Interleukin-10 inhibits neutrophil phagocytic and bactericidal activity

Abstract Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells, which is dependent upon the coordinated expression of both pro- and anti-inflammatory cytokines. Interleukin-10 (IL-10) is a recently described cytokine with potent anti-inflammatory properties in vivo and in vitro. In this study we investigated whether IL-10 could directly regulate the ability of neutrophils (PMN) to phagocytose and kill bacteria. Initial studies demonstrated that human recombinant IL-10 (hrIL-10) inhibited the ability of PMN to phagocytose Escherichia coli in vitro. Inhibition of phagocytosis occurred in the absence of changes in CR1 (C3b) or Fc receptor expression, as treatment of PMN with IL-10 failed to induce significant changes in FcγIIR, FcγIIIR or CR1 cell surface expression. However, incubation of PMN with IL-10 resulted in a dose-dependent decrease in CD11b (Mac-1) expression. In addition to effects on PMN phagocytosis, hrIL-10 significantly attenuated PMN microbicidal activity, as bactericidal assays revealed that co-incubation of PMN with hrIL-10 resulted in a marked decrease in killing of phagocytosed bacteria. Furthermore, IL-10 inhibited the production of superoxide from PMA-stimulated PMN, suggesting that the detrimental effects of IL-10 on PMN microbicidal activity were due, in part, to suppression of respiratory burst. In summary, our studies indicate that IL-10 inhibits PMN-dependent phagocytosis and killing of E. coli in vitro, and suggest that this cytokine may impair effective antibacterial host defense in vivo.     

The effect of cytokines on bactericidal activity of murine neutrophils

Abstract A range of recombinant cytokines have now been shown to modify aspects of the phenotype and function of human and murine neutrophils. However, few reports describe modification of the bactericidal activity of neutrophils. We therefore examined the recombinant murine cytokines tumor necrosis factor-α (TNF-α, 10–1000 ng ml⁻¹) and granulocyte macrophage-colony stimulating factor (GM-CSF, 10–1000 U ml⁻¹) for their ability to increase the bacterial killing capacity of murine neutrophils. Neutrophils from either bone marrow (fresh or cultured), or peritoneal exudates, or abscesses, were pre-incubated with either cytokine for 30–60 min and the killing of Proteus mirabilis, Escherichia coli , or Bacteriodes fragilis was examined in the presence or absence of serum over a 90 min period. Only for one combination was a small but significantly enhanced level of bacterial killing observed, the phagocytic killing of P. mirabilis by peritoneal exudate neutrophils in the presence of GM-CSF and serum. With this exception there was no enhancement of bacterial killing for the range of combinations of neutrophils and bacterial species tested. In contrast, at the concentrations tested for effect on bactericidal activity, TNF-α and GM-CSF were able to significantly upregulate CR3(but not FcγRII) expression on mouse neutrophils. There results indicate that upregulation of CR3 as an index of neutrophil activation does not necessarily correlate with increased bactericidal activity.     

琥珀酸对酵母细胞转录组的影响

寻找抗衰老活性小分子并研究其作用机制是衰老药物学研究的重点和热点。本文报道了一种新的抗衰老活性小分子琥珀酸,发现琥珀酸可以显著延缓芽殖酵母细胞的衰老并增强细胞的压力抗性。随后,利用DNA Microarray技术及生物信息学手段较系统分析了琥珀酸处理对基因表达谱、基因本体聚类及相关信号通路的影响。结果显示,琥珀酸处理对细胞转录组产生了显著影响,共导致3 485个基因的差异表达(P 0.05),其中1 335个基因显著上调,2 150个基因显著下调。进一步对基因本体聚类及信号通路分析显示,线粒体及核糖体生物合成相关的分子功能、细胞组分、生物学过程和信号通路可能是琥珀酸作用的主要靶点,其他可能的作用靶点还包括蛋白酶体、细胞内吞、过氧化物酶体代谢及细胞自噬等。本研究为进一步阐明琥珀酸介导的寿命及压力调控机制提供了理论参考和研究线索。     

一株耐受低pH、高浓度乳酸及琥珀酸菌株的筛选与鉴定

【目的】通过构建的人工耐酸系统,筛选耐受低pH值、乳酸及琥珀酸的菌株。【方法】构建人工耐酸系统长期驯化菌株,利用不同p H的酸性平板进行筛选,从环境中筛选出一株对低p H值、高浓度乳酸以及琥珀酸有很好耐受性的菌株。通过形态学特征、生理生化特征研究,并结合18S rDNA基因序列分析及分子系统发育树的构建结果,确定菌株的种类。【结果】经过酸性人工系统的长期驯化,筛选分离出一株耐受低pH值、高浓度乳酸以及琥珀酸的菌株WJ-2,经鉴定该菌株为酿酒酵母(Saccharomyces cerevisiae),其最适生长温度为30°C。酸性平板实验显示该菌株能够耐受pH2.5的酸性环境,同时对9%的乳酸及8%的琥珀酸也有很好的耐受性。另外,耐酸菌株WJ-2在pH 2.5、9%乳酸和8%琥珀酸的培养环境中仍能保持相对中性的细胞内pH值。【结论】通过构建人工酸性系统,成功筛选出一株对低pH值、高浓度乳酸以及琥珀酸具有耐受性的菌株——酿酒酵母菌WJ-2,该方法可为筛选具有特定耐受能力菌株提供一个新思路。     

Effect of valproic acid on the urinary metabolic profile of a patient with succinic semialdehyde dehydrogenase deficiency

The metabolic changes in a patient with succinic semialdehyde dehydrogenase deficiency were investigated following valproate administration using urease pretreatment and gas chromatography-mass spectrometry. A stable isotope dilution technique was used for quantification of urinary 4-hydroxybutyrate. Urinary levels of 4-hydroxybutyrate were 4-fold higher after 1-month valproate therapy. 4,5-Dihydrohexanoate, 2-deoxytetronate and 3-deoxytetronate were also 1.7-2.7-fold higher. The urinary excretions of 4-hydroxybutyrate in valproate non-medicated controls were age dependence and decreased with age. Relationships between 4-hydroxybutyrate excretion and 4-hydroxyvalproate or 5-hydroxyvalproate excretion were observed in valproate medicated controls. It seems that 4-hydroxyvalproate and 5-hydroxyvalproate as well as valproate are involved with increased excretion of 4-hydroxybutyrate following valproate administrations.     

Bioreactors for succinic acid production processes

Succinic acid (SA) has been recognized as one of the most important bio-based building block chemicals due to its numerous potential applications. Fermentation SA production from renewable carbohydrate feedstocks can have the economic and sustainability potential to replace petroleum-based production in the future, not only for existing markets, but also for new larger volume markets. Design and operation of bio-reactors play a key role. During the last 20 years, many different fermentation strategies for SA production have been described in literature, including utilization of immobilized biocatalysts, integrated fermentation and separation systems and batch, fed-batch, and continuous operation modes. This review is an overview of different fermentation process design developed over the past decade and provides a perspective on remaining challenges for an economically feasible succinate production processes. The analysis stresses the idea of improving the efficiency of the fermentation stage by improving bioreactor design and by increasing bioreactor performance.     

Rapid screening procedures for identification of succinic acid producers

Succinic acid, an intermediate of tricarboxylic acid cycle, is produced and accumulated by anaerobic microorganisms. The long-standing interest in the production of this organic acid is because it is a key compound in producing more than 30 commercially important products. The detection of succinic acid is generally carried out by gas chromatography (GC), enzymatic assays, ion-exclusion chromatography (IEC) or by high performance liquid chromatography (HPLC). However, these methods are time consuming, require sophisticated instrumentation and are expensive. In the present investigation we are reporting two rapid, cost effective screening methods for the detection of this important organic acid. These methods can be utilized to screen a large number of microbes producing succinic acid in a very short span of time.     

Effects of redox potential control on succinic acid production by engineered Escherichia coli under anaerobic conditions

The effects of oxido-reduction potential (ORP) control on succinic acid production have been investigated in Escherichia coli LL016. In LL016, two CO₂ fixation pathways were achieved and NAD⁺ supply was enhanced by co-expression of heterologous pyruvate carboxylase (PYC) and nicotinic acid phosphoribosyltransferase (NAPRTase). During anaerobic fermentation, cell growth and metabolite distribution were changed with redox potential levels in the range of −200 to −400 mV. From the results, the ORP level of −400 mV was preferable, which resulted in the high succinic acid concentration (28.6 g/L) and high succinic acid productivity (0.33 g/L/h). Meanwhile, the yield of succinic acid at the ORP level of −400 mV was 39% higher than that at the ORP level of −200 mV. In addition, a higher NADH/NAD⁺ ratio and increased enzyme activities were also achieved by regulating the culture to a more reductive environment, which further enhanced the succinic acid production.     

Adaptive evolution improves acid tolerance and succinic acid production in Actinobacillus succinogenes

Fermentation at low pH is an efficient way to improve the competitiveness of biological succinic acid-producing process. Actinobacillus succinogenes shows good performance of succinic acid production under anaerobic conditions, but its succinic acid production capability at the low-pH is inefficient due to the poor acid resistance. Herein, a mutant A. succinogenes BC-4 with improved cell growth and succinic acid production under weak acid conditions was obtained by adaptive evolution. The specific growth rate and succinic acid production of BC-4 reached 0.13 g/L/h and 20.77 g/L, which were increased by 3.25- and 2.95- fold, respectively compared with the parent strain under anaerobic condition at pH 5.8. The activities of specific enzymes with ATP generation were significantly enhanced under weak acidic conditions, resulting in 1.28-fold increase in the maximum ATP level. Membrane fatty acid composition analysis demonstrated that the ratio of saturated to unsaturated fatty acids was decreased from 1.62 to 1.44 in mutant BC-4, leading to improved intracellular pH homeostasis. Furthermore, the change from long-chain to median-chain fatty acid might lower the permeability of H⁺ into cytoplasm for survival under acid stress. These results indicated that A. succinogenes BC-4 is a promising candidate for succinic acid production under weak acid condition.     

Polychromatic luminescence and improved antifungal performance of succinic acid in the lattice of L-Lysine monohydrochloride

The incorporation of succinic acid (SA) in the lattice of L-Lysine monohydrochloride (LM) has opened the new avenue in the field of production and application of scintillator materials such as LED and antifungal drug. Crystalline trait and monoclinic structure were scanned by XRD. The existence of carbonyl, carboxylate and protonated amine group were confirmed through FTIR and UV spectra predicted the transmittance of SA: LM crystal. Polychromatic luminescence behaviour had achieved through the incorporation of SA instead of blue luminescence, which is a new result. Also SA: LM exhibited good response towards pathogenic fungi which causes numerous types of infections and diseases in both humans and animals. The high inhibitory zone at 16 mm was formed by the grown SA: LM crystal against the life threatening fungi like Candida albicans. Also fungal inhibition against candida parapsilosis and Aspergillus flaves, respectively, were tuned by the inclusion of succinic acid.     

The possible formation of nano-clusters of succinic acid and maleic acid in tetrahydrofuran due to incomplete solvation

In continuation of our work on the conformational analysis of succinic acid (SA) and maleic acid (MA) in different solvents, we present here the experimental dielectric and IR and also the ab initio Hartree–Fock calculations of the two dicarboxylic acids in tetrahydrofuran (THF). The dielectric measurements are carried out at microwave X-band frequency of 9.7 GHz and the calculations are performed at STO-3G and 6-31G(d) basis sets. The dielectric data and the dipole moment determined experimentally are compared with the dipole moment determined from the conformal analysis. It is seen that the dielectric properties of SA/MA in THF are much different from that of SA/MA in 1-4, dioxane (1-4D) that we had reported previously. The IR spectra of SA–THF system is also reported here. The present study indicates the possible formation of nano-clusters of SA/MA in THF due to incomplete solvation by THF.     

Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes

The inhibition of substrate and products on the growth of Actinobacillus succinogenes in fermentation using glucose as the major carbon source was studied. A. succinogenes tolerated up to 143 g/L glucose and cell growth was completely inhibited with glucose concentration over 158 g/L. Significant decrease in succinic acid yield and prolonged lag phase were observed with glucose concentration above 100 g/L. Among the end-products investigated, formate was found to have the most inhibitory effect on succinic acid fermentation. The critical concentrations of acetate, ethanol, formate, pyruvate and succinate were 46, 42, 16, 74, 104 g/L, respectively. A growth kinetic model considering both substrate and product inhibition is proposed, which adequately simulates batch fermentation kinetics using both semi-defined and wheat-derived media. The model accurately describes the inhibitory kinetics caused by both externally added chemicals and the same chemicals produced during fermentation. This paper provides key insights into the improvement of succinic acid production and the modelling of inhibition kinetics.     

Salting-out extraction and crystallization of succinic acid from fermentation broths

In this study, salting-out extraction (SOE) and crystallization were combined to recover succinic acid from fermentation broths. Of the different SOE systems investigated, the system consisting of organic solvents and acidic salts appeared to be more favorable. A system using acetone and ammonium sulfate was investigated to determine the effect of phase composition and pH. The highest partition coefficient (8.64) and yield of succinic acid (90.05%) were obtained by a system composed of 30% (w/w) acetone and 20% (w/w) ammonium sulfate at a pH of 3.0. Additionally, 99.03% of cells, 90.82% of soluble proteins, and 94.89% of glucose could be simultaneously removed from the fermentation broths. Interestingly, nearly 40% of the pigment was removed using the single-step salting-out extraction process. The analysis of the effect of pH on salting-out extraction indicates that a pH lower than the pK of succinic acid is beneficial for the recovery of succinic acid in an SOE system. Crystallization was performed for the purification of succinic acid at 4 °C and pH 2.0. By combining salting-out extraction with crystallization, an identical total yield (65%) and a higher purity (97%) of succinic acid were obtained using a synthetic fermentation broth compared with the actual fermentation broth (65% and 91%, respectively).     

Flow cytometric measurements of neutrophil functions: the dependence on the stimulus to cell ratio

Phagocytosis and antimicrobial killing of neutrophils has been quantitatively determined as a function of the stimulus (Candida albicans) to cell ratio R using two donor collectives containing a total of 115 blood samples. Analysis of the collectives in two different laboratories according to the same flow cytometric protocol for simultaneous measurement of neutrophil functions did not produce statistically significant differences. The number of phagocytosing leukocytes as well as that of killed fungi per leukocyte depends strongly on R. While each phagocytosing neutrophil kills one fungus at low values of R, each neutrophil kills on average 2.5 fungi for large R.     

Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH

Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7 g/L succinic acid with a yield of 0.53 g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.     

The effect of glutamine supplementation and physical exercise on neutrophil function

Glutamine is the most abundant free amino acid in the body. Its primary source is skeletal muscle, from where it is released into the bloodstream and transported to a variety of tissues. Several studies have shown that glutamine is important for rat and human neutrophil function and that these cells utilize glutamine at high rates. Physical exercise has also been shown to induce considerable changes in neutrophil metabolism and function. As neutrophils represent 50-60% of the total circulating leukocyte pool and play a key role in inflammation, both physical exercise and glutamine might be expected to regulate the inflammatory process. In this review, the changes in neutrophil function induced by physical exercise and glutamine supplementation are compared.     

Fermentation process for continuous production of succinic acid in a fibrous bed bioreactor

Succinic acid (SA) was produced from Actinobacillus succinogenes with high cell density by continuous fermentation using fibrous bed bioreactor (FBB). The effects of feeding glucose concentration, dilution rate, and pH on continuous production of SA were examined to achieve an efficient and economical bioprocess. The optimum feeding glucose concentration, dilution rate, and pH were 80 g/L, 0.05 1/h, and 6.0–6.5, respectively. A SA concentration of 55.3 ± 0.8 g/L, productivity of 2.77 ± 0.04 g/L/h, and yield of 0.8 ± 0.02 g/g were obtained, and the continuous fermentation exhibited long-term stability for as long as 18 days (440 h) with no obvious fluctuations in both SA and biomass levels. The Jerusalimsky equation for the specific rate of SA production presented the inhibition phenomenon of the product, demonstrating that 60 g/L SA might be a critical concentration in this continuous FBB system. The results obtained could be beneficial for future fermentor designs and improvements in SA production.     

CO2 fixation for succinic acid production by engineered Escherichia coli co-expressing pyruvate carboxylase and nicotinic acid phosphoribosyltransferase

In wild-type Escherichia coli, 1 mol of CO₂ was fixated in 1 mol of succinic acid generation anaerobically. The key reaction in this sequence, catalyzed by phosphoenolpyruvate carboxylase (PPC), is carboxylation of phosphoenolpyruvate to oxaloacetate. Although inactivation of pyruvate formate-lyase and lactate dehydrogenase is found to enhance the PPC pathway for succinic acid production, it results in excessive pyruvic acid accumulation and limits regeneration of NAD⁺ from NADH formed in glycolysis. In other organisms, oxaloacetate is synthesized by carboxylation of pyruvic acid by pyruvate carboxylase (PYC) during glucose metabolism, and in E. coli, nicotinic acid phosphoribosyltransferase (NAPRTase) is a rate-limiting enzyme of the NAD(H) synthesis system. To achieve the NADH/NAD⁺ ratio decrease as well as carbon flux redistribution, co-expression of NAPRTase and PYC in a pflB, ldhA, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production under anaerobic conditions. After 72 h, 14.5 g L⁻¹ of glucose was consumed to generate 12.08 g L⁻¹ of succinic acid. Furthermore, under optimized condition of CO₂ supply, the succinic acid productivity and the CO₂ fixation rate reached 223.88 mg L⁻¹ h⁻¹ and 83.48 mg L⁻¹ h⁻¹, respectively.     

Modeling of batch fermentation kinetics for succinic acid production by Mannheimia succiniciproducens

Kinetic models are proposed for the batch production of succinic acid from glucose by Mannheimia succiniciproducens MBEL55E. The models include terms accounting for both substrate and product inhibitions. Experimental data collected from a series of batch fermentations with different initial glucose concentrations were used to estimate parameters and also to validate the models proposed. The optimal values of the parameters were approximated by minimizing the discrepancy between the model predictions and corresponding experimental data. The growth of M. succiniciproducens could be expressed by a modified Monod model incorporating inhibitions of glucose and organic acids accumulated in the culture broth. The Luedeking–Piret model was able to describe the formation of organic acids as the fermentation proceeded, in which succinic, acetic, and formic acids followed a mixed-growth-associated pattern. However, unexpectedly, lactic acid fermentation by M. succiniciproducens was nearly nongrowth-associated. In all cases, the model simulation matched well with the experimental observations, which made it possible to elucidate the fermentation characteristics of M. succiniciproducens during efficient succinic acid production from glucose. These models thus can be employed for the development and optimization of biobased succinic acid production processes.