Thus, the demonstration that HBO, a therapy used in the management of different diseases, is toxic for both L. amazonensis life stages and can alter macrophage susceptibility to the infection encourages further studies of this therapy in animal models of Leishmania infection.     

Cre-mediated recombination in cell lineages that express the progesterone receptor

Using gene-targeting methods, a progesterone receptor Cre knockin (PR-Cre) mouse was generated in which Cre recombinase was inserted into exon 1 of the PR gene. The insertion positions the Cre gene downstream (and under the specific control) of the endogenous PR promoter. As for heterozygotes for the progesterone receptor knockout (PRKO) mutation, mice heterozygous for the Cre knockin insertion are phenotypically indistinguishable from wildtype. Crossing the PR-Cre with the ROSA26R reporter revealed that Cre excision activity is restricted to cells that express PR in progesterone-responsive tissues such as the uterus, ovary, oviduct, pituitary gland, and mammary gland. Initial characterization of the PR-Cre mouse underscores the utility of this model to precisely ablate floxed target genes specifically in cell lineages that express the PR. In the wider context of female reproductive tissue ontology, this model will be indispensable in tracing the developmental fate of cell lineages that descend from PR positive progenitors.     

Plectin-RACK1 (receptor for activated C kinase 1) scaffolding: a novel mechanism to regulate protein kinase C activity

Agonist-induced translocation of protein kinase C (PKC) isozymes is mediated by receptors for the activated form of the kinase, shuttling it from one intracellular site to another and enhancing its catalytic activity. It is however unknown whether the receptors themselves are anchored to certain intracellular structures prior to their engagement with PKC. We show here sequestering of receptor for activated C kinase 1 (RACK1) to the cytoskeleton through the cytoskeletal linker protein plectin during the initial stages of cell adhesion. We found that upon PKC activation, RACK1 was released from the cytoskeleton and transferred to the detergent-soluble cell compartment, where it formed an inducible triple complex with one of the PKC isozymes, PKCdelta, and with plectin. In plectin-deficient cells the cytoskeleton-associated RACK1 fraction was reduced, and the protein was found predominantly at sites to which it normally translocated upon PKC activation. Concomitantly, dislocation of PKCdelta and elevated enzymatic activity were observed in these cells. PKCdelta was also more rapidly degraded, likely due to its overactivation. We propose a previously unrecognized function of plectin as cytoskeletal regulator of PKC signaling, and possibly other signaling events, through sequestration of the scaffolding protein RACK1.     

Extracellular High Mobility Group Box-1 (HMGB1) Inhibits Enterocyte Migration via Activation of Toll-like Receptor-4 and Increased Cell-Matrix Adhesiveness

Toll-like receptor-4 (TLR4) is the receptor for bacterial lipopolysaccharide, yet it may also respond to a variety of endogenous molecules. Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in newborn infants and is characterized by intestinal mucosal destruction and impaired enterocyte migration due to increased TLR4 signaling on enterocytes. The endogenous ligands for TLR4 that lead to impaired enterocyte migration remain unknown. High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. We thus hypothesize that extracellular HMGB1 inhibits enterocyte migration via activation of TLR4 and sought to define the pathways involved. We now demonstrate that murine and human NEC are associated with increased intestinal HMGB1 expression, that serum HMGB1 is increased in murine NEC, and that HMGB1 inhibits enterocyte migration in vitro and in vivo in a TLR4-dependent manner. This finding was unique to enterocytes as HMGB1 enhanced migration of inflammatory cells in vitro and in vivo. In seeking to understand the mechanisms involved, TLR4-dependent HMGB1 signaling increased RhoA activation in enterocytes, increased phosphorylation of focal adhesion kinase, and increased phosphorylation of cofilin, resulting in increased stress fibers and focal adhesions. Using single cell force traction microscopy, the net effect of HMGB1 signaling was a TLR4-dependent increase in cell force adhesion, accounting for the impaired enterocyte migration. These findings demonstrate a novel pathway by which TLR4 activation by HMGB1 delays mucosal repair and suggest a novel potential therapeutic target in the amelioration of intestinal inflammatory diseases like NEC.     

Leishmania infantum chagasi: A genome-based approach to identification of excreted/secreted proteins

The parasitic protozoan, Leishmania, survives in harsh environments within its mammalian and sand fly hosts. Secreted proteins likely play critical roles in the parasite’s interactions with its environment. As a preliminary identification of the spectrum of potential excreted/secreted (ES) proteins of Leishmania infantum chagasi (Lic), a causative agent of visceral leishmaniasis, we used standard algorithms to screen the annotated L. infantum genome for genes whose predicted protein products have an N-terminal signal peptide and lack transmembrane domains and membrane anchors. A suite of 181 candidate ES proteins were identified. These included several that were documented in the literature to be released by other Leishmania spp. Six candidate ES proteins were selected for further validation of their expression and release by different parasite stages. We found both amastigote-specific and promastigote-specific released proteins. The ES proteins of Lic are candidates for future studies of parasite virulence determinants and host protective immunity.     

Novel purification strategy for human PON1 and inhibition of the activity by cephalosporin and aminoglikozide derived antibiotics

Human serum paraoxonase (PON1, EC 3.1.8.1.) is a high-density lipid (HDL)-associated, calcium-dependent enzyme; its physiological substrates are not known. In this study, a new purification strategy for human PON1 enzyme was developed using two-step procedures, namely ammonium sulfate precipitation and sepharose-4B-l-tyrosine-1-napthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43 kDa. Overall purification rate of our method was found 227-fold. The V(max) and K(m) of the purified enzyme were determined 227.27 EU and 4.16 mM, respectively. The in vitro effects of commonly used antibiotics, namely gentamycin sulfate and cefazolin sodium was also investigated on the purified human serum PON1 enzyme and human liver PON1 enzyme from human hepatoma cell (HepG2). Gentamycin sulfate and cefazolin sodium caused a dose- and time-dependent decrease on PON1 activity in HepG2 cells. Moreover, gentamycin sulfate and cefazolin sodium were effective inhibitors on purified human serum PON1 activity with IC(50) of 0.887 and 0.0084 values, respectively. The kinetics of interaction of gentamycin sulfate and cefazolin sodium with the purified human serum PON1 indicated a different inhibition pattern. Cefazolin sodium showed a competitive inhibition with K(i) of 0.012+/-0.00065 mM. However, Gentamycin sulfate was inhibited in non-competitive manner with K(i) of 0.026+/-0.015. In order to determine the inhibition statue of these drugs on a living system, the effects of same antibiotics on PON1 enzyme activity of mouse serum PON1 and liver PON1 were investigated in vivo. Gentamycin sulfate (3.2 mg/kg) and cefazolin sodium (106.25 mg/kg) leads to the significant decrease in mouse serum PON1 after 2, 4, 6 h and 2, 4 h drug administration, respectively. Cefazolin sodium did not exhibit any inhibition effect for the liver PON1, in vivo.