Influence of protein extraction and texturization on odor-active compounds of pea proteins.

BACKGROUND: The use of plant proteins as food ingredients might be limited due to the presence of foreign or 'off' flavors, which may evolve during extraction and subsequent processing. In this study, the influence of dry (TVP) and wet (WTP) texturization on characteristic volatile compounds of two different pea protein isolates was assessed using gas chromatography-mass spectrometry-olfactometry (GC-MS-O) after direct immersion stir bar sorptive extraction (DI-SBSE). RESULTS: Twenty-four odor-active compounds were found, with a prevalence of carbonyls from fat oxidation. Nine of these compounds which are also known as major (off-) flavor contributors in peas were distinctively impacted in all texturates: hexanal, nonanal, 2-undecanone, (E)-2-octenal, (E, Z)-3,5-octadiene-2-one, (E, E)-2,4-decadienal, 2-pentyl-furan, 2-pentyl-pyridine, and γ-nonalactone. For example, hexanal, a characteristic green odorant, was reduced by up to sixfold by wet texturization, from 3.29 ± 1.05% (Pea Protein I) to 0.52 ± 0.02% (Pea WTP I). Furthermore, (E,Z)-3,5-Octadiene-2-one and (E,E)-2,4-decadienal were decreased by 1.5- and 1.8-fold when Pea Protein I and Pea TVP I were compared. CONCLUSION: An overall reduction in fat oxidation products and of green and fatty odor-active compounds was observed. The results represent a first insight into the process-related modulation of pea protein (off-) flavors to broaden the applicability of pea proteins as food ingredients.

Monitoring of Soft Deposition Layers in Liquid-Filled Tubes with Guided Acoustic Waves Excited by Clamp-on Transducers.

The monitoring of liquid-filled tubes with respect to the formation of soft deposition layers such as biofilms on the inner walls calls for non-invasive and long-term stable sensors, which can be attached to existing pipe structures. For this task a method is developed, which uses an ultrasonic clamp-on device. This method is based on the impact of such deposition layers on the propagation of circumferential guided waves on the pipe wall. Such waves are partly converted into longitudinal compressional waves in the liquid, which are back-converted to guided waves in a circular cross section of the pipe. Validating this approach, laboratory experiments with gelatin deposition layers on steel tubes exhibited a distinguishable sensitivity of both wave branches with respect to the thickness of such layers. This allows the monitoring of the layer growth.

Meat hybrids-An assessment of sensorial aspects, consumer acceptance, and nutritional properties.

So-called meat hybrids are a new class of products where a fraction of the meat product (e.g., 20%) is replaced with alternative protein sources, such as plant-based ones. Research suggests that these products could serve as a low-threshold offer for a specific target group that wants to cut down on meat, thereby facilitating the transition toward a more healthy and sustainable diet. Nonetheless, data demonstrate that meat hybrids with a high substantial meat substitution level often fail in the market. This study summarises findings on the physicochemical properties, sensory, and acceptance of six different meat hybrids (70% meat and 30% plant proteins) that were collected in the framework of a case study in the project AiF 196 EN. For this purpose, sensory characteristics were collected via two QDA sessions and a hedonic consumer test. Furthermore, the hybrid recipes were analysed in their proximate composition. The respective recipes varied in protein source (soybean, pumpkin, and pea) and mode of incorporation [textured vegetable protein (TVP), high moisture extrudate (HME)]. It was shown that a meat hybrid with a relatively high share of 30% plant-based proteins with peas as a protein source and TVP as a processing method can still attract consumers.

Acidification behavior of mixtures of pork meat and wet texturized plant proteins in a minced model system.

The increasing use of wet texturized plant proteins as meat substitutes requires a characterization of their functional properties, especially in terms of pH-behavior when being mixed with meat proteins to create so-called hybrid products. In this study, a minced model system containing pork meat, curing salt, and various amounts (0-100 wt%) of wet extruded proteins from pea (Pea I, II), pumpkin (Pumpkin I, II, III), and sunflower was used to evaluate the effect of mixing on pH and time-dependent pH-changes upon the addition of glucono-delta-lactone (GDL). Increasing concentrations of plant extrudates resulted in a linear increase of the initial (pH0h ), intermediate (pH6h ), and final pH48h for all samples and higher slopes at higher native pH of extrudates were found. Acidification kinetics of all samples were similar with a distinct pH-drop by 0.3 to 0.8 pH-units per wt% GDL in the first 6 h, followed by a plateau where pH remained constant. At extrudate concentrations of 5 wt% (Pea I, II, Pumpkin I, II) or 15 wt% (Pumpkin III, Sunflower), a sufficient acidification with typically used GDL-amounts ( = 1 wt%) could be achieved, while higher plant protein contents required higher GDL-concentrations in order to reach a pH value of 5.0; a common target value in dry-cured sausages. A mathematical model was proposed to correlate pH, time, acidifier, extrudate concentration, and plant protein origin, to aid in the adjustment of dry-cured hybrid meat formulations, and to describe thresholds of the feasible extrudate and acidifier concentrations. PRACTICAL APPLICATION: Despite the increasing relevance of texturized plant proteins as meat mimetics, little is known about their functional and process-related properties. This study shows that plant protein origin, the level of meat replacement, and the amount of acidifier are linked to the time-dependent pH-value on the basis of a mathematical model. This brings food developers one step closer in creating tailored formulations and estimating the effects of these novel ingredients in the final product characteristics of hybrid meats and analogues.

Effect of varying pH on solution interactions of soluble meat proteins with different plant proteins.

The exchange of animal-based for plant-based proteins is becoming more and more popular due to an increasing demand for alternative and more sustainable protein sources. In this study, solubilized water- (ws) or salt-and-water (sws) meat proteins were evaluated in their pH-dependent interactions with soluble protein fractions from wheat, pumpkin, sunflower, rapeseed, or potato proteins. For this purpose, 1 : 1 (v/v) mixtures of 1.0 wt% meat (ws or sws) and plant proteins were prepared at a sodium chloride concentration of 1.8 wt% (ionic strength: 0.31 mol L-1) and adjusted to different pH-values in between 4.5-7.0. While only slight differences were found upon comparison of interactions of ws and sws batches (p > 0.05), interactions among these animal-based and soluble plant proteins took place. First, optical observations, light microscopy, and SDS-PAGE revealed increasing protein solubility with increasing pH. Second, particle size distributions (PSDs) revealed a shift towards slightly larger particle sizes e.g. at pH 5.3 and 7.0 with d4,3 of 43.2 and 21.3 µm (sws) to 45.4 and 23.9 µm (sws + potato), respectively. Furthermore, heat-induced gel formation was improved at pH > 6.0, in particular in mixtures of meat and wheat or rapeseed proteins that formed a homogenous gel structure. Based on the obtained results, protein-protein complexations mainly by electrostatic forces are suggested which occur due to various pI of meat and plant proteins e.g. pH 7.5 (wheat), 7.2 (potato), and 6.6 (rapeseed) in comparison to 5.1 (ws) and 5.6 (sws). The filamentous microstructure of some gels (soluble fraction of rapeseed, potato and wheat proteins) led to the assumption that meat proteins, mainly at pH values greater than 5.8 (optimally ≥6.5), had a structuring effect on plant proteins.

PavB is a surface-exposed adhesin of Streptococcus pneumoniae contributing to nasopharyngeal colonization and airways infections.

The genomic analysis of Streptococcus pneumoniae strains identified the Pneumococcal adherence and virulence factor B (PavB), whose repetitive sequences, designated Streptococcal Surface REpeats (SSURE), interact with human fibronectin. Here, we showed the gene in all tested pneumococci and identified that the observed differences in the molecular mass of PavB rely on the number of repeats, ranging from five to nine SSURE. PavB interacted with fibronectin and plasminogen in a dose-dependent manner as shown by using various SSURE peptides. In addition, we identified PavB as colonization factor. Mice infected intranasally with DeltapavB pneumococci showed significantly increased survival times compared with wild-type bacteria. Importantly, the pavB-mutant showed a delay in transmigration to the lungs as observed in real-time using bioluminescent pneumococci and decreased colonization rates in a nasopharyngeal carriage model. In co-infection experiments the wild-type out-competed the pavB-mutant and infections of epithelial cells demonstrated that PavB contributes to adherence to host cell. Blocking experiments suggested a function of PavB as adhesin, which was confirmed by direct binding of SSURE peptides to host cells. Finally, PavB may represent a new vaccine candidate as SSURE peptides reacted with human sera. Taken together, PavB is a surface-exposed adhesin, which contributes to pneumococcal colonization and infections of the respiratory airways.

Buffering capacity of wet texturized plant proteins in comparison to pork meat.

There is an increasing demand to develop and characterize high moisture extrudates from alternative plant proteins due to their increased use in various foods. In this study, wet texturized proteins from two pea isolates and four oilseed flours from pumpkin and sunflower were subjected to an acid titration to gain insights into their buffering capacity. Results were compared to pork meat with a special emphasis on compositional differences. Wet texturized pumpkin and sunflower proteins had the highest buffering capacity, especially in between pH7.0 and pH4.5, while pea protein extrudates and pork meat were more prone to acidification and similar in buffering capacity. A multiple linear regression model further revealed that ash and select minerals and amino acids are key influencing factors on the overall buffering capacity, while the effect of protein and non-protein nitrogen depends on the evaluated pH-regime. The obtained results underline the importance for a more in-depth physicochemical characterization of texturized plant proteins and their raw materials and suggest a need for recipe and process adjustment to achieve stable pH values.

Toll-like receptor stimulation enhances phagocytosis and intracellular killing of nonencapsulated and encapsulated Streptococcus pneumoniae by murine microglia.

Toll-like receptors (TLRs) are crucial pattern recognition receptors in innate immunity that are expressed in microglia, the resident macrophages of the brain. TLR2, -4, and -9 are important in the responses against Streptococcus pneumoniae, the most common agent causing bacterial meningitis beyond the neonatal period. Murine microglial cultures were stimulated with agonists for TLR1/2 (Pam(3)CSK(4)), TLR4 (lipopolysaccharide), and TLR9 (CpG oligodeoxynucleotide) for 24 h and then exposed to either the encapsulated D39 (serotype 2) or the nonencapsulated R6 strain of S. pneumoniae. After stimulation, the levels of interleukin-6 and CCL5 (RANTES [regulated upon activation normal T-cell expressed and secreted]) were increased, confirming microglial activation. The TLR1/2, -4, and -9 agonist-stimulated microglia ingested significantly more bacteria than unstimulated cells (P < 0.05). The presence of cytochalasin D, an inhibitor of actin polymerizaton, blocked >90% of phagocytosis. Along with an increased phagocytic activity, the intracellular bacterial killing was also increased in TLR-stimulated cells compared to unstimulated cells. Together, our data suggest that microglial stimulation by these TLRs may increase the resistance of the brain against pneumococcal infections.

Fibronectin stimulates Escherichia coli phagocytosis by microglial cells.

Microglia express Toll-like receptors (TLRs) that recognize invading pathogens as well as endogenous proteins such as fibronectin under nonphysiological conditions. Here, we demonstrated that fibronectin stimulates murine microglia in culture in a dose-dependent manner: microglial cells secreted proinflammatory cytokines and chemokines and increased phagocytosis of Escherichia coli DH5alpha and E. coli K1 strains. Low levels of fibronectin exerted a synergistic effect on the release of proinflammatory compounds by microglia co-stimulated with agonists for TLR1/2 (Pam(3)CSK(4)) or TLR9 (CpG DNA), but not in combination with the TLR4 agonist lipopolysaccharide (LPS). Phagocytosis of bacterial strains was moderately enhanced when microglia was co-stimulated with high concentrations of fibronectin and one pathogen-derived TLR agonist. In conclusion, fibronectin increased proinflammatory and phagocytotic functions in microglia and partially synergized with microbial TLR agonists.

Recurrent systemic infections with Streptococcus pneumoniae do not aggravate the course of experimental neurodegenerative diseases.

Neurological symptoms of patients suffering from neurodegenerative diseases such as Alzheimer's dementia (AD), Parkinson's disease (PD), or amyotrophic lateral sclerosis (ALS) often worsen during infections. We assessed the disease-modulating effects of recurrent systemic infections with the most frequent respiratory pathogen, Streptococcus pneumoniae, on the course of AD, PD, and ALS in mouse models of these neurodegenerative diseases [transgenic Tg2576 mice, (Thy1)-[A30P]alpha SYN mice, and Tg(SOD1-G93A) mice]. Mice were repeatedly challenged intraperitoneally with live S. pneumoniae type 3 and treated with ceftriaxone for 3 days. Infection caused an increase of interleukin-6 concentrations in brain homogenates. The clinical status of (Thy1)-[A30P]alpha SYN mice and Tg(SOD1-G93A) mice was monitored by repeated assessment with a clinical score. Motor performance was controlled by the tightrope test and the rotarod test. In Tg2576 mice, spatial memory and learning deficits were assessed in the Morris water maze. In none of the three mouse models onset or course of the disease as evaluated by the clinical tests was affected by the recurrent systemic infections performed. Levels of alpha-synuclein in brains of (Thy1)-[A30P]alpha SYN mice did not differ between infected animals and control animals. Plaque sizes and concentrations of A beta 1-40 and A beta 1-42 were not significantly different in brains of infected and uninfected Tg2576 mice. In conclusion, onset and course of disease in mouse models of three common neurodegenerative disorders were not influenced by repeated systemic infections with S. pneumoniae, indicating that the effect of moderately severe acute infections on the course of neurodegenerative diseases may be less pronounced than suspected.

Toll-like receptor prestimulation increases phagocytosis of Escherichia coli DH5alpha and Escherichia coli K1 strains by murine microglial cells.

Meningitis and meningoencephalitis caused by Escherichia coli are associated with high rates of mortality. When an infection occurs, Toll-like receptors (TLRs) expressed by microglial cells can recognize pathogen-associated molecular patterns and activate multiple steps in the inflammatory response that coordinate the brain's local defense, such as phagocytosis of invading pathogens. An upregulation of the phagocytic ability of reactive microglia could improve the host defense in immunocompromised patients against pathogens such as E. coli. Here, murine microglial cultures were stimulated with the TLR agonists Pam(3)CSK(4) (TLR1/TLR2), lipopolysaccharide (TLR4), and CpG oligodeoxynucleotide (TLR9) for 24 h. Upon stimulation, levels of tumor necrosis factor alpha and the neutrophil chemoattractant CXCL1 were increased, indicating microglial activation. Phagocytic activity was studied after adding either E. coli DH5alpha or E. coli K1 strains. After 60 and 90 min of bacterial exposure, the number of ingested bacteria was significantly higher in cells prestimulated with TLR agonists than in unstimulated controls (P < 0.01). Addition of cytochalasin D, an inhibitor of actin polymerization, blocked >90% of phagocytosis. We also analyzed the ability of microglia to kill the ingested E. coli strains. Intracellularly surviving bacteria were quantified at different time points (90, 150, 240, and 360 min) after 90 min of phagocytosis. The number of bacteria killed intracellularly after 6 h was higher in cells primed with the different TLR agonists than in unstimulated microglia. Our data suggest that microglial stimulation by the TLR system can increase bacterial phagocytosis and killing. This approach could improve central nervous system resistance to infections in immunocompromised patients.

Enriched environment fails to increase meningitis-induced neurogenesis and spatial memory in a mouse model of pneumococcal meningitis.

An increase in adult neurogenesis was observed after exposure to enriched environment (EE) and during reconvalescence from experimental pneumococcal meningitis. This study investigated neurogenesis and spatial learning performance 5 weeks after bacterial meningitis and exposure to EE. C57BL/6 mice were infected by intracerebral injection of Streptococcus pneumoniae and treated with ceftriaxone for 5 days. Forty-eight hours after infection, one group (n = 22) was exposed to EE and the other group (n = 23) housed under standard conditions. Another set of mice was kept under either enriched (n = 16) or standard (n = 15) conditions without bacterial meningitis. Five weeks later, the Morris water maze was performed, and neurogenesis was evaluated by means of immunohistochemistry. Mice housed in EE without prior bacterial infection displayed both increased neurogenesis and improved water maze performance in comparison with uninfected control animals. Bacterial meningitis stimulated neurogenesis in the granular cell layer of the dentate gyrus: with standard housing conditions, we observed a higher density of BrdU-immunolabeled and TUC-4-expressing cells 5 weeks after induction of bacterial meningitis than in the noninfected control group. EE did not further increase progenitor cell proliferation and neuronal differentiation in the subgranular cell layer of the dentate gyrus after bacterial meningitis in comparison with infected mice housed under standard conditions. Moreover, the Morris water maze showed no significant differences between survivors of meningitis exposed to EE and animals kept in standard housing. In summary, exposure to EE after pneumococcal meningitis did not further increase meningitis-induced neurogenesis or improve spatial learning.

Emulsifying properties of water-soluble proteins extracted from the microalgae Chlorella sorokiniana and Phaeodactylum tricornutum.

This study investigated the formation and stability of emulsions with lyophilized water-soluble protein extracts from two different microalgae species. Lyophilized soluble protein extracts from Chlorella sorokiniana and Phaeodactylum tricornutum with a protein content of 39.2 and 37.2 wt%, respectively, were used. Drop-shape analysis showed them to have considerable interfacial activity at the oil-water interface. The application in emulsions, prepared by high-pressure homogenization (1000 bar, 3 passes, 5.0 wt% oil) further revealed that a concentration of 1.0 wt% soluble protein from Chlorella sorokiniana was sufficient to manufacture an emulsion with a monomodal droplet size distribution and a small volume based mean particle diameter (d43 = 232 ± 22 nm). Emulsions remained stable throughout 7 days of storage (d43,7d = 265 ± 4 nm). In contrast, 3.7 wt% of the respective proteins from Phaeodactylum tricornutum were needed to obtain a stable emulsion (d43 = 334 ± 12 nm and d43,7d = 325 ± 8 nm). Emulsions prepared with both algae fractions showed unusually high salt stabilities up to 500 mM of sodium chloride, with no appreciable changes in volume based mean particle diameter, appearance, or microstructure. Furthermore, model emulsions with soluble lyophilized proteins from Chlorella sorokiniana had a very high stability toward changes in pH (pH ≥ 5), whereas soluble proteins of Phaeodactylum tricornutum showed only a moderate pH stability with the smallest volume based particle size at pH 7.

Formation and Stability of Emulsions Prepared with a Water-Soluble Extract from the Microalga Chlorella protothecoides.

Unicellular microalgae are a valuable source of macro- and micronutrients. They contain, for example, proteins that are potentially useful as new emulsifiers. The aim of this study was to investigate the emulsifying properties of a less-refined lyophilized crude water-soluble extract (WSE), obtained from the heterotrophically cultivated microalga Chlorella protothecoides. Interfacial tension measurements indicated that mainly the proteins in the extract showed interfacial activity. O/W emulsions were prepared by high-pressure homogenization (1 000 bar, 3 passes) with 5.0 wt % of oil and 2.5 wt % of protein from Chlorella protothecoides, resulting in emulsions having a volume-based mean droplet diameter of d43 ≤ 1 µm and being stable for at least 7 days. Two different stress tests showed that ( i) protein-stabilized emulsions were resistant to very high salt concentrations (up to 500 mM NaCl), and ( ii) emulsions were stable over a very broad pH range of 2-9, with only minor changes in the particle size d43 (e.g. with an increase of only 300 nm when the pH was lowered from 5 to 4) compared to whey protein-stabilized emulsions. All WSE emulsions had monomodal particle size distributions and were macro- and microscopically stable during a storage of up to 7 days. The results indicate that the WSE of Chlorella protothecoides has remarkably good emulsifying properties and might be of use as a new emulsifier in various applications in which emulsions are exposed to a broad range of ionic strengths and pH values.

Microglial cells and peritoneal macrophages release activin A upon stimulation with Toll-like receptor agonists.

Activin A levels are elevated in the cerebrospinal fluid (CSF) of patients with meningitis and in the sera of patients with sepsis. The source(s) of the elevated concentrations of activin A in CSF and serum have not yet been discovered. Here we demonstrate that primary mouse microglial cells and peritoneal macrophages release activin A after treatment with agonists of Toll-like receptor (TLR) 2, 4, and 9. These findings provide further evidence for a role of activin in the innate immune response and suggest that microglial cells and macrophages are a source of elevated activin A concentrations observed in the CSF during bacterial meningitis and in the systemic circulation during sepsis.

Expression of a Cu,Zn superoxide dismutase typical for familial amyotrophic lateral sclerosis increases the vulnerability of neuroblastoma cells to infectious injury.

BACKGROUND: Infections can aggravate the course of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Mutations in the anti-oxidant enzyme Cu,Zn superoxide dismutase (EC 1.15.1.1, SOD1) are associated with familial ALS. Streptococcus pneumoniae, the most frequent respiratory pathogen, causes damage by the action of the cholesterol-binding virulence factor pneumolysin and by stimulation of the innate immune system, particularly via Toll-like-receptor 2. METHODS: SH-SY5Y neuroblastoma cells transfected with the G93A mutant of SOD1 typical for familial ALS (G93A-SOD1) and SH-SY5Y neuroblastoma cells transfected with wildtype SOD1 were both exposed to pneumolysin and in co-cultures with cultured human macrophages treated with the Toll like receptor 2 agonist N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine x 3 HCl (Pam3CSK4). Cell viability and apoptotic cell death were compared morphologically and by in-situ tailing. With the help of the WST-1 test, cell viability was quantified, and by measurement of neuron-specific enolase in the culture supernatant neuronal damage in co-cultures was investigated. Intracellular calcium levels were measured by fluorescence analysis using fura-2 AM. RESULTS: SH-SY5Y neuroblastoma cells transfected with the G93A mutant of SOD1 typical for familial ALS (G93A-SOD1) were more vulnerable to the neurotoxic action of pneumolysin and to the attack of monocytes stimulated by Pam3CSK4 than SH-SY5Y cells transfected with wild-type human SOD1. The enhanced pneumolysin toxicity in G93A-SOD1 neuronal cells depended on the inability of these cells to cope with an increased calcium influx caused by pores formed by pneumolysin. This inability was caused by an impaired capacity of the mitochondria to remove cytoplasmic calcium. Treatment of G93A-SOD1 SH-SY5Y neuroblastoma cells with the antioxidant N-acetylcysteine reduced the toxicity of pneumolysin. CONCLUSION: The particular vulnerability of G93A-SOD1 neuronal cells to hemolysins and inflammation may be partly responsible for the clinical deterioration of ALS patients during infections. These findings link infection and motor neuron disease and suggest early treatment of respiratory infections in ALS patients.

Continuous production of core-shell protein nanoparticles by antisolvent precipitation using dual-channel microfluidization: Caseinate-coated zein nanoparticles.

Antisolvent precipitation is commonly used to fabricate protein nanoparticles using a simple batch method that involves injecting a protein-solvent mixture into an antisolvent. In this study, the potential of producing core-shell protein nanoparticles by antisolvent precipitation using a continuous dual-channel microfluidization method was investigated. The solvent phase (zein in ethanol) and antisolvent phase (casein in water) were made to impinge on each other at high velocity, which generates intense shear, turbulent, and cavitation forces that ensure thorough mixing and breakup of the phases. Relatively small core-shell protein nanoparticles (d<125nm) could be produced using this method when the conditions were optimized. The mean particle diameter decreased with increasing antisolvent-to-solvent ratio, increasing homogenization pressure, increasing ethanol content in the solvent phase, and decreasing zein content in the solvent phase. Depending on the processing conditions employed, zein particles in the range of about 120nm to over 1000nm could be produced. The operating conditions were further optimized to increase the final zein concentration and decrease the organic solvent content while still obtaining small particles. The surface potential of the core-shell protein nanoparticles went from positive at low pH to negative at high pH, with a point of zero charge around pH5. Electron microscopy indicated that the protein particles formed had a roughly spherical shape. The results suggest that the dual-channel microfluidizer could be used to continuously form protein nanoparticles by antisolvent precipitation. Nevertheless, when the microfluidization method was compared with the simple batch method the size of the particles produced under similar conditions were fairly similar.

Activin A concentrations in human cerebrospinal fluid are age-dependent and elevated in meningitis.

OBJECTIVE: Activin A, and its binding protein, follistatin (FS), are expressed in the central nervous system (CNS). We have previously shown elevated concentrations of FS in the cerebrospinal fluid (CSF) of patients with meningitis and increased concentrations of activin A in the CSF of rabbits with bacterial meningitis. METHODS: We measured CSF and serum concentrations of activin A and FS in normal subjects and in patients with various neurological diseases using previously validated immunoassays specific for activin A or FS. RESULTS: In healthy persons, serum concentrations of both activin A and FS were age-dependent. In CSF, concentrations of activin A ranged from 0.03 to 0.33 ng/ml and were strongly correlated with age in both sexes, whereas FS CSF concentrations were below the assay detection limit in most of the patients. Activin A concentrations in CSF of patients with various neurological diseases, including meningitis, chronic inflammatory CNS diseases, neurodegenerative diseases, tumors in the CNS, cerebral ischemia, intracerebral/subarachnoid hemorrhages, subdural hemorrhages and epileptic seizures, were compared with age- and sex-matched control patients. The comparisons revealed significantly elevated concentrations of activin A in patients with meningitis (P=0.017). Serum concentrations of activin A or FS were not affected by any of the neurological diseases examined. CONCLUSIONS: Our results show for the first time that in normal subjects concentrations of activin A in CSF are correlated with age, and furthermore, that activin A CSF concentrations are elevated in patients with meningitis. The latter underlines a role for activin A in acute inflammatory processes within the CNS.

Dose-dependent activation of microglial cells by Toll-like receptor agonists alone and in combination.

Microglial cells express Toll-like receptors (TLRs) recognising exogenous and endogenous ligands. Upon stimulation with agonists of TLR2, TLR4, and TLR9, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) were released by primary mouse microglial cell cultures. Endotoxin was most potent in stimulating microglia followed by pneumolysin, cytosine-guanosine (CpG) oligodesoxynucleotide (ODN), and Tripalmitoyl-S-glyceryl-cysteine. Maximum stimulation of TLR2, TLR4, and TLR9 resulted in approximately equal amounts of nitric oxide release. Pneumolysin was a potent activator of microglial cells; at high concentrations, it reduced cell viability. No cytotoxicity was noted with the other TLR agonists. Costimulation with maximum concentrations of two TLR agonists did not further increase nitric oxide release. Costimulation with submaximum concentrations was additive or supraadditive, suggesting that even low concentrations of products of infectious agents can lead to microglial activation via TLRs.

Limited protection of TAT-Bcl-X(L) against pneumolysin-induced neuronal cell death.

Severe brain damage in patients with pneumococcal meningitis is in part caused by the cytosolic pneumococcal protein pneumolysin. The devastating effect of this neurotoxin might be alleviated by interfering with the cell death pathways that it sets in motion. An important player in these pathways is Bcl-X(L), an antiapoptotic protein of the Bcl-2 family, which is neuroprotective in various in vitro and in vivo models of cell death. We investigated whether its membrane-permeable form, the TAT-Bcl-X(L) fusion protein, is capable of protecting human SH-SY5Y neuroblastoma cells against pneumolysin-induced cell death. Under mild pneumolysin-induced neuronal injury, TAT-Bcl-X(L) increased cell viability significantly by approximately 40% (82.7 +/- 16.1% versus 70.0+/-8.2%; p = 0.04). When the cells were exposed to a more rigorous pneumolysin treatment, TAT-Bcl-X(L) had no protective effects. This suggests the involvement of additional neuronal death pathways in pneumolysin-induced cell death, which are not controlled by Bcl-X(L). Therefore, Bcl-X(L), a promising therapeutic candidate for ischemia and neurodegenerative diseases, is only of partial efficacy in preventing the direct neurotoxicity of pneumolysin.