Time-dependent reversal of sepsis-induced PMN uptake and lung vascular injury by expression of CD18 antagonist

We determined the time-dependent effects of conditional expression of neutrophil inhibitory factor (NIF), a specific 41-kDa CD18 integrin antagonist, on the time course of NIF expression and lung PMN (polymorphonuclear leukocyte) infiltration and vascular injury in a model of Escherichia coli-induced sepsis in mice. Studies were made in mice transduced with the E-selectin (ES) promoter-NIF construct (using liposomes) in which the NIF cDNA was driven by the inflammation- and endothelial cell-specific ES promoter. We observed time-dependent expression of NIF in pulmonary vascular endothelium that paralleled the ES expression. Expression of both was evident at 1 h after E. coli challenge, peaked at 3-6 h, and returned to basal level within 48 h. We observed that increases in PMN uptake and transalveolar PMN migration induced by E. coli challenge were reversed in a time-dependent manner following NIF expression in mice. NIF expression also prevented the progression of lung vascular injury and edema formation following E. coli challenge. Thus the conditional expression of NIF using the ES promoter can reverse, in a time-dependent manner, lung PMN infiltration and vascular injury induced by gram-negative sepsis. The results support the model that initial engagement of CD18 integrins enables the further recruitment of additional PMN into lung tissues such that PMN continue to sequester and migrate after E. coli challenge.     

Effect on prolactin secretion of Echinacea purpurea, Hypericum perforatum and Eleutherococcus senticosus

It has been recently reported that prolactin (PRL) plays an important role in immune system regulation. In this study we investigated the activity of three natural drugs with immunomodulatory activity: Echinacea purpurea (EP), Hypericum perforatum (HP) and Eleutherococcus senticosus (ES) on PRL production. Male rats were orally treated with two different doses (30 and 100 mg/kg) of extract of these drugs for 3 or 15 days. A 3-day treatment was not able to modify PRL serum levels, whereas a 15-day treatment with EP and HP at the higher dose significantly inhibits PRL production. A treatment with ES was always ineffective. A possible mechanism for this effect could be that both HP and EP extracts display a direct dopaminergic activity, although an involvement of the GABA-ergic system cannot be excluded.     

Eosinophil differentiation in response to Fasciola hepatica and its excretory/secretory antigens

Bone marrow cells from mice infected with Fasciola hepatica, from mice injected with F. hepatica excretory/secretory (ES) antigens, and from uninfected or uninjected control animals were cultured in the presence of F. hepatica ES antigens or the eosinophil differentiation cytokine IL-5. Eosinophil maturation in cultures was assessed quantitatively by measuring eosinophil peroxidase (EPO) activity and qualitatively by visual appraisal in stained preparations over a week. It was found that the presence in all cultures (including those from control animals) of either ES antigens at an optimal concentration of 100 μml⁻¹ (established in preliminary trials) or IL-5 at 500 units ml⁻¹ led to enhanced EPO activity. EPO activity in cultures without IL-5 or ES antigens remained static or fell over the culture period. At day 3 in all cultures containing IL-5 or ES antigens, there was maintenance of, or only a slight decline in, the number of eosinophils that were present when cultures were initiated, and more of them were mature than at day 0 as evidenced by their EPO activity. However, there was a marked fall in eosinophil numbers in all cultures in the absence of IL-5 or ES antigens. The results indicate that F. hepatica ES antigens, like IL-5, stimulate eosinophil maturation in bone marrow with a consequent rise in EPO activity in the cells. Whether the antigen(s) acts directly or indirectly on the eosinophils or their precursors has yet to be established. Nevertheless, it seems clear that F. hepatica produces a molecule with a functionally similar effect to that of IL-5.     

Forced Expression of the Homeobox-Containing Gene Pem Blocks Differentiation of Embryonic Stem Cells

Similarities in the differentiation of mouse embryos and ES cell embryoid bodies suggest that aspects of early mammalian embryogenesis can be studied in ES cell embryoid bodies. In an effort to understand the regulation of cellular differentiation during early mouse embryogenesis, we altered the expression of the Pem homeobox-containing gene in ES cells. Pem is normally expressed in the preimplantation embryo and expressed in a lineage-restricted fashion following implantation, suggesting a role for Pem in regulating cellular differentiation in the early embryo. Here, we show that the forced expression of Pem from the mouse Pgk-1 promoter in ES cells blocks the in vitro and in vivo differentiation of the cells. In particular, embryoid bodies produced from these Pgk-Pem ES cells do not differentiate into primitive endoderm or embryonic ectoderm, which are prominent features of early embryoid bodies from normal ES cells. This Pgk-Pem phenotype is also different from the null phenotype, as embryoid bodies derived from ES cells in which endogenous Pem gene expression has been blocked show a pattern of differentiation similar to that of normal ES cells. When the Pgk-Pem ES cells were introduced into subcutaneous sites of nude mice, only undifferentiated EC-like cells were found in the teratomas derived from the injected cells. The Pem-dependent block of ES cell differentiation appears to be cell autonomous;Pgk-Pem ES cells did not differentiate when mixed with normal, differentiating ES cells. A block to ES cell differentiation, resulting from the forced expression of Pem, can also be produced by the forced expression of the nonhomeodomain region of Pem. These studies are consistent with a role for Pem in regulating the transition between undifferentiated and differentiated cells of the early mouse embryo.     

A highly purified allergen from excretory and secretory products of Dirofilaria immitis

Preceding data revealed that the allergen concentrated mainly in excretory and secretory (ES) products exhausted by adult Dirofilaria immitis. The present paper reported that a highly purified allergen was obtainable from ES products more easily and effectively. An allergen in ES products was purified by a combination of DEAE-Sephadex A-50 chromatography, Sephadex G-200 and Sephadex G-100 gel filtration. The purified preparation was proved to be one protein by sodium dodecyl sulfate (SDS) gel electrophoresis and to be exact the same allergen with the one obtained from the crude extract of adult Dirofilaria worms. The molecular weight of the purified allergen was estimated to be 15,000, and the allergen was inclined to aggregate in the buffered solution.     

The CD11/CD18 (beta2) integrins modulate neutrophil caspase activation and survival following TNF-alpha or endotoxin induced transendothelial migration

Neutrophils (PMN) are short-lived cells but their survival is often prolonged in inflammation. The beta2 (CD11/CD18) integrins are involved in PMN migration into inflammation but their role in PMN survival is not well understood. We investigated the role of beta2 integrins in PMN caspase activation, a key enzyme cascade in apoptosis. After 20 h, caspase activation (Western blotting) was markedly decreased in PMN cultured on fibrinogen, a ligand for Mac-1 (CD11b/CD18), but not on fibronectin or albumin. In the presence of TNF-alpha or endotoxin (LPS), blockade of CD18 (beta2 chain) with mAb markedly increased caspase activation in PMN on fibrinogen. PMN which migrated through endothelium in vitro in response to TNF-alpha, LPS, IL-1alpha, IL-8 or C5a contained 58% fewer active caspase positive PMN after 20 h than non-migrated PMN remaining on the endothelium. When beta2 (CD18) integrin or lymphocyte function antigen (LFA)-1 (CD11a) plus Mac1 (CD11b) were blocked by mAb (intact or Fab'), the proportion of migrated PMN (but not of non-migrated PMN) with active caspases was significantly increased (2-4-fold) and this was associated with accelerated PMN apoptosis and death. Thus, engagement of ligands on extracellular matrix and endothelium by the beta2 integrins Mac-1 and LFA-1 plays a role in delaying apoptosis in PMN recruited in response to LPS and TNF-alpha. Inhibition of beta2 integrin function may not only inhibit PMN infiltration, but also accelerate PMN clearance from inflamed tissue.     

Degradation of low rank coal by Trichoderma atroviride ES11

A new isolate of Trichoderma atroviride has been shown to grow on low rank coal as the sole carbon source. T. atroviride ES11 degrades ∼82% of particulate coal (10 g l⁻¹) over a period of 21 days with 50% reduction in 6 days. Glucose (5 g l⁻¹) as a supplemented carbon source enhanced the coal solubilisation efficiency of T. atroviride ES11, while 10 and 20 g l⁻¹ glucose decrease coal solubilisation efficiency. Addition of nitrogen [1 g l⁻¹ (NH₄)₂SO₄] to the medium also increased the coal solubilisation efficiency of T. atroviride ES11. Assay results from coal-free and coal-supplemented cultures suggested that several intracellular enzymes are possibly involved in coal depolymerisation processes some of which are constitutive (phenol hydroxylase) and others that were activated or induced in the presence of coal (2,3-dihydrobiphenyl-2,3-diol dehydrogenase, 3,4-dihydro phenanthrene-3,4-diol dehydrogenase, 1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase, 1,2-dihydro-1,2-dihydroxyanthracene dehydrogenase). GC-MS analysis of chloroform extracts obtained from coal degrading T. atroviride ES11 cultures showed the formation of only a limited number of specific compounds (4-hydroxyphenylethanol, 1,2-benzenediol, 2-octenoic acid), strongly suggesting that the intimate association between coal particles and fungal mycelia results in rapid and near-quantitative transfer of coal depolymerisation products into the cell. An erratum to this article can be found at     

Ostertagia ostertagi: Isolation and partial characterization of somatic and metabolic antigens

One metabolic (ES) and two somatic extracts (AS and MS) were prepared from Ostertagia ostertagi. Partial characterization of the three preparations was accomplished by sodium dodecyi sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot techniques. In immunoblots, AS showed a low number of bands that reacted weakly with sera collected from infected calves. MS reacted strongly with the homologous sera, and a sharp group of bands appeared from 12 to 14 kDa, increasing in intensity as the infection progressed. ES showed a group of strongly immunogenic bands in the range of 16–22 kDa. The three preparations were also tested for reactivity with specific antt-Ostertagia antibodies in an enzyme-linked immunosorbant assay (ELISA). The ELISA results showed that all three worm extracts contained antigen epitopes recognized by circulating antibody in sera taken from O. ostertagi-infected calves. The strongest response was seen when antibodies of the IgGl isotype were reacted with MS and ES. When sera taken from O. radiatum-infected calves were used both somatic extracts showed high levels of cross-reactivity.     

In vitro differentiation of mouse ES cells into hepatocytes with coagulation factors VIII and IX expression profiles

Coagulation factors II, V, VII, VIII, IX and X are produced by hepatocytes. So factors VIII and IX deficiencies, which result in hemophilia A and B, have the potential to respond to cellular replacement therapy. Embryonic stem (ES) cells provide a unique source for therapeutic applications. Here, E14 mouse ES cells have been induced into hepatocytes in vitro. Morphology revealed that ES-derived hepatic-like cells were round or polyhedral shaped with distinct boundary of individual cells, and some arranged in trabeculae. These cells expressed endodermal-or liver-specific mRNA—transthyretin (TTR), α1-anti-trypsin (AAT), α-fetoprotein (AFP), albumin (ALB), glucose-6-phoshpatase (G6P) and tyrosine aminotransferase (TAT). Approximately (85.1±0.5)% of the ES-derived cells was stained positive green with ICG uptake. These cells were also stained magenta as a result of PAS reaction. In this paper, expression of coagulation factors VIII and IX mRNA in the ES-derived cells is documented. Therefore, ES cells might be developed as substitute donor cells for the therapy of coagulation factor deficiencies.     

The genetic analysis of F1 hybrid larvae between female Trichinella spiralis and male Trichinella britovi

Hybrids between female Trichinella spiralis and male Trichinella britovi were constructed. Then, hybrid genotype was characterized by DNA markers including mitochondrial cytochrome c oxidase subunit I (CO I) gene, the gene encoding the 43-kDa excretory–secretory (ES) protein, and genomic DNA fragments specific for T. spiralis and T. britovi identified from random amplified polymorphism DNA (RAPD). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial CO I gene revealed that all hybrids carried a T. spiralis pattern. The same analysis of the gene encoding the 43-kDa ES protein showed that each hybrid carried both T. spiralis and T. britovi gene type simultaneously. In the analysis of genomic DNA using RAPD-derived PCR primers, some hybrids carried T. spiralis and T. britovi-specific RAPD markers, while others carried the RAPD marker of T. spiralis only.     

Effect of mechanical deformation of neutrophils on their CD18/ICAM-1-dependent adhesion.

Mechanical deformation of polymorphonuclear leukocytes (PMN) changes their expression of the surface adhesion molecule CD11b/CD18. We tested the hypothesis that mechanical deformation of PMN enhances their adhesiveness. Purified human PMN were deformed through either 5- or 3-microm polycarbonate membrane filters and allowed to adhere to 96-well plates coated with human recombinant intercellular adhesion molecule-1 (ICAM-1). Flow cytometric studies showed that deformation of PMN increased CD11b/CD18 expression (P < 0.01). PMN adhesion to ICAM-1-coated plates was dependent on the magnitude of cell deformation (5 microm, 63.8 +/- 8.1%, P < 0.04; 3 microm, 232.4 +/- 20.9%, P < 0.01). Priming of PMN (0.5 nM N-formyl-methionyl-leucyl-phenylalanine) before deformation (5 microm) increased PMN adhesion (63.8 +/- 8.1 vs. 105.3 +/- 16.4%; P < 0.04). Stimulation (5% zymosan-activated plasma) of PMN after deformation resulted in increased adhesion, and the degree of increase was dependent on the magnitude of PMN deformation (stimulation, 50.6 +/- 4%; 5-microm filtration and stimulation, 62.9 +/- 6.6%; 3-microm filtration and stimulation, 249.9 +/- 24.2%; P < 0.01). This study shows that mechanical deformation of PMN causes an increase in PMN adhesiveness to ICAM-1 that was enhanced by both priming of PMN before deformation and stimulation after cell deformation.     

Human neutrophil adherence to thrombospondin occurs through a CD11/CD18-independent mechanism.

Thrombospondin (TSP), a 450-kDa trimeric glycoprotein secreted by platelets and endothelial cells at sites of tissue injury or inflammation, may play an important role in polymorphonuclear leukocyte (PMN) adherence to blood vessel walls before diapedesis. We have examined the adherence of PMN to TSP and compared it to adherence to other extracellular matrix proteins. PMN adherence to TSP-coated plastic was complete by 60 min with spreading completed by 2 h. The kinetics of adhesion and spreading on TSP were similar to that of vitronectin (VN), laminin (LN), and fibronectin (FN). Activation of PMN with the calcium ionophore A23187 or the chemotactic peptide FMLP increased PMN adherence to LN and FN, but not to TSP or VN, suggesting that PMN activation may differentially regulate expression of TSP and VN receptors as compared to LN and FN receptors. The specificity of PMN adherence to TSP was confirmed by competition with saturating amounts of TSP and inhibition with anti-TSP antibodies. mAb A6.1, which binds to the protease-resistant core of TSP, was the most effective in blocking PMN adherence to TSP. Using TSP proteolytic fragments, we demonstrated that the primary interaction of PMN with TSP was mediated through the 140-kDa COOH-terminal domain. Inasmuch as the 140-kDa fragment of TSP contains an Arg-Gly-Asp sequence similar to the cell recognition site of FN and VN, we determined whether RGDS peptides would inhibit PMN adhesion. RGDS did not significantly inhibit PMN adhesion to TSP, VN, or LN, but reduced PMN adhesion to FN by 50%. To determine if PMN adhesion to TSP was mediated by a beta 2 integrin receptor such as LFA-1, MO-1, or p150,95, we performed adhesion assays using PMN isolated from patients with leukocyte adhesion deficiency that lack beta 2 receptors. Leukocyte adhesion deficiency PMN exhibited normal adherence to TSP. In contrast, adherence to VN, LN, and FN was reduced by 95%. Therefore, adherence to TSP is probably not mediated by a beta 2 integrin receptor. These data contribute to the accumulating evidence that PMN can interact with extracellular matrix proteins through a CD11/CD18-independent process.     

Reduced ligand affinity leads to an impaired function of the adenosine A2A receptor of human granulocytes in sepsis

The enhanced release of reactive oxygen species by excessively activated polymorphonuclear leucocytes (PMN) is a key step in the pathogenesis of sepsis. Potent action of adenosine in inhibiting cytotoxic PMN functions has been documented. Recent data, however provide evidence that in sepsis a diminished capability of adenosine to inhibit the generation of oxygen radicals by PMN occurs. Here, we investigated the underlying mechanisms in an in vitro sepsis model and in PMN of sepsis patients. We report that lipopolysaccharide (LPS)-incubation of human PMN elicited the same increase in the half-maximal inhibitory concentration (IC₅₀) of adenosine as observed in patients with septic shock. Coupling to adenylyl cyclase was impaired as well, as indicated by a decreased potency of adenosine to stimulate cyclic adenosine monophosphate (cAMP) accumulation. Ligand-binding studies conducted with native, LPS-stimulated PMN, and with PMN of sepsis patients revealed that, despite an increased adenosine A_2A receptor (A_2AR) expression, the receptor function declines due to a diminished ligand-binding affinity most likely caused by allosteric modulators within the inflammatory environment. A_2AR function obviously is highly dependent upon the cellular environment and thus, further functional characterization of A_2AR responses in sepsis may be a promising approach to develop new adenosine or A_2AR agonists based therapeutic strategies.     

Cytokine-induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins and CD11/CD18 integrins

Human polymorphonuclear leukocytes (PMN) released large quantities of hydrogen peroxide in response to tumor necrosis factor, but only when the cells were adherent to surfaces coated with extracellular matrix proteins. The PMN did not respond when exposed to cytokines and matrix proteins in suspension, or when exposed to cytokines while adherent to surfaces coated with stearic acid. PMN from children with genetic deficiency of the CD11/CD18 integrins underwent a normal respiratory burst upon adherence to uncoated polystyrene, but not in response to tumor necrosis factor when tested on polystyrene that was coated with serum, fibronectin, vitronectin, fibrinogen, thrombospondin, or laminin. Anti-CD18 antibodies, alone of sixteen antibodies tested, induced a similar defect in PMN from normal donors, when the PMN were tested on surfaces coated with serum, fibrinogen, thrombospondin, or laminin; no defect was induced by the anti-CD18 monoclonal antibody IB4 in normal PMN tested on surfaces coated with fibronectin or vitronectin. Thus, for cytokines to induce a respiratory burst in PMN, the cells must be able to use CD11/CD18 integrins and must interact with matrix proteins in the solid phase. CD11/CD18, which is already known to serve as a receptor for fibrinogen, may also be a receptor for thrombospondin and laminin. Finally, receptor(s) exist on PMN for fibronectin and vitronectin which are not blocked by the anti-CD18 antibody IB4 but which are nonetheless CD11/CD18 dependent.     

Detection of anti-oxidant enzymatic activities and purification of glutathione transferases from Angiostrongylus cantonensis

There are several anti-oxidant enzyme families that play pivotal roles in facilitating the survival of parasites. Glutathione transferases (GSTs) are members of the anti-oxidant family that can detoxify a broad range of exogenous or endogenous compounds including reactive oxidative species. GSTs have been studied as vaccine candidates, immunodiagnostic markers and as treatment targets. Helminths of the genus Angiostrongylus live inside arteries of vertebrates and two main species are associated with accidental human infections: Angiostrongylus costaricensis adult worms live inside the mesenteric arteries and larvae of Angiostrongylus cantonensis become trapped in the central nervous system vasculature. Since the interactions between angiostrongylid nematodes and their vertebrate hosts are poorly understood, this study characterized the anti-oxidant enzymatic activities of A. cantonensis from female worms by collecting excreted and secreted (ES) and total extract (TE) molecules. Catalase (CAT) and superoxide dismutase (SOD) activities were found both in the ES and TE while glutathione peroxidase (GPX) and GST were found only in the TE. GSTs were purified by glutathione agarose affinity column (AcGST) and the pool of eluted GSTs was analyzed by mass spectrometry (LC-MS/MS) and de novo sequencing (Masslynx software). Sequences from two peptides (AcGSTpep1 and AcGSTpep2) present high identity to the N-terminal and C-terminal from sigma class GSTs of nematodes. It is known that these GST enzymes are associated with host immune regulation. Furthermore, understanding the role of parasite-derived anti-oxidant molecules is important in understanding host-parasite interactions.     

Combined activin A/LiCl/Noggin treatment improves production of mouse embryonic stem cell-derived definitive endoderm cells

Induction of definitive endoderm (DE) cells is a prerequisite for the whole process of embryonic stem (ES) cells differentiating into hepatic or pancreatic progenitor cells. We have established an efficient method to induce mouse ES cell-derived DE cells in suspension embryonic body (EB) culture. Similar to previous studies, mouse ES cell-derived DE cells, which were defined as Cxcr4(+) c-Kit(+) , Cxcr4(+) E-cadherin(+) cells or Cxcr4(+) PDGFRa(-) cells, could be induced in the serum-free EBs at Day 4 of induction. The activations of Wnt, Nodal, and FGF signaling pathways in differentiating EBs promoted DE cell differentiation, while activation of BMP4 signaling inhibited the process. In the present study, we found that chemical activation of canonical Wnt signaling pathway by LiCl could synergize with Activin A-mediated Nodal signaling pathway to promote induction of DE cells, and inhibition of Bmp4 signaling by Noggin along with Activin A/LiCl further improved the efficiency of DE cell differentiation. The derived DE cells were proved for their capacities to become hepatic progenitor cells or pancreatic progenitor cells. In conclusion, we significantly improved the efficiency of generating mouse ES cell-derived DE cells by combined Activin A/LiCl/Noggin treatment. Our work will be greatly helpful to generate ES cell-derived hepatic cells and ES cell-derived pancreatic cells for future regenerative medicine.     

Sulfite induces adherence of polymorphonuclear neutrophils to immobilized fibrinogen through activation of Mac-1 beta2-integrin (CD11b/CD18)

Sulfite is a major air pollutant which can cause respiratory tract inflammation characterized by an influx of polymorphonuclear neutrophils (PMN). We have previously shown that human PMN can produce sulfite either spontaneously or in response to stimulation with lipopolysaccharide. We now demonstrate that sulfite activates PMN to adhere to immobilized fibrinogen via the beta2-integrin Mac-1 (CD11b/CD18). Mac-1 expression is not altered by treatment with this agent. Although unaffected by pertussis toxin, sulfite-triggered PMN adhesion was abrogated by pretreating cells with the membrane-impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a modifier of thiol groups on the cell surface. These results suggest that sulfite-induced PMN adhesion is dependent on a modification of thiols at the cell surface. Given its potent antioxidant and antimicrobial activities, sulfite may act as an endogenous mediator in host defense and/or inflammation.     

Mac-1 (CD11b/CD18) as accessory molecule for Fc alpha R (CD89) binding of IgA

IgA, the principal ligand for FcalphaRI, exists in serum as monomeric IgA and at mucosal sites as secretory IgA (SIgA). SIgA consists of dimeric IgA linked by joining chain and secretory components. Human polymorphonuclear leukocytes (PMN) and mouse PMN transgenic for human FcalphaRI exhibited spreading and elicited respiratory burst activity upon interaction with either serum or SIgA. However, PMN devoid of the beta(2) integrin Mac-1 (Mac-1(-/-)) were unable to bind SIgA, despite expression of FcalphaRI. Consistent with this, serum IgA stimulated Mac-1(-/-) PMN oxygen radical production, in contrast to SIgA. Binding studies showed the secretory component, by itself, to interact with Mac-1-expressing PMN, but not with Mac-1(-/-) PMN. These data demonstrate an essential role for Mac-1 in establishing SIgA-FcalphaRI interactions.