Nitric oxide controls proliferation of Leishmania major by inhibiting the recruitment of permissive host cells.

Nitric oxide (NO) is an important antimicrobial effector but also prevents unnecessary tissue damage by shutting down the recruitment of monocyte-derived phagocytes. Intracellular pathogens such as Leishmania major can hijack these cells as a niche for replication. Thus, NO might exert containment by restricting the availability of the cellular niche required for efficient pathogen proliferation. However, such indirect modes of action remain to be established. By combining mathematical modeling with intravital 2-photon biosensors of pathogen viability and proliferation, we show that low L. major proliferation results not from direct NO impact on the pathogen but from reduced availability of proliferation-permissive host cells. Although inhibiting NO production increases recruitment of these cells, and thus pathogen proliferation, blocking cell recruitment uncouples the NO effect from pathogen proliferation. Therefore, NO fulfills two distinct functions for L. major containment: permitting direct killing and restricting the supply of proliferation-permissive host cells.

Natural particles can armor emulsions against lipid oxidation and coalescence.

Traditional functional ingredients, such as conventional emulsifiers (surfactants, animal-derived proteins), and synthetic antioxidants may become obsolete in the development of clean-label, plant-based, sustainable food emulsions. Previously, we showed that tailor-made antioxidant-loaded particles can yield both physically and oxidatively stable emulsions, and we expected that natural particles with related properties could also show these beneficial effects. Here, we investigated Pickering emulsions prepared with natural plant particulate materials. Particles that showed weak aggregation in acidic aqueous media, indicating a relatively hydrophobic surface, were able to physically stabilize oil-in-water emulsions, through either Pickering stabilization (powders of matcha tea, spinach leaves, and spirulina cake), or an increase in viscosity (pineapple fibers). Matcha tea and spinach leaf particle-stabilized emulsions were highly stable to lipid oxidation, as compared to emulsions stabilized by conventional emulsifiers. Taking this dual particle functionality as a starting point for emulsion design is, in our view, essential to achieve clean-label food emulsions.

Percutaneous transhepatic biliary drainage (PTBD) in patients with dilated vs. nondilated bile ducts: technical considerations and complications.

OBJECTIVES: The aim of this study was to compare success, technical complexity, and complication rates of percutaneous transhepatic biliary drainage (PTBD) in patients with dilated vs. nondilated bile ducts. METHODS: In a retrospective analysis, we evaluated all consecutive PTBD performed in our department over a period of 5 years. Technical success, technical data (side, fluoroscopy time, radiation dose, amount of contrast media, use of disposable equipment), procedure-related complications and peri-interventional mortality were compared for patients with dilated vs. non-dilated bile ducts. Independent t test and χ2 test were used to evaluate the statistical significance. RESULTS: A total of 253 procedures were performed on 187 patients, of whom 101/253 had dilated bile ducts and 152/253 not. In total, 243/253 procedures were successful. PTBD was significantly more often successful in patients with dilated vs. nondilated bile ducts (150/153 vs. 93/101; p 0.02). Overall complication rate (13%) did not differ significantly between patients with dilated vs. nondilated bile ducts. Procedures in patients with normal, nondilated bile ducts were associated with a significantly higher rate of post-interventional bleeding (5/101 vs. 0/152). Mean fluoroscopy time (42:36 ± 35:39 h vs. 30:28 ± 25:10 h; p 0.002) and amount of contrast media (66 ± 40 ml vs. 52 ± 24 ml; p 0.07) or use of disposables were significantly higher in patients with nondilated ducts. A significantly lower fluoroscopy time and amount of contrast medium were used in left hepatic PTBD. CONCLUSION: Despite the higher technical complexity, PTBD with nondilated bile ducts was associated with similar overall complication rates but higher bleeding complications compared with PTBD with dilated bile ducts. KEY POINTS: • PTBD was associated with similar overall complication rates in patients with dilated vs. nondilated bile ducts. • Although overall complication rates were low, PTBD in patients with nondilated bile ducts was associated with a higher incidence of post-interventional bleeding. • PTBD in patients with nondilated bile ducts is technically more complex.

Pickering particles as interfacial reservoirs of antioxidants.

HYPOTHESIS: Emulsions are common structures encapsulating lipophilic bioactive molecules, both in biological systems and in manufactured products. Protecting these functional molecules from oxidation is essential; Nature excels at doing so by placing antioxidants at the oil-water interface, where oxidative reactions primarily occur. We imagined a novel approach to boost the activity of antioxidants in designer emulsions by employing Pickering particles that act both as physical emulsion stabilizers and as interfacial reservoirs of antioxidants. EXPERIMENTS: α-Tocopherol or carnosic acid, two model lipophilic antioxidants, were entrapped in colloidal lipid particles (CLPs) that were next used to physically stabilize sunflower oil-in-water emulsions ("concept" Pickering emulsions). We first assessed the physical properties and stability of the CLPs and of the Pickering emulsions. We then monitored the oxidative stability of the concept emulsions upon incubation, and compared it to that of control emulsions of similar structure, yet with the antioxidant present in the oil droplet interior. FINDINGS: Both tested antioxidants are largely more effective when loaded within Pickering particles than when solubilized in the oil droplet interior, thus confirming the importance of the interfacial localization of antioxidants. This approach revisits the paradigm for lipid oxidation prevention in emulsions and offers potential for many applications.

Can we prevent lipid oxidation in emulsions by using fat-based Pickering particles?

Interest has recently been rising in the development of food-compatible Pickering emulsions, i.e., particle-stabilized emulsions, and various biobased particles have been demonstrated as useful for such a purpose. Most of the related work has focused on the physical stability of the emulsions, but whether such particles can be advantageous in terms of chemical stability, and in particular, with regard to lipid oxidation, is largely unexplored. Recently, we found that colloidal lipid particles (CLPs) are efficient Pickering stabilizers, and the objective of the present study was to investigate the oxidative stability of emulsions stabilized with those particles. Three types of sunflower oil-in-water (O/W) emulsions were considered: Pickering emulsions stabilized with colloidal lipid particles (CLPs) made of high melting point (HMP) fat (tripalmitin or palm stearin), adsorbed onto the liquid oil droplets; and, as references, two conventional sodium caseinate-stabilized emulsions, of which one contained only liquid oil, and the other liquid oil mixed with HMP fat as the core of the emulsion droplets. In the presence of iron, the latter oxidized faster than conventional liquid oil and Pickering emulsions, resulting in 2- to 3-fold higher amounts of primary and secondary lipid oxidation products. This may be due to intra-droplet HMP fat pushing oxidizable lipids towards the oil-water interface, which would promote lipid oxidation. This shows that the localization of solid fat in O/W emulsions affects lipid oxidation. We also found that CLP-stabilized Pickering emulsions had similar oxidation rates as conventional sodium caseinate-stabilized emulsions containing only liquid oil. This suggests that the potential of such Pickering particles to prevent lipid oxidation is limited. This could be because diffusion of small pro-oxidant molecules is not hindered by Pickering particles, as they cannot form an interfacial barrier that is structurally homogeneous at such a small scale.

Tailored microstructure of colloidal lipid particles for Pickering emulsions with tunable properties.

Sub-micron colloidal lipid particles (CLPs) can successfully be used as Pickering stabilizers in oil-in-water (O/W) emulsions, leading to an enhanced physical stability compared to conventional emulsifier-stabilized emulsions. Varying the lipid solid-liquid ratio leads to particles with distinct nanostructure and morphology, resulting in tunable emulsion stabilization performance. Our CLPs are produced by hot high pressure homogenization of high melting point fats in water, and subsequent cooling to induce lipid crystallization. Lath-like tripalmitin and palm stearin CLPs form jammed, cohesive interfacial layers that prevent relaxation of emulsion droplets, and form a three-dimensional network in the continuous aqueous phase. CLPs consisting of a mixture of solid tripalmitin and liquid tricaprylin are polycrystalline platelet-like particles that form O/W emulsions with spherical and bridged droplets covered by a thin particle layer. Our results present a versatile approach to interfacial design that also opens up new perspectives for development of novel delivery systems for active ingredients.

Early type II fiber atrophy in intensive care unit patients with nonexcitable muscle membrane.

OBJECTIVE: Intensive care unit-acquired weakness indicates increased morbidity and mortality. Nonexcitable muscle membrane after direct muscle stimulation develops early and predicts intensive care unit-acquired weakness in sedated, mechanically ventilated patients. A comparison of muscle histology at an early stage in intensive care unit-acquired weakness has not been done. We investigated whether nonexcitable muscle membrane indicates fast-twitch myofiber atrophy during the early course of critical illness. DESIGN: Prospective observational study. SETTING: Two intensive care units at Charité University Medicine, Berlin. PATIENTS: Patients at increased risk for development of intensive care unit-acquired weakness, indicated by Sepsis-related Organ Failure Assessment scores ≥8 on 3 of 5 consecutive days within their first week in the intensive care unit. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Electrophysiological compound muscle action potentials after direct muscle stimulation and muscle biopsies were obtained at median days 7 and 5, respectively. Patients with nonexcitable muscle membranes (n = 15) showed smaller median type II cross-sectional areas (p < .05), whereas type I muscle fibers did not compared with patients with preserved muscle membrane excitability (compound muscle action potentials after direct muscle stimulation ≥3.0 mV; n = 9). We also observed decreased mRNA transcription levels of myosin heavy chain isoform IIa and a lower densitometric ratio of fast-to-slow myosin heavy chain protein content. CONCLUSION: We suggest that electrophysiological nonexcitable muscle membrane predicts preferential type II fiber atrophy in intensive care unit patients during early critical illness.

Susceptibility to tuberculosis is associated with TLR1 polymorphisms resulting in a lack of TLR1 cell surface expression.

Human TLR1 plays an important role in host defense against Mycobacterium tuberculosis. Our aim was to analyze the association of the loss of TLR1 surface expression and TLR1 SNPs with susceptibility to TB. TLR1neg and TLR1pos cells from healthy individuals were identified by flow cytometry and compared by sequencing. TLR1 expression was measured using quantitative real-time PCR and immunoblotting. TLR1 SNP analyses of healthy individuals and TB patients from EU-C and Ghana were performed, and association of the TLR1 genotypes with increased risk of developing TB was statistically evaluated. Lack of TLR1 surface expression accompanied by impaired function was strongly associated with TLR1 SNP G743A. Genotyping of EU-C controls and TB patients revealed an association of TLR1 743A/1805G alleles [OR 2.37 (95% CI 1.13, 4.93), P=0.0219; OR 2.74 (95% CI 1.26, 6.05), P=0.0059] as well as TLR1neg 743AA/1805GG versus TLR1pos genotypes 743AG/1805TG [OR 4.98 (95% CI 1.64, 15.15), P=0.0034; OR 5.70 (95% CI 1.69, 20.35), P=0.0015] and 743AG + GG/1805TG + TT [OR 3.54 (95% CI 1.29, 9.90), P=0.0086; OR 4.17 (95% CI 1.52, 11.67), P=0.0025] with increased susceptibility to TB. No association of G743A with TB was found in Ghana as a result of a low frequency of genotype 743AA. Our data gain new insights in the role of TLR1 in M. tuberculosis defense and provide the first evidence that TLR1 variants are associated with susceptibility to TB in a low-incidence country.

T-helper type 1 cytokine release is enhanced by in vitro zinc supplementation due to increased natural killer cells.

OBJECTIVE: We examined the influence of zinc on T-helper type 1 (Th1)/T-helper type 2 (Th2) balance in human lymphocytes. METHODS: Human peripheral blood mononuclear cells or diluted whole blood were cultured for 8 d in the presence of zinc (30 or 60 microM) or 1 microM of N, N, N', N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) (a zinc-specific chelator). Phytohemagglutinin-induced cytokine release was measured by enzyme-linked immunosorbent assay, and expression of CD56/CD69, CCR4/CD3, and CCR5/CD3 and intracellular labile zinc were detected by flow cytometry. RESULTS: We found that our in vitro supplementation resulted in an increase of intracellular labile zinc comparable to that of a 7-wk administration of 10 mg of zinc per day in vivo. Zinc triggered interferon-gamma release and impaired interleukin-10 release. Phenotypically, a Th2/Th1 shift could not be confirmed after detecting the Th1-specific chemokine receptor CCR5 or CCR4 for Th2 cells. Surprisingly, we detected a larger amount of CD56+ cells after zinc stimulation, leading us to the conclusion that the amount of interferon-gamma release after zinc supplementation might be attributed to the upregulation of natural killer cells after in vitro zinc supplementation rather than to a Th2/Th1 shift. CONCLUSION: We suggest that a nutritional intake of 10 mg of zinc increases the quantity of interferon-gamma-producing natural killer cells and strengthens the immune system against neoplasms and viral infections.

The superantigen staphylococcal enterotoxin A (SEA) and monoclonal antibody L243 share a common epitope but differ in their ability to induce apoptosis via MHC-II.

Crosslinking of MHC class II (MHC-II) molecules by antibodies or by superantigens (SAg) induces a variety of functional responses in the antigen presenting cell. We were able to allocate K39 as the residue that is essential for binding of antibody L243 to the alpha chain of HLA-DR. K39 is also essential for binding of staphylococcal enterotoxin A (SEA). However, the functional responses of the two ligands differ considerably exemplified by the ability of L243 to induce apoptosis in monocytic cells and in B cells, whereas SEA is unable to activate the apoptosis pathway. Despite the differences in functional responses, both ligands induce cell aggregation in MonoMac-1 cells. The SEA molecule with its two different binding sites associates one MHC alpha chain with one beta chain as opposed to two alpha chains that are brought into close proximity by the two identical antigen binding sites of L243. We therefore conclude that the spatial orientation of dimerized MHC-II and their associated proteins is an important factor for the nature of the transduced signal and consequently the outcome of functional responses.

Polymorphonuclear leucocytes selectively produce anti-inflammatory interleukin-1 receptor antagonist and chemokines, but fail to produce pro-inflammatory mediators.

The role of neutrophils in the immune response has long been regarded as mainly phagocytic, but recent publications have indicated the production of several cytokines by polymorphonuclear leucocytes (PMN). The results of the individual reports, however, vary considerably. In this study, we established a cytokine profile of pure human neutrophils and demonstrated that minor contamination of peripheral blood mononuclear cells (PBMCs) in PMN preparations can lead to false-positive results. In our hands, peripheral blood PMN fail to produce the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha). Instead, they secrete large amounts of the chemokine IL-8 and the anti-inflammatory IL-1 receptor antagonist (IL-1ra). Additionally, PMN preparations of a high purity show production of the chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and growth-related oncogene-alpha (GRO-alpha), as well as macrophage colony-stimulating factor (M-CSF). The neutrophil therefore represents a novelty by producing the antagonist of IL-1beta (i.e. IL-1ra) in the absence of IL-1beta itself. To support our results, we differentiated stem cells from human cord blood into PMN and monocytes, respectively. These in vitro-differentiated PMN showed the same cytokine profile as peripheral blood PMN lacking IL-1beta, while differentiated monocytes produced the expected IL-1beta in addition to IL-1ra. The clear anti-inflammatory nature of their cytokine profile enables PMN to antagonize pro-inflammatory signals in experimental conditions. It is therefore possible that PMN play a key role in immune regulation by counteracting a dysregulation of the inflammatory process. Clinical studies, in which administration of recombinant G-CSF had a favourable effect on the outcome of severe infections and even sepsis without worsening inflammation, could thus be explained by our results.

Crosslinking of CD66B on peripheral blood neutrophils mediates the release of interleukin-8 from intracellular storage.

Crosslinking of CD66 antigens on the neutrophil surface induces functional responses such as aggregation of the cells and protein kinase activity. Although CD66b (carcinoembryonic antigen-related cell adhesion molecule-8) has been reported as a candidate receptor for galectin-3, its natural ligand is still unknown and therefore its physiologic function remains to be elucidated. We were able to detect the storage of intracellular interleukin-8 (IL-8) in unstimulated human neutrophils and its secretion in response to the crosslinking of CD66b. In contrast to lipopolysaccharide (LPS), the stimulation via CD66b does not induce a de novo synthesis of cytokines but rather a directed release of the preformed IL-8. This process may represent a very low state of activation for the neutrophil. As it extravasates into the tissue, the neutrophil might interact with the extracellular matrix via CD66b. In response to this interaction, polymorphonuclear neutrophils (PMN) release their preformed IL-8, establishing a chemotactic track for other cells to follow. By contact with pathogenic stimuli such as LPS in the infected tissue, the neutrophil then becomes fully activated and is able to synthesize cytokines de novo to release greater quantities.

Differential synthesis of two interleukin-1 receptor antagonist variants and interleukin-8 by peripheral blood neutrophils.

With a short lifespan and containing only few ribosomes and endoplasmic reticulum structures, neutrophils are thought to have a limited capacity for protein synthesis. We here show that peripheral blood polymorphonuclear neutrophils (PMN) are able react to stimulants with differential production of two interleukin (IL)-1 receptor antagonist (IL-1ra) isoforms, secreted IL-1ra (sIL-1ra) and the 16kDa intracellular form of IL-1ra (icIL-1ra3), as well as IL-8. Neutrophils of a high purity and with a low degree of preactivation upregulate mRNA and de novo synthesize protein of both IL-1ra variants and IL-8 in response to granulocyte-macrophage colony-stimulating factor and lipopolysaccharide. The cytokines are differentially regulated and distributed in two intracellular compartments. In comparison with peripheral blood mononuclear cells (PBMC), PMN produce distinctly more sIL-1ra but significantly less IL-8. This may indicate an anti-inflammatory role, enabling PMN to antagonize proinflammatory signals. It is therefore possible that PMN play an important role in immune regulation by counteracting a dysregulation of the inflammatory process.

Neutrophil immunity of the elderly.

Due to age-related changes of the immune system, elderly people are more susceptible to microbial infections than the young. Most research concerning immune senescence has been done on T and B cells, yet the first cells to migrate into microbe-infected tissue are neutrophils, which phagocytose and kill the pathogens. Long regarded as mere phagocytes, the neutrophils' importance for the immune response has been recognized in current publications, which acknowledge them an active participation in the cytokine network. Similarities in the symptoms of patients with genetical neutrophil deficiencies and those of the elderly indicate a leading role of neutrophils in the effects of immune senescence. While the number of circulating neutrophils remains unaltered in the elderly compared with young controls, phagocytosis and intracellular killing have been reported impaired. Oddly enough, the results for various stimuli differed: while some showed a decrease in neutrophil activation, the response to others remained unaltered. More research needs to be done on this, preferably using preparations of high purity to exclude monocytic interventions. Elucidation of immune deficiencies caused by neutrophil senescence can be an important contribution to a healthier elderly population.

VP5 and the N terminus of VP2 are not responsible for the different pathotype of serotype I and II infectious bursal disease virus.

Two serotypes have been identified in infectious bursal disease virus (IBDV), a member of the family BIRNAVIRIDAE: A reverse genetics system was used for generation of chimeras in genome segment A of the two serotypes, in which the complete viral VP5 gene and 3' noncoding region (NCR), or parts thereof, were exchanged. The engineered viruses were characterized in vitro and in vivo in comparison to serotype I and II IBDV. Our results show that IBDV chimeras exhibit a different phenotype in cell culture compared to the wild-type viruses. In in vitro-cultivated bursal-derived cells, chimeric viruses infected B lymphocytes, as does serotype I IBDV. Surprisingly, serotype II virus was also able to infect in vitro-cultivated bursal cells, but these were neither B lymphocytes nor macrophages. After infection of susceptible chickens all chimeras replicated in the bursa of Fabricius (BF), and three chimeric viruses caused mild depletion of bursal cells. In contrast, after infection of chickens with a chimeric IBDV containing exchanged VP5 as well as 3'-NCR, no depletion was detectable. The serotype II strain did not replicate in the BF nor did it cause depletion of bursal cells. Thus, the origin of VP5 does not explain the different pathotype of IBDV serotype I and II.