High level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin

To characterize the structural and functional properties of viral interleukin 10 (vIL-10), its cDNA was cloned into the bacterial expression vector pMAL-c2, which directs the synthesis of the inserted gene as a fusion protein with maltose binding protein (MBP). The MBP-vIL-10 fusion protein was expressed in Escherichia coli and purified from cell lysates using amylose resin chromatography. Viral interleukin 10 (IL-10) was released from the fusion protein by cleavage with the proteolytic enzyme factor Xa. We show that vIL-10 will bind to heparin and use this property to purify vIL-10 from factor Xa cleaved products and trace contaminants using heparin agarose chromatography. A simple one-step procedure is described for the removal of endotoxins from heavily contaminated vIL-10 preparations. The protocol exploits the high binding affinity of MBP for amylose resin or vIL-10 for heparin and the ability of Triton-X114 to dissociate endotoxins from proteins. The biological activity of purified vIL-10 was demonstrated through its ability to inhibit interferon gamma (IFN-gamma) production by mitogen activated peripheral blood mononuclear cells and to down-regulate HLA-class II expression on activated monocytes/macrophages. The availability of an efficient expression and purification strategy for vIL-10 together with appropriate assays will contribute to a greater understanding of how vIL-10 has evolved to retain and modify those activities of cellular IL-10 best suited for Epstein-Barr virus (EBV)'s specialized niche within the host.     

Construction, Expression, and Characterization of a Baculovirally Expressed Catalytic Domain of Human Matrix Metalloproteinase-9

We report DNA construction, baculovirus expression, and partial characterization of a minienzyme form of the human matrix metalloproteinase-9 (MMP-9). The MMP-9 minienzyme gene construct consisting of the pre, pro, and catalytic domains of the MMP-9 was introduced into Sf9 insect cells using a baculovirus expression system. The expression of the recombinant MMP-9 minienzyme was estimated to be approximately 0.8 mg/L of cell medium. The recombinant protein was purified using a single-step gelatin-Sepharose affinity column and yielded a highly stable and active minienzyme with gelatinolytic activity. Moreover, two interesting findings related to MMP-9 interactions with heparin and TIMP-1 resulted from our studies. First, the pro and catalytic domains of the human MMP-9 are not sufficient for heparin affinity. Second, in contrast to the prevailing consensus, TIMP-1 blockade of the enzymatic activity of MMP-9 does not require prior binding to the C-terminus of its MMP-9 protein substrate.     

Trace Element Level in Different Tissues of <Emphasis Type="Italic">Rutilus frisii kutum</Emphasis> Collected from Tajan River,Iran

Tajan River is among the most significant rivers of the Caspian Sea water basin. In this study, the concentration of Cr, Cu, Fe, Mn, Ni, Pb, Cd, and Zn were determined in brain, heart, liver, gill, bile, and muscle of Rutilus frisii kutum which has great economic value in the Mazandaran state. Trace element levels in fish samples were analyzed by means of atomic absorption spectrometry. Nearly all non-essential metals levels (Ni, Pb, Cd) detected in tissues were higher than limits for fish proposed by FAO/WHO, EU, and TFC. Generally, non-essential metals (Ni, Pb) were so much higher in muscle than the essential metals (Cu, Zn, and Mn) except Fe, which was higher than other metals in nearly all parts, except in gills. Fe distribution pattern in tissues was in order of heart > brain > liver > muscle > bile > gill. Distribution patterns of metal concentrations in the muscle of fish as a main edible part followed the sequence: Fe > Pb > Ni > Cu > Mn > Zn > Cd.     

The Novel Secreted Adipokine WNT1-inducible Signaling Pathway Protein 2 (WISP2) Is a Mesenchymal Cell Activator of Canonical WNT

WNT1-inducible-signaling pathway protein 2 (WISP2) is primarily expressed in mesenchymal stem cells, fibroblasts, and adipogenic precursor cells. It is both a secreted and cytosolic protein, the latter regulating precursor cell adipogenic commitment and PPARγ induction by BMP4. To examine the effect of the secreted protein, we expressed a full-length and a truncated, non-secreted WISP2 in NIH3T3 fibroblasts. Secreted, but not truncated WISP2 activated the canonical WNT pathway with increased β-catenin levels, its nuclear targeting phosphorylation, and LRP5/6 phosphorylation. It also inhibited Pparg activation and the effect of secreted WISP2 was reversed by the WNT antagonist DICKKOPF-1. Differentiated 3T3-L1 adipose cells were also target cells where extracellular WISP2 activated the canonical WNT pathway, inhibited Pparg and associated adipose genes and, similar to WNT3a, promoted partial dedifferentiation of the cells and the induction of a myofibroblast phenotype with activation of markers of fibrosis. Thus, WISP2 exerts dual actions in mesenchymal precursor cells; secreted WISP2 activates canonical WNT and maintains the cells in an undifferentiated state, whereas cytosolic WISP2 regulates adipogenic commitment.     

Radioprotective effects of hawthorn against genotoxicity induced by gamma irradiation in human blood lymphocytes

The radioprotective effect of hawthorn (Crataegus microphylla) fruit extract was investigated in cultured blood lymphocytes from human volunteers. Peripheral blood samples were collected from five human volunteers 10 min before and 1, 2 and 3 h after a single oral ingestion of 500 mg hawthorn powder extract. At each time point, the whole blood was exposed in vitro to 150 cGy of cobalt-60 gamma irradiation, and then the lymphocytes were cultured with mitogenic stimulation to determine the micronuclei in cytokinesis-blocked binucleated cell. The lymphocytes in the blood samples collected after extract ingestion exhibited a significant decrease in the incidence of binucleated cells containing micronuclei as compared to similarly irradiated lymphocytes collected prior to extract ingestion. The maximum decrease in the frequency of micronuclei-containing cells was observed at 1 h after ingestion of Hawthorn extract (on average a 44% decrease). These data suggest that it may be possible to use Hawthorn extracts in personnel exposed to radiation in order to protect lymphocytes from radiation effects.     

A Novel Probiotic Mixture Exerts a Therapeutic Effect on Experimental Autoimmune Encephalomyelitis Mediated by IL-10 Producing Regulatory T Cells

Background

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). One potential therapeutic strategy for MS is to induce regulatory cells that mediate immunological tolerance. Probiotics, including lactobacilli, are known to induce immunomodulatory activity with promising effects in inflammatory diseases. We tested the potential of various strains of lactobacilli for suppression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS.

Methodology/Principal Findings

The preventive effects of five daily-administered strains of lactobacilli were investigated in mice developing EAE. After a primary screening, three Lactobacillus strains, L. paracasei DSM 13434, L. plantarum DSM 15312 and DSM 15313 that reduced inflammation in CNS and autoreactive T cell responses were chosen. L. paracasei and L. plantarum DSM 15312 induced CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) and enhanced production of serum TGF-β1, while L. plantarum DSM 15313 increased serum IL-27 levels. Further screening of the chosen strains showed that each monostrain probiotic failed to be therapeutic in diseased mice, while a mixture of the three lactobacilli strains suppressed the progression and reversed the clinical and histological signs of EAE. The suppressive activity correlated with attenuation of pro-inflammatory Th1 and Th17 cytokines followed by IL-10 induction in MLNs, spleen and blood. Additional adoptive transfer studies demonstrated that IL-10 producing CD4⁺CD25⁺ Tregs are involved in the suppressive effect induced by the lactobacilli mixture.

Conclusions/Significance

Our data provide evidence showing that the therapeutic effect of the chosen mixture of probiotic lactobacilli was associated with induction of transferable tolerogenic Tregs in MLNs, but also in the periphery and the CNS, mediated through an IL-10-dependent mechanism. Our findings indicate a therapeutic potential of oral administration of a combination of probiotics and provide a more complete understanding of the host-commensal interactions that contribute to beneficial effects in autoimmune diseases.     

Cholestasis induced nephrotoxicity: The role of endogenous opioids

AimsElevated levels of endogenous opioids play a pivotal role in several deleterious consequences of cholestasis. Renal dysfunction occurs in cholestasis but its exact mechanism is still unknown. In this study, we investigated the role of endogenous opioids in cholestasis induced nephrotoxicity.Main methodsThirty-five rats were divided into five groups. In groups 1 and 2 BDL rats received either daily subcutaneous 20 mg/kg of naltrexone or its vehicle, for 7 days after BDL. In groups 3 and 4, BDL or Sham rats received no injections. In group 5, normal rats received subcutaneous injections of 20 mg/kg/day of naltrexone for 7 days. At the 7th day, 24 h urine was collected to measure urinary N-acetyl-β-D-glucosaminidase (NAG) as an early marker of renal tubular injury. Kidney samples were then collected for light and electron microscopic studies.Key findingsBDL significantly increased NAG activity compared to sham groups. Naltrexone significantly reversed NAG activity to normal levels in BDL animals. Naltrexone treatment in BDL animals also significantly reversed ALT and AST to their normal levels. In light and electron microscopic studies, there were significant structural alterations in BDL samples, which were mostly prevented in naltrexone treated BDL animals.SignificanceSignificant changes in urinary NAG activity and renal morphology of cholestatic rats were reversed by naltrexone treatment. These results suggest a possible role for endogenous opioids in inducing cholestatic nephrotoxicity.     

Effects of epidural lidocaine analgesia on labor and delivery: A randomized, prospective, controlled trial

Background

Epidural anaesthesia is used extensively for cardiothoracic and vascular surgery in some centres, but not in others, with argument over the safety of the technique in patients who are usually extensively anticoagulated before, during, and after surgery. The principle concern is bleeding in the epidural space, leading to transient or persistent neurological problems.

Methods

We performed an extensive systematic review to find published cohorts of use of epidural catheters during vascular, cardiac, and thoracic surgery, using electronic searching, hand searching, and reference lists of retrieved articles.

Results

Twelve studies included 14,105 patients, of whom 5,026 (36%) had vascular surgery, 4,971 (35%) cardiac surgery, and 4,108 (29%) thoracic surgery. There were no cases of epidural haematoma, giving maximum risks following epidural anaesthesia in cardiac, thoracic, and vascular surgery of 1 in 1,700, 1 in 1,400 and 1 in 1,700 respectively. In all these surgery types combined the maximum expected rate would be 1 in 4,700. In all these patients combined there were eight cases of transient neurological injury, a rate of 1 in 1,700 (95% confidence interval 1 in 3,300 to 1 in 850). There were no cases of persistent neurological injury (maximum expected rate 1 in 4,600).

Conclusion

These estimates for cardiothoracic epidural anaesthesia should be the worst case. Limitations are inadequate denominators for different types of surgery in anticoagulated cardiothoracic or vascular patients more at risk of bleeding.     

Giardia duodenalis: The double-edged sword of immune responses in giardiasis

Giardiasis is one of the most common intestinal protozoan infections worldwide. The etiological agent, Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis), is a flagellated, binucleated protozoan parasite which infects a wide array of mammalian hosts (Adam, 2001). The symptoms of giardiasis include abdominal cramps, nausea, and acute or chronic diarrhea, with malabsorption and failure of children to thrive occurring in both sub-clinical and symptomatic disease (Thompson et al., 1993). Infections are transmitted by cysts which are excreted in the feces of infected humans and animals. Human giardiasis is distributed worldwide, with rates of detection between 2-5% in the developed world and 20-30% in the developing nations (Farthing, 1994). There is significant variation in the outcome of Giardia infections. Most infections are self-limiting, although re-infection is common in endemic areas and chronic infections also occur. Moreover, some individuals suffer from severe cramps, nausea and diarrhea while others escape these overt symptoms. This review will describe recent advances in parasite genetics and host immunity that are helping to shed light on this variability.     

Separation of bacteria smaller than 4 µm from other blood components using insulator-based dielectrophoresis: numerical simulation approach

Biomechanics and Modeling in Mechanobiology - Bloodstream infection (BSI) is a life-threatening infection that causes more than 80,000 deaths and more than 500,000 infections annually in North...