Mycobacterium Tuberculosis Infection in Rhesus Monkeys (Macaca mulatta) and Evaluation of ESAT-6 and CFP10 as Immunodiagnostic Antigens#

Tuberculosis (TB) in nonhuman primates is a serious menace to the welfare of the animals and human who come into contact with them, while the rapid, accurate, and robust diagnosis is challenging. In this study, we first sought to establish an appropriate primate TB model resembling natural TB in nonhuman primates. Four rhesus monkeys (Macaca mulatta) of Chinese origin were infected intratracheally with two low doses of M. tuberculosis H37Rv. Regardless of the infectious doses, all monkeys were demonstrated to be successfully infected by clinical assessments, tuberculin skin test conversions, peripheral immune responses, gross observations, histopathology analysis, and M. tuberculosis burdens. Furthermore, we extended the usefulness of this model for assessing the following immunodiagnostic antigens: CFP10, ESAT-6, CFP10-ESAT-6, and an antigen cocktail of CFP10 and ESAT-6. The data showed that CFP10 was an M. tuberculosis-specific, “early” antigen used for serodiagnosis of TB in nonhuman primates. In conclusion, we established a useful primate TB model depending on low doses of M .tuberculosis and affording new opportunities for studies of M. tuberculosis disease and diagnostics.     

Morphogenesis beyond Cytokinetic Arrest in Saccharomyces cerevisiae

The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37°C, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother–daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.     

Topography of Extracellular Matrix Mediates Vascular Morphogenesis and Migration Speeds in Angiogenesis

The extracellular matrix plays a critical role in orchestrating the events necessary for wound healing, muscle repair, morphogenesis, new blood vessel growth, and cancer invasion. In this study, we investigate the influence of extracellular matrix topography on the coordination of multi-cellular interactions in the context of angiogenesis. To do this, we validate our spatio-temporal mathematical model of angiogenesis against empirical data, and within this framework, we vary the density of the matrix fibers to simulate different tissue environments and to explore the possibility of manipulating the extracellular matrix to achieve pro- and anti-angiogenic effects. The model predicts specific ranges of matrix fiber densities that maximize sprout extension speed, induce branching, or interrupt normal angiogenesis, which are independently confirmed by experiment. We then explore matrix fiber alignment as a key factor contributing to peak sprout velocities and in mediating cell shape and orientation. We also quantify the effects of proteolytic matrix degradation by the tip cell on sprout velocity and demonstrate that degradation promotes sprout growth at high matrix densities, but has an inhibitory effect at lower densities. Our results are discussed in the context of ECM targeted pro- and anti-angiogenic therapies that can be tested empirically.     

中国对虾非包涵体杆状病毒在体内的感染与发生

在感染发病成虾的肝胰腺和中肠上皮细胞的胞核内,出现大量病毒发生基质,核衣壳,套膜和完全的病毒粒子。病毒粒子为短杆状,两端呈钝园形,平均大小约为２５０－３００×ｌ１０ｎｍ,在核内无包涵体形成。在胞质内发现伴随病毒拉子并具有双层膜的蛋白质结构,这种结构建议称为该病毒的“封入体”。同时,我们认为这种无包涵体的杆状病毒,有可能应归属于杆状病毒科的第三个亚组。     

Choriodecidual group B streptococcal inoculation induces fetal lung injury without intra-amniotic infection and preterm labor in Macaca nemestrina

Background

Early events leading to intrauterine infection and fetal lung injury remain poorly defined, but may hold the key to preventing neonatal and adult chronic lung disease. Our objective was to establish a nonhuman primate model of an early stage of chorioamnionitis in order to determine the time course and mechanisms of fetal lung injury in utero.

Methodology/Principal Findings

Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118–125 days gestation (term = 172 days) received one of two treatments: 1) choriodecidual and intra-amniotic saline (n = 5), or 2) choriodecidual inoculation of Group B Streptococcus (GBS) 1×10⁶ colony forming units (n = 5). Cesarean section was performed regardless of labor 4 days after GBS or 7 days after saline infusion to collect fetal and placental tissues. Only two GBS animals developed early labor with no cervical change in the remaining animals. Despite uterine quiescence in most cases, blinded review found histopathological evidence of fetal lung injury in four GBS animals characterized by intra-alveolar neutrophils and interstitial thickening, which was absent in controls. Significant elevations of cytokines in amniotic fluid (TNF-α, IL-8, IL-1β, IL-6) and fetal plasma (IL-8) were detected in GBS animals and correlated with lung injury (p<0.05). Lung injury was not directly caused by GBS, because GBS was undetectable in amniotic fluid (∼10 samples tested/animal), maternal and fetal blood by culture and polymerase chain reaction. In only two cases was GBS cultured from the inoculation site in low numbers. Chorioamnionitis occurred in two GBS animals with lung injury, but two others with lung injury had normal placental histology.

Conclusions/Significance

A transient choriodecidual infection can induce cytokine production, which is associated with fetal lung injury without overt infection of amniotic fluid, chorioamnionitis or preterm labor. Fetal lung injury may, thus, occur silently without symptoms and before the onset of the fetal systemic inflammatory response syndrome.     

Ras GTPase Activating Protein CoIra1 Is Involved in Infection-Related Morphogenesis by Regulating cAMP and MAPK Signaling Pathways through CoRas2 in Colletotrichum orbiculare

Colletotrichum orbiculare is the causative agent of anthracnose disease on cucurbitaceous plants. Several signaling pathways, including cAMP–PKA and mitogen-activating protein kinase (MAPK) pathways are involved in the infection-related morphogenesis and pathogenicity of C. orbiculare. However, upstream regulators of these pathways for this species remain unidentified. In this study, CoIRA1, encoding RAS GTPase activating protein, was identified by screening the Agrobacterium tumefaciens-mediated transformation (AtMT) mutant, which was defective in the pathogenesis of C. orbiculare. The coira1 disrupted mutant showed an abnormal infection-related morphogenesis and attenuated pathogenesis. In Saccharomyces cerevisiae, Ira1/2 inactivates Ras1/2, which activates adenylate cyclase, leading to the synthesis of cAMP. Increase in the intracellular cAMP levels in coira1 mutants and dominant active forms of CoRAS2 introduced transformants indicated that CoIra1 regulates intracellular cAMP levels through CoRas2. Moreover, the phenotypic analysis of transformants that express dominant active form CoRAS2 in the comekk1 mutant or a dominant active form CoMEKK1 in the coras2 mutant indicated that CoRas2 regulates the MAPK CoMekk1–Cmk1 signaling pathway. The CoRas2 localization pattern in vegetative hyphae of the coira1 mutant was similar to that of the wild-type, expressing a dominant active form of RFP–CoRAS2. Moreover, we demonstrated that bimolecular fluorescence complementation (BiFC) signals between CoIra1 and CoRas2 were detected in the plasma membrane of vegetative hyphae. Therefore, it is likely that CoIra1 negatively regulates CoRas2 in vegetative hyphae. Furthermore, cytological analysis of the localization of CoIraI and CoRas2 revealed the dynamic cellular localization of the proteins that leads to proper assembly of F-actin at appressorial pore required for successful penetration peg formation through the pore. Thus, our results indicated that CoIra1 is involved in infection-related morphogenesis and pathogenicity by proper regulation of cAMP and MAPK signaling pathways through CoRas2.     

mzGroupAnalyzer-Predicting Pathways and Novel Chemical Structures from Untargeted High-Throughput Metabolomics Data

The metabolome is a highly dynamic entity and the final readout of the genotype x environment x phenotype (GxExP) relationship of an organism. Monitoring metabolite dynamics over time thus theoretically encrypts the whole range of possible chemical and biochemical transformations of small molecules involved in metabolism. The bottleneck is, however, the sheer number of unidentified structures in these samples. This represents the next challenge for metabolomics technology and is comparable with genome sequencing 30 years ago. At the same time it is impossible to handle the amount of data involved in a metabolomics analysis manually. Algorithms are therefore imperative to allow for automated m/z feature extraction and subsequent structure or pathway assignment. Here we provide an automated pathway inference strategy comprising measurements of metabolome time series using LC- MS with high resolution and high mass accuracy. An algorithm was developed, called mzGroupAnalyzer, to automatically explore the metabolome for the detection of metabolite transformations caused by biochemical or chemical modifications. Pathways are extracted directly from the data and putative novel structures can be identified. The detected m/z features can be mapped on a van Krevelen diagram according to their H/C and O/C ratios for pattern recognition and to visualize oxidative processes and biochemical transformations. This method was applied to Arabidopsis thaliana treated simultaneously with cold and high light. Due to a protective antioxidant response the plants turn from green to purple color via the accumulation of flavonoid structures. The detection of potential biochemical pathways resulted in 15 putatively new compounds involved in the flavonoid-pathway. These compounds were further validated by product ion spectra from the same data. The mzGroupAnalyzer is implemented in the graphical user interface (GUI) of the metabolomics toolbox COVAIN (Sun & Weckwerth, 2012, Metabolomics 8: 81–93). The strategy can be extended to any biological system.     

Oxidative Stress Alters Syndecan-1 Distribution in Lungs with Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by severe, progressive fibrosis. Roles for inflammation and oxidative stress have recently been demonstrated, but despite advances in understanding the pathogenesis, there are still no effective therapies for IPF. This study investigates how extracellular superoxide dismutase (EC-SOD), a syndecan-binding antioxidant enzyme, inhibits inflammation and lung fibrosis. We hypothesize that EC-SOD protects the lung from oxidant damage by preventing syndecan fragmentation/shedding. Wild-type or EC-SOD-null mice were exposed to an intratracheal instillation of asbestos or bleomycin. Western blot was used to detect syndecans in the bronchoalveolar lavage fluid and lung. Human lung samples (normal and IPF) were also analyzed. Immunohistochemistry for syndecan-1 and EC-SOD was performed on human and mouse lungs. In vitro, alveolar epithelial cells were exposed to oxidative stress and EC-SOD. Cell supernatants were analyzed for shed syndecan-1 by Western blot. Syndecan-1 ectodomain was assessed in wound healing and neutrophil chemotaxis. Increases in human syndecan-1 are detected in lung homogenates and lavage fluid of IPF lungs. Syndecan-1 is also significantly elevated in the lavage fluid of EC-SOD-null mice after asbestos and bleomycin exposure. On IHC, syndecan-1 staining increases within fibrotic areas of human and mouse lungs. In vitro, EC-SOD inhibits oxidant-induced loss of syndecan-1 from A549 cells. Shed and exogenous syndecan-1 ectodomain induce neutrophil chemotaxis, inhibit alveolar epithelial wound healing, and promote fibrogenesis. Oxidative shedding of syndecan-1 is an underlying cause of neutrophil chemotaxis and aberrant wound healing that may contribute to pulmonary fibrosis.Idiopathic pulmonary fibrosis (IPF)² is an interstitial lung disease characterized by severe and progressive fibrosis. IPF patients have a mean survival of 3–5 years (1, 2) and no effective therapies (3, 4), other than orthotopic lung transplantation, have proven to improve survival. The pathogenesis of IPF is poorly understood; however, inflammation and oxidant/antioxidant imbalances in the lung are thought to play important roles (5–7). A better understanding of the molecular mechanisms involved in oxidative injury and fibrosis could lead to the development of novel therapeutic targets.Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme bound to heparan sulfate in the lung extracellular matrix (8–10), which can inhibit inflammation (11, 12) and prevent subsequent development of fibrosis (13–16). Despite its beneficial role, the mechanisms through which EC-SOD protects the lung remain unknown.The extracellular matrix (ECM) is essential for tissue homeostasis and changes in the ECM microenvironment can be detrimental to cell function during inflammation and wound healing. Heparan sulfate proteoglycans (HSPG) contain a membrane-bound core protein and extracellular carbohydrate side chains. Syndecans are the most abundant HSPG in humans; there are 4 isoforms with variable cell expression (17, 18). Both syndecan-1 and -4 are expressed in the lung, with epithelial cell and ubiquitous expression, respectively (19). Syndecans are essential for ECM homeostasis by binding cytokines and growth factors, acting as co-receptors and soluble effectors. They also have potential roles in inflammation (18, 20, 21), fibrosis (22, 23), and wound healing (24–26). Syndecans are shed under physiological and pathological conditions but the function of shed syndecans is poorly understood (22). Reactive oxygen species (ROS) are capable of fragmenting HSPG (27) and other ECM components. Notably, EC-SOD has been shown to prevent oxidative damage to many ECM components (23, 28, 29). Within the lung, EC-SOD binds to syndecan-1 on the cell surface via a heparin-binding domain (8, 30). Because of the known functions of syndecans and its close interaction with EC-SOD, syndecan-1 is a key target that may contribute to the anti-inflammatory and anti-fibrotic effects of EC-SOD in the lung and in the pulmonary fibrosis.This study was conducted to determine the role of EC-SOD in protecting the ECM from oxidative stress and to investigate our hypothesis that EC-SOD protects the lung from inflammation and fibrosis by inhibiting oxidant-induced shedding of syndecan-1. Our findings suggest that a loss of EC-SOD in the lung leaves syndecan-1 vulnerable to oxidative stress and that oxidatively shed syndecan-1 ectodomain induces neutrophil chemotaxis, impairs epithelial wound healing, and promotes fibrogenesis. The discovery that oxidative stress alters the distribution of syndecan-1 in the lung microenvironment is a novel finding in the context of pulmonary fibrosis. These findings advance the understanding of the pathogenesis of idiopathic pulmonary fibrosis and provide a potential new therapeutic target for intervention in IPF.     

Enterococcus faecalis from Healthy Infants Modulates Inflammation through MAPK Signaling Pathways

Colonizing commensal bacteria after birth are required for the proper development of the gastrointestinal tract. It is believed that bacterial colonization pattern in neonatal gut affects gut barrier function and immune system maturation. Studies on the development of faecal microbiota in infants showed that the neonatal gut was first colonized with enterococci followed by other microbiota such as Bifidobacterium. Other studies showed that babies who developed allergy were less often colonized with Enterococcus during the first month of life as compared to healthy infants. Many studies have been conducted to elucidate how bifidobacteria or lactobacilli, some of which are considered probiotic, regulate infant gut immunity. However, fewer studies have been focused on enterococi. In our study, we demonstrate that E. faecalis, isolated from healthy newborns, suppress inflammatory responses activated in vivo and in vitro. We found E. faecalis attenuates proinflammatory cytokine secretions, especially IL-8, through JNK and p38 signaling pathways. This finding shed light on how the first colonizer, E.faecalis, regulates inflammatory responses in the host.     

Age- and Sex-Based Hematological and Biochemical Parameters for Macaca fascicularis

Background

The cynomolgus monkey (Macaca fascicularis) has been increasingly used in biomedical research, making knowledge of its blood-based parameters essential to support the selection of healthy subjects and its use in preclinical research. As age and sex affect these blood-based parameters, it is important to establish baseline indices for these parameters on an age and sex basis and determine the effects of age and sex on these indices.

Methods

A total of 917 cynomolgus monkeys (374 males and 543 females) were selected and segregated by age (five groups) and sex. A total of 30 hematological and 22 biochemical parameters were measured, and the effects of age and sex were analyzed.

Results

Baseline indices for hematological and biochemical parameters were separately established by age and sex. Significant effects by age, sex, and age-sex interaction were observed in a number of blood parameters. In the 49–60 months and 61–72 months age groups, red blood cell count, hemoglobulin, and hematocrit showed significantly lower values (P<0.01) in females than males. Serum alkaline phosphatase varied with age in both sexes (P<0.01) and was significantly higher in females than males (P<0.05) in the groups aged 13–24 months and 25–36 months; however, in the three groups aged over 25–36 months, serum alkaline phosphatase was significantly lower in females than males (P<0.01). Creatinine concentration increased with age (P<0.01) in all age groups; specifically in the groups aged 49–60 months and 61–72 months, creatinine was significantly higher (P<0.01) in males than females. Total protein and globulin both increased with age (P<0.01).

Conclusion

The baseline values of hematological and biochemical parameters reported herein establish reference indices of blood-based parameters in the cynomolgus monkey by age and sex, thereby aiding researchers in selecting healthy subjects and evaluating preclinical studies using this species.     

Enterovirus 74 Infection in Children

Enterovirus 74 (EV74) is a rarely detected viral infection of children. In 2010, EV74 was identified in New Zealand in a 2 year old child with acute flaccid paralysis (AFP) through routine polio AFP surveillance. A further three cases of EV74 were identified in children within six months. These cases are the first report of EV74 in New Zealand. In this study we describe the near complete genome sequence of four EV74 isolates from New Zealand, which shows only limited sequence identity in the non-structural proteins when compared to the other two known EV74 sequences. As is typical of enteroviruses multiple recombination events were evident, particularly in the P2 region and P3 regions. This is the first complete EV74 genome sequenced from a patient with acute flaccid paralysis.     

Cryptococcus neoformans Infection in Mice Lacking Type I Interferon Signaling Leads to Increased Fungal Clearance and IL-4-Dependent Mucin Production in the Lungs

Type I interferons (IFNs) are secreted by many cell types upon stimulation via pattern recognition receptors and bind to IFN-α/β receptor (IFNAR), which is composed of IFNAR1 and IFNAR2. Although type I IFNs are well known as anti-viral cytokines, limited information is available on their role during fungal infection. In the present study, we addressed this issue by examining the effect of IFNAR1 defects on the host defense response to Cryptococcus neoformans. In IFNAR1KO mice, the number of live colonies was lower and the host immune response mediated not only by Th1 but also by Th2 and Th17-related cytokines was more accelerated in the infected lungs than in WT mice. In addition, mucin production by bronchoepithelial cells and expression of MUC5AC, a major core protein of mucin in the lungs, were significantly higher in IFNAR1KO mice than in WT mice. This increase in mucin and MUC5AC production was significantly inhibited by treatment with neutralizing anti-IL-4 mAb. In contrast, administration of recombinant IFN-αA/D significantly suppressed the production of IL-4, but not of IFN-γ and IL-17A, in the lungs of WT mice after cryptococcal infection. These results indicate that defects of IFNAR1 led to improved clearance of infection with C. neoformans and enhanced synthesis of IFN-γ and the IL-4-dependent production of mucin. They also suggest that type I IFNs may be involved in the negative regulation of early host defense to this infection.