Serotype Identification of Group B Streptococci by PCR and Sequencing

Group B streptococcus (GBS; Streptococcus agalactiae) is the most common cause of neonatal and obstetric sepsis and is an increasingly important cause of septicemia in elderly individuals and immunocompromised patients. Ongoing surveillance to monitor GBS serotype distribution will be needed to guide the development and use of GBS conjugate vaccines. We designed sequencing primers based on the previously published sequences of the capsular polysaccharide (cps) gene clusters to further define partial cps gene clusters for eight of the nine GBS serotypes (serotypes Ia to VII). Subsequently, we designed and evaluated primers to identify serotypes Ia, Ib, III, IV, V, and VI directly by PCR and all eight serotypes (serotypes Ia to VII) by sequence heterogeneity. A total of 206 clinical GBS isolates were used to compare our molecular serotype (MS) identification method with conventional serotyping (CS). All clinical isolates were assigned an MS, whereas 188 of 206 (91.3%) were assigned a serotype by use of antisera. A small number of isolates (serosubtypes III-3 and III-4) showed different serotype specificities between PCR and sequencing, but the PCR results correlated with those obtained by CS. The overall agreement between the MS identification method and CS for isolates for which results of both tests were available was 100% (188 of 188 isolates). The MS identification method is a specific and practical alternative to conventional GBS serotyping and will facilitate epidemiological studies.     

Identification of Fusarium Species Involved in Human Infections by 28S rRNA Gene Sequencing

Fusarium spp. have emerged as major opportunistic fungal agents. Since new antifungal agents exhibit variable activity against Fusarium isolates depending on the species, rapid identification at the species level is required. Conventional culture methods are difficult, fastidious, and sometimes inconclusive. In this work, we sequenced a 440-bp fragment encoding the 28S rRNA from 33 Fusarium isolates belonging to six Fusarium species associated with human infections. The data were then analyzed by the neighbor-joining method. By using distance matrix analysis and constructing the phylogram, we could easily distinguish the different species for all but one isolate. The method also allowed differentiation between the closely related genera Acremonium and Cylindrocarpon. In contrast to the case with conventional methods, the results could be obtained within 48 h from a 3-day culture and are independent of mycologist experience, making this method rapid and reliable for identification of Fusarium species isolated from patients.     

Rapid Identification of Clinically Relevant Nocardia Species to Genus Level by 16S rRNA Gene PCR

Two regions of the gene coding for 16S rRNA in Nocardia species were selected as genus-specific primer sequences for a PCR assay. The PCR protocol was tested with 60 strains of clinically relevant Nocardia isolates and type strains. It gave positive results for all strains tested. Conversely, the PCR assay was negative for all tested species belonging to the most closely related genera, including Dietzia, Gordona, Mycobacterium, Rhodococcus, Streptomyces, and Tsukamurella. Besides, unlike the latter group of isolates, all Nocardia strains exhibited one MlnI recognition site but no SacI restriction site. This assay offers a specific and rapid alternative to chemotaxonomic methods for the identification of Nocardia spp. isolated from pathogenic samples.     

Inhibition of Phagocytosis-Associated Chemiluminescence by Superoxide Dismutase

During the process of phagocytosis, human leukocytes emit a burst of luminescence which can be measured in a liquid scintillation spectrometer. The enzyme superoxide dismutase, which removes superoxide anions (O(2.)), inhibited this chemiluminescence by 70% at a concentration of 100 mug/ml. The enzyme did not inhibit phagocytosis. These results support other studies indicating that O(2.) is elaborated by phagocytizing leukocytes. They also indicate that O(2.) plays a major role in phagocytosis-associated chemiluminescence, though not necessarily as the luminescing agent. Catalase and benzoate inhibited the chemiluminescence of phagocytosis to a slight extent, suggesting that hydrogen peroxide and hydroxyl radical, respectively, might also be involved in this phenomenon. The relationship between the mediators of chemiluminescence and those responsible for phagocytic bactericidal activity remains to be defined.     

Phylogeny and Identification of Pantoea Species and Typing of Pantoea agglomerans Strains by Multilocus Gene Sequencing

Pantoea agglomerans and other Pantoea species cause infections in humans and are also pathogenic to plants, but the diversity of Pantoea strains and their possible association with hosts and disease remain poorly known, and identification of Pantoea species is difficult. We characterized 36 Pantoea strains, including 28 strains of diverse origins initially identified as P. agglomerans, by multilocus gene sequencing based on six protein-coding genes, by biochemical tests, and by antimicrobial susceptibility testing. Phylogenetic analysis and comparison with other species of Enterobacteriaceae revealed that the genus Pantoea is highly diverse. Most strains initially identified as P. agglomerans by use of API 20E strips belonged to a compact sequence cluster together with the type strain, but other strains belonged to diverse phylogenetic branches corresponding to other species of Pantoea or Enterobacteriaceae and to probable novel species. Biochemical characteristics such as fosfomycin resistance and utilization of d-tartrate could differentiate P. agglomerans from other Pantoea species. All 20 strains of P. agglomerans could be distinguished by multilocus sequence typing, revealing the very high discrimination power of this method for strain typing and population structure in this species, which is subdivided into two phylogenetic groups. PCR detection of the repA gene, associated with pathogenicity in plants, was positive in all clinical strains of P. agglomerans, suggesting that clinical and plant-associated strains do not form distinct populations. We provide a multilocus gene sequencing method that is a powerful tool for Pantoea species delineation and identification and for strain tracking.The genus Pantoea includes several species that are generally associated with plants, either as epiphytes or as pathogens, and some species can cause disease in humans. Pantoea agglomerans, the Pantoea species most commonly isolated from humans, is widely distributed in nature and has been isolated from numerous ecological niches, including plants, water, soil, humans, and animals. It is frequently associated with plants as an epiphyte or an endophyte, and some isolates have been reported to be tumorogenic pathogens (20, 51). As an opportunistic human pathogen, P. agglomerans can occur sporadically or in outbreaks. At the beginning of the 1970s, P. agglomerans (then called Enterobacter agglomerans) was implicated in a large U.S. and Canadian outbreak of septicemia caused by contaminated closures on bottles of infusion fluids; 25 hospitals were involved, with 378 cases (34). Since then, P. agglomerans bacteremia has also been described in association with the contamination of intravenous fluid, parenteral nutrition, the anesthetic agent propofol, blood products, and transference tubes used for intravenous hydration (2-4, 22, 36). P. agglomerans has been recovered from joint fluids of patients with arthritis, synovitis, or osteomyelitis (7). Infection often occurs after injuries with plant thorns, wood slivers, or wooden splinters (7, 8, 16, 30, 40, 49). Cases of peritonitis due to P. agglomerans have also been reported (15, 31). Finally, P. agglomerans, which is known to colonize cotton and cotton plants heavily, is associated with cotton fever, a benign febrile syndrome seen in intravenous drug abusers (14).Seven Pantoea species are currently distinguished: P. agglomerans, the type species of the genus; Pantoea ananatis; Pantoea stewartii (divided into the two subspecies Pantoea stewartii subsp. stewartii, the agent of Stewart''s vascular wilt in maize and sweet corn, and Pantoea stewartii subsp. indologenes); Pantoea dispersa; Pantoea citrea; Pantoea punctata; and Pantoea terrea (18, 20, 28, 37). The Pantoea agglomerans complex was previously designated Erwinia herbicola or Enterobacter agglomerans (18). The biochemical heterogeneity of P. agglomerans and related strains and species renders identification difficult, even if several biochemical or nutritional characteristics distinguish the “Japanese group” (20) of Pantoea species (P. citrea, P. punctata, and P. terrea). Currently, confident identification is not achieved routinely.Precise knowledge of the phylogenetic relationships and the degree of genetic distinctness among Pantoea species is a prerequisite for their correct identification. Phylogenetic relationships among Pantoea species were initially based on 16S rRNA analysis (24, 39, 53), which showed that P. agglomerans, P. ananatis, and P. stewartii were closely related. The same result was obtained based on the three protein-coding genes atpD, carA, and recA (53). However, only one or a few strains per species were analyzed, and the phylogenetic relationships of the three “Japanese” species with the other Pantoea species have, to our knowledge, never been described. Hence, it is not clear whether Pantoea species are clearly demarcated and if the genus Pantoea forms only one phylogenetic branch.Defining bacterial species remains a challenge, especially given the fact that homologous recombination or lateral gene transfer can disturb species boundaries. Currently, the approach to defining bacterial species uses both genomic and phenotypic characteristics. The pragmatic values of 70% DNA-DNA reassociation and a difference of <5°C in the melting temperature have been proposed as a cutoff for species definition (50) but are technically challenging to obtain. Multilocus sequence analysis (MLSA) provides an alternative way to define species and to explore sequence discontinuities among them (19). Phylogenetic analysis of concatenated multiple protein-coding genes sometimes allow one to clearly separate species into sequence clusters that can be used to define species, even if borders may be fuzzy for highly recombinogenic species (17, 23). Moreover, the MLSA approach can be used to estimate homologous recombination among species, which is important for determining the reliability of molecular identification based on one or a few genes and for estimating the impact of homologous recombination or lateral gene transfer on the speciation process. So far, no MLSA approach has been reported for Pantoea species.Strain typing and population genetics studies are necessary for epidemiological purposes and to identify strains with important phenotypes such as virulence to plants or humans (11, 47). For example, it is important to determine if P. agglomerans strains differ in their abilities to infect humans or to cause specific diseases in plants. Currently, P. agglomerans strains can be differentiated using fluorescent amplified fragment length polymorphism (5) or pulsed-field gel electrophoresis (51). However, these methods do not provide unambiguous definition of clones and clonal families, and the results are generally difficult to compare among laboratories. Currently, the amount of diversity within P. agglomerans and its population structure are unknown. A widely accepted method for studying strain relationships is multilocus sequence typing (MLST) (33). It consists of sequencing internal portions of several protein-coding genes. This method provides unambiguous and portable data and allows one to compare data worldwide, which is necessary in order to achieve a comprehensive overview of strain diversity and epidemiological distribution (32). In addition, this method is suitable for studying strain phylogeny and allows one to address evolutionary questions at the strain level within species (11). In contrast to MLSA, MLST relies on the comparison of allelic profiles of strains within species, whereas MLSA uses concatenation of gene sequences to define boundaries and phylogenetic relationships between species.There are few data concerning the susceptibility of P. agglomerans to antimicrobial agents. In 1986, Muytjens et al. reported in vitro susceptibility data for eight species of Enterobacter, including 27 strains of E. agglomerans (38). The strains exhibited very variable susceptibilities to β-lactams, aminoglycosides, and quinolones. Hieng et al. (25) described a case of septicemia due to an Erwinia herbicola strain that was resistant to ampicillin, carbenicillin, and cephalothin (cefalotin) and susceptible to other antibiotics usually active on gram-negative bacilli. Cruz et al. (7) reported similar results. No β-lactamase was found among the Pantoea species. In 2000, a clinical isolate of P. agglomerans recovered from a patient with septic arthritis was reported to be highly resistant to fosfomycin (8).The objectives of this study were (i) to define the phylogenetic relationships among P. agglomerans strains, or strains that may be misidentified as P. agglomerans, and other species of Enterobacteriaceae; (ii) to characterize Pantoea species or phylogenetic clusters biochemically and for their susceptibilities to antimicrobial agents; and (iii) to develop and evaluate MLST for strain discrimination and determination of the population structure of P. agglomerans.     

人铜锌超氧化物歧化酶的基因工程研究——Ⅰ.人铜锌超氧化物歧化酶基因的合成与克隆

本文报道人铜锌超氧化物歧化酶(Cu/Zn superoxide Dismutase, SOD)基因的合成与克隆。采用大肠杆菌偏爱密码子,按入Cu/Zu SOD一级结构顺序编码。基因全长495个碱基对,合成时,分成26个基因片段分别合成。采用分级退火的方法,在T4 DNA连接酶作用下,将基因片段组装成完整的基因,经序列分析证明,合成的基因顺序与设计一致。     

Memory Decline of Aging Reduced by Extracellular Superoxide Dismutase Overexpression

Extracellular superoxide dismutase (EC-SOD) plays an important role in controlling oxidative stress as well as intercellular signaling. In the current study, we tested the effect of EC-SOD overexpression over the lifespan of a set of mice and their wild-type controls to determine the time scale over which EC-SOD overexpression might attenuate aging-induced memory impairment. Mice with overexpression of EC-SOD and wild-type controls were initially trained on the radial-arm maze as young adults (3–5 months) and then retrained during middle age (12–14 months) and retested in old age at 27 and 30 months. There was little EC-SOD effect during the young adult middle age periods. EC-SOD overexpression prevented the decline in choice accuracy when the mice were 27–30 months of age. The EC-SOD overexpressing mice maintained their performance, while the wild-type mice declined to naïve levels of performance by 30 months of age. Enhancement of EC-SOD activity appears to improve memory performance specifically in aging mice. EC-SOD mimetic treatment during the course of aging may hold promise for aging-induced cognitive impairment.     

比色法测定超氧化物歧化酶的临床评价及其应用研究

对比色法测定超氧化物歧化酶试剂盒进行临床评价;比较健康人群与多种疾病患者的血清超氧化物歧化酶（Super-oxide dismutase,SOD）浓度,探索血清SOD对多种疾病的预警价值。用罗氏全自动生化分析仪测定,分析评价比色法测定超氧化物歧化酶试剂盒的准确性、精密度及线性等指标。选取明确诊断的多种疾病（包括冠心病、肝脏疾病和脑部疾病）共105例作为疾病组及144名健康查体者作为对照组,测定两组血清SOD浓度。结果显示,用该方法测定SOD高、低两种浓度血清的准确度分别为99.8%和97.8%,批内变异系数分别为0.59%和0.43%,批间变异系数分别为1.45%和1.09%;在12.5～200U/mL范围内,线性良好,与理论值的相关系数为0.9999（R2=0.9998）。疾病组的血清SOD水平为97.77±28.31U/mL,显著低于对照组139.79±13.88 U/mL（P〈0.01）;疾病组内的每种疾病血清SOD水平均明显低于对照组（P〈0.01）,但各种疾病之间血清SOD水平无差异;比色法测定血清SOD的ROC曲线下面积为90.5%;当界值为116.15 U/mL时,约登指数最大（0.86）,此时灵敏度为97.22%,特异性为74.3%。以上结果表明,超氧化物歧化酶比色法试剂盒操作简便、稳定、快速,符合临床应用要求,适合进行临床大样本的常规检测。对多种疾病具有良好的预警价值。     

Identification and Molecular Analysis of the Gene Encoding Rickettsia typhi Hemolysin

Rickettsia typhi, the causative agent of murine typhus, grows directly within the host cell cytoplasm, accumulating a large number of progeny, and eventually lyses the cells. Typhus group rickettsiae (R. typhi and R. prowazekii) adhere to and lyse human and sheep erythrocytes. However, the molecular mechanism underlying erythrocyte lysis by R. typhi has not been defined. Here we describe the cloning and nucleotide sequence analysis of the gene (tlyC) encoding a hemolysin from R. typhi. DNA sequence analysis of R. typhi tlyC revealed an open reading frame of 912 bp, which encodes a protein of 304 amino acids with a predicted molecular mass of 38 kDa. To associate the R. typhi tlyC gene product with hemolytic activity, we performed complementation studies with hemolysin-negative Proteus mirabilis WPM111 (a HpmA(-) mutant of BA6163) transformed with R. typhi tlyC or R. typhi GFPuv-tlyC constructs. We demonstrated that the cloned tlyC gene conferred a hemolytic phenotype on an otherwise nonhemolytic mutant of P. mirabilis. The availability of the cloned R. typhi tlyC will permit further characterization and definition of its role in rickettsial virulence.     

Neutralization of Mitochondrial Superoxide by Superoxide Dismutase 2 Promotes Bacterial Clearance and Regulates Phagocyte Numbers in Zebrafish

Mitochondria are known primarily as the location of the electron transport chain and energy production in cells. More recently, mitochondria have been shown to be signaling centers for apoptosis and inflammation. Reactive oxygen species (ROS) generated as by-products of the electron transport chain within mitochondria significantly impact cellular signaling pathways. Because of the toxic nature of ROS, mitochondria possess an antioxidant enzyme, superoxide dismutase 2 (SOD2), to neutralize ROS. If mitochondrial antioxidant enzymes are overwhelmed during severe infections, mitochondrial dysfunction can occur and lead to multiorgan failure or death. Pseudomonas aeruginosa is an opportunistic pathogen that can infect immunocompromised patients. Infochemicals and exotoxins associated with P. aeruginosa are capable of causing mitochondrial dysfunction. In this work, we describe the roles of SOD2 and mitochondrial ROS regulation in the zebrafish innate immune response to P. aeruginosa infection. sod2 is upregulated in mammalian macrophages and neutrophils in response to lipopolysaccharide in vitro, and sod2 knockdown in zebrafish results in an increased bacterial burden. Further investigation revealed that phagocyte numbers are compromised in Sod2-deficient zebrafish. Addition of the mitochondrion-targeted ROS-scavenging chemical MitoTEMPO rescues neutrophil numbers and reduces the bacterial burden in Sod2-deficient zebrafish. Our work highlights the importance of mitochondrial ROS regulation by SOD2 in the context of innate immunity and supports the use of mitochondrion-targeted ROS scavengers as potential adjuvant therapies during severe infections.     

Contribution of Mn-Cofactored Superoxide Dismutase (SodA) to the Virulence of Streptococcus agalactiae

Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide, which, in turn, is metabolized by catalases and/or peroxidases. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and hence play a role in the pathogenesis of certain bacteria. We previously demonstrated that group B streptococci (GBS) possess a single Mn-cofactored superoxide dismutase (SodA). To analyze the role of this enzyme in the pathogenicity of GBS, we constructed a sodA-disrupted mutant of Streptococcus agalactiae NEM316 by allelic exchange. This mutant was subsequently cis complemented by integration into the chromosome of pAT113/Sp harboring the wild-type sodA gene. The SOD specific activity detected by gel analysis in cell extracts confirmed that active SODs were present in the parental and complemented strains but absent in the sodA mutant. The growth rates of these strains in standing cultures were comparable, but the sodA mutant was extremely susceptible to the oxidative stress generated by addition of paraquat or hydrogen peroxide to the culture medium and exhibited a higher mutation frequency in the presence of rifampin. In mouse bone marrow-derived macrophages, the sodA mutant showed an increased susceptibility to bacterial killing by macrophages. In a mouse infection model, after intravenous injection the survival of the sodA mutant in the blood and the brain was markedly reduced in comparison to that of the parental and complemented strains whereas only minor effects on survival in the liver and the spleen were observed. These results suggest that SodA plays a role in GBS pathogenesis.     

Identification of Group a Streptococci by Reverse Passive Hemagglutination

A method for preparing sensitized human 0 erythrocytes with specific antibody is reported. Whole sera or 40% ammonium sulfate insoluble antibody globulin fractions did not satisfactorily sensitize erythrocytes to agglutinate in the presence of either group specific polysaccharide of group A beta-hemolytic streptococci or antigen produced during colony formation. Antibody purified-by affinity chromatography sensitized the erythrocytes to rapidly agglutinate in the presence of either antigen. No spontaneous or “pseudo-immune” agglutination occurred when the sensitized erythrocytes were suspended in human serum. Such sensitized human cells, while more difficult to prepare than sensitized staphylococci, should be suitable for detecting bacterial or viral antigens in vivo.     

人超氧化物歧化酶用于细胞毒活性的检测

为了探讨人超氧化物歧化酶(SOD)值测定,作为一种新的细胞毒活性的检测方法及其临床应用的可行性,本文对含有不同细胞数的正常人外周血单个核细胞,反复冻融后,用放免方法(RIA)测定上清中的SOD值,观察四组不同浓度细胞数量与所测SOD值的相关性.测定20份标本SOD值均值±标准差分别为:细胞数为4×106组:SOD值为490±26.3ng/mL;2×106组;249±20.8ng/mL;1×106组:132.55±13.03ng/mL;5×105组:102.65±48.5ng/mL.四组细胞数与SOD均值的相关系数为0.91,绘制散点图,发现5×105组的均值偏离另三组的直线趋势,予以去除后,剩余的三组的相关系数为1.03,呈现完全相关.结果显示:SOD值在同一类型细胞中的含量相对恒定,在个体间一致性较高;SOD值与单个核细胞量存在直线相关性,提示这个参数可用于了解某类细胞被破坏的情况.值得注意的是,在细胞浓度较低时,SOD值可能会存在误差,建议实验时标本的细胞数宜控制在1×106以上.     

矽肺患者抗氧化指标超氧化物歧化酶检测的分析

探讨血清抗氧化应激指标超氧化物歧化酶( SOD)在不同期别矽肺患者和矽肺合并感染的患者中的变化,为煤工尘肺早期损伤的监测和防治提供可能的线索.选取矽肺病人210例,其中I期94例,Ⅱ期 95例,Ⅲ 期21例.分组后检测血清超氧化物歧化酶( SOD) 的水平.结果显示:矽肺组及矽肺合并其它疾病组血清SOD水平均较对照组明...     

Identification of Group a Streptococci by Reverse Passive Hemagglutination

A method for preparing sensitized human 0 erythrocytes with specific antibody is reported. Whole sera or 40% ammonium sulfate insoluble antibody globulin fractions did not satisfactorily sensitize erythrocytes to agglutinate in the presence of either group specific polysaccharide of group A beta-hemolytic streptococci or antigen produced during colony formation. Antibody purified-by affinity chromatography sensitized the erythrocytes to rapidly agglutinate in the presence of either antigen. No spontaneous or “pseudo-immune” agglutination occurred when the sensitized erythrocytes were suspended in human serum. Such sensitized human cells, while more difficult to prepare than sensitized staphylococci, should be suitable for detecting bacterial or viral antigens in vivo.     

Comparison of MALDI-TOF MS,Housekeeping Gene Sequencing,and 16S rRNA Gene Sequencing for Identification of Aeromonas Clinical Isolates

PurposeThe genus Aeromonas is a pathogen that is well known to cause severe clinical illnesses, ranging from gastroenteritis to sepsis. Accurate identification of A. hydrophila, A. caviae, and A. veronii is important for the care of patients. However, species identification remains difficult using conventional methods. The aim of this study was to compare the accuracy of different methods of identifying Aeromonas at the species level: a biochemical method, matrix-assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF MS), 16S rRNA sequencing, and housekeeping gene sequencing (gyrB, rpoB).ResultsThe conventional biochemical method and 16S rRNA sequencing identified Aeromonas at the genus level very accurately, although species level identification was unsatisfactory. MALDI-TOF MS system correctly identified 60 (92.3%) isolates at the species level and an additional four (6.2%) at the genus level. Overall, housekeeping gene sequencing with phylogenetic analysis was found to be the most accurate in identifying Aeromonas at the species level.ConclusionThe most accurate method of identification of Aeromonas to species level is by housekeeping gene sequencing, although high cost and technical difficulty hinder its usage in clinical settings. An easy-to-use identification method is needed for clinical laboratories, for which MALDI-TOF MS could be a strong candidate.     

Comparison of Three Methods for Identification of Streptococcus milleri Group Isolates to Species Level

 A collection of 180 clinical isolates of Streptococcus milleri group were identified to species level using two phenotypic methods (a commercial system and the reference method based on differential phenotypic reactions) and a genotypic method (hybridisation of the 16S rRNA gene with species-specific probes) in order to evaluate the performance of the respective methods. A high level of agreement (80%) was observed between the results of the reference method and the genotypic method. The highest level of agreement was found for the species Streptococcus anginosus (83%), a high level of agreement (76%) also being achieved for Streptococcus constellatus and Streptococcus intermedius. The sensitivity of the commercial system compared to the genotypic method was 76% overall, but it was low (57.5%) for Streptococcus intermedius. Twenty-five strains belonged to the recently described CI strains.     

Inflammatory Cytokine Induced Regulation of Superoxide Dismutase 3 Expression by Human Mesenchymal Stem Cells

Increasing evidence suggests that bone marrow derived-mesenchymal stem cells (MSCs) have neuroprotective properties and a major mechanism of action is through their capacity to secrete a diverse range of potentially neurotrophic or anti-oxidant factors. The recent discovery that MSCs secrete superoxide dismutase 3 (SOD3) may help explain studies in which MSCs have a direct anti-oxidant activity that is conducive to neuroprotection in both in vivo and in vitro. SOD3 attenuates tissue damage and reduces inflammation and may confer neuroprotective effects against nitric oxide-mediated stress to cerebellar neurons; but, its role in relation to central nervous system inflammation and neurodegeneration has not been extensively investigated. Here we have performed a series of experiments showing that SOD3 secretion by human bone marrow-derived MSCs is regulated synergistically by the inflammatory cytokines TNF-alpha and IFN-gamma, rather than through direct exposure to reactive oxygen species. Furthermore, we have shown SOD3 secretion by MSCs is increased by activated microglial cells. We have also shown that MSCs and recombinant SOD are able to increase both neuronal and axonal survival in vitro against nitric oxide or microglial induced damage, with an increased MSC-induced neuroprotective effect evident in the presence of inflammatory cytokines TNF-alpha and IFN-gamma. We have shown MSCs are able to convey these neuroprotective effects through secretion of soluble factors alone and furthermore demonstrated that SOD3 secretion by MSCs is, at least, partially responsible for this phenomenon. SOD3 secretion by MSCs maybe of relevance to treatment strategies for inflammatory disease of the central nervous system.