A Randomised Trial Evaluating the Safety and Immunogenicity of the Novel Single Oral Dose Typhoid Vaccine M01ZH09 in Healthy Vietnamese Children

Background

The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile.

Objectives

We conducted a randomised placebo controlled, single-blind trial to evaluate the safety and immunogenicity of M01ZH09 in healthy Vietnamese children aged 5 to 14 years.

Methods

Subjects were randomly assigned to receive either a nominal dose of 5×10⁹ CFU of M01ZH09 or placebo and were followed up for 28 days. The primary safety outcome was the proportion of subjects with any adverse event attributed to M01ZH09. The primary immunogenicity endpoint was the proportion of subjects who showed a positive immune response to M01ZH09 in the Salmonella Typhi lipopolysaccharide (LPS) specific serum IgA and IgG ELISA.

Principal Findings

One hundred and fifty-one children were enrolled, 101 subjects received M01ZH09 and 50 subjects received placebo. An intention to treat analysis was conducted. There were no serious adverse events and no bacteraemias. In the M01ZH09 group, 26 (26%; 95% CI, 18–5%) of 101 subjects experienced adverse events compared to 11 (22%; 95% CI, 12–36%) of 50 subjects in the placebo group (odds ratio (OR) [95%CI]  = 1.23 [0.550–2.747]; p = 0.691). Faecal shedding of S. Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 was detected in 51 (51%; 95% CI, 41–61%) of 100 M01ZH09 subjects. No shedding was detected beyond day 3. A positive immune response, defined as 70% increase (1.7 fold change) in LPS specific serum IgG (day 14 or 28) and/or 50% increase (1.5 fold change) in LPS specific serum IgA (day 7 or 14) from baseline was detected in 98 (97%; 95% CI, 92–99%) of 101 M01ZH09 recipients and 8 (16%; 95% CI, 7–29%) of 50 placebo recipients. Twenty-eight (100%; 95% CI, 88–100%) of 28 vaccine recipients who were evaluated in the LPS specific IgA ELISPOT assay showed a positive response compared to none of the 14 placebo recipients tested.

Conclusions

This was the first phase II trial of a novel oral candidate typhoid vaccine in children in an endemic country. M01ZH09 had an appropriate safety profile and was immunogenic in children.

Trial Registration

Controlled-trials.comISRCTN91111837     

Discovery of novel metabolites from marine actinomycetes

Recent findings from culture-dependent and culture-independent methods have demonstrated that indigenous marine actinomycetes exist in the oceans and are widely distributed in different marine ecosystems. There is tremendous diversity and novelty among the marine actinomycetes present in marine environments. Progress has been made to isolate novel actinomycetes from samples collected at different marine environments and habitats. These marine actinomycetes produce different types of new secondary metabolites. Many of these metabolites possess biological activities and have the potential to be developed as therapeutic agents. Marine actinomycetes are a prolific but underexploited source for the discovery of novel secondary metabolites.     

Mobile Phone Based Clinical Microscopy for Global Health Applications

Light microscopy provides a simple, cost-effective, and vital method for the diagnosis and screening of hematologic and infectious diseases. In many regions of the world, however, the required equipment is either unavailable or insufficiently portable, and operators may not possess adequate training to make full use of the images obtained. Counterintuitively, these same regions are often well served by mobile phone networks, suggesting the possibility of leveraging portable, camera-enabled mobile phones for diagnostic imaging and telemedicine. Toward this end we have built a mobile phone-mounted light microscope and demonstrated its potential for clinical use by imaging P. falciparum-infected and sickle red blood cells in brightfield and M. tuberculosis-infected sputum samples in fluorescence with LED excitation. In all cases resolution exceeded that necessary to detect blood cell and microorganism morphology, and with the tuberculosis samples we took further advantage of the digitized images to demonstrate automated bacillus counting via image analysis software. We expect such a telemedicine system for global healthcare via mobile phone – offering inexpensive brightfield and fluorescence microscopy integrated with automated image analysis – to provide an important tool for disease diagnosis and screening, particularly in the developing world and rural areas where laboratory facilities are scarce but mobile phone infrastructure is extensive.     

CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage

CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.     

Shc depletion stimulates brown fat activity in vivo and in vitro

Adipose tissue is an important metabolic organ that integrates a wide array of homeostatic processes and is crucial for whole‐body insulin sensitivity and energy metabolism. Brown adipose tissue (BAT) is a key thermogenic tissue with a well‐established role in energy expenditure. BAT dissipates energy and protects against both hypothermia and obesity. Thus, BAT stimulation therapy is a rational strategy for the looming pandemic of obesity, whose consequences and comorbidities have a huge impact on the aged. Shc‐deficient mice (ShcKO) were previously shown to be lean, insulin sensitive, and resistant to high‐fat diet and obesity. We investigated the contribution of BAT to this phenotype. Insulin‐dependent BAT glucose uptake was higher in ShcKO mice. Primary ShcKO BAT cells exhibited increased mitochondrial respiration; increased expression of several mitochondrial and lipid‐oxidative enzymes was observed in ShcKO BAT. Levels of brown fat‐specific markers of differentiation, UCP1, PRDM16, ELOVL3, and Cox8b, were higher in ShcKO BAT. In vitro, Shc knockdown in BAT cell line increased insulin sensitivity and metabolic activity. In vivo, pharmacological stimulation of ShcKO BAT resulted in higher energy expenditure. Conversely, pharmacological inhibition of BAT abolished the improved metabolic parameters, that is the increased insulin sensitivity and glucose tolerance of ShcKO mice. Similarly, in vitro Shc knockdown in BAT cell lines increased their expression of UCP1 and metabolic activity. These data suggest increased BAT activity significantly contributes to the improved metabolic phenotype of ShcKO mice.     

The differentiation of Staphylococcus aureus from other Micrococcaceae isolated from bovine mammary glands

Haemolysin production, the slide coagulase test and the tube coagulase test were assessed for their capability to differentiate Staphylococcus aureus among other Micrococcaceae in 199 isolates from udders of cows in herds with a low bulk milk somatic cell count. The API-Staph test was used as a reference.
Haemolysin production was less effective in identifying Staph. aureus among Micrococcaceae than a combination of other tests. Differences were found in the predictive values of results from diagnostic protocols in which the slide coagulase test was performed on all Micrococcaceae, or on β-haemolysin-negative Micrococcaceae only. Diagnostic protocols in which haemolysin production was combined with the results of the other tests resulted in excellent diagnostic performance and a reduction in diagnostic procedures. Recommendations for routine Staph. aureus identification in bovine mastitis bacteriology are given.     

Unidirectional Movement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in Mucilage Extrusion,Adherence, and Ray Formation

CELLULOSE SYNTHASE5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed, in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion and mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella, providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence.The epidermal cells of Arabidopsis (Arabidopsis thaliana) seed coats produce two distinct secondary cell walls: pectin-rich mucilage and cellulose-rich columellae (Western et al., 2000). When seeds are hydrated, mucilage expands rapidly, rupturing the outer tangential cell wall and forming a mucilage capsule that surrounds the seed. Seed coat mucilage is composed primarily of rhamnogalacturonan I (RG I) and also contains homogalacturonan (HG), hemicelluloses (such as xylans and glucomannans), and cellulose (for review, see Haughn and Western, 2012). Extruded mucilage consists of an outer, nonadherent fraction and an inner, adherent fraction (Western et al., 2000, 2001; Macquet et al., 2007a). The adherent and nonadherent mucilage layers differ in the amount of methylesterified HG (Rautengarten et al., 2008; Saez-Aguayo et al., 2013; Voiniciuc et al., 2013), galactans (Dean et al., 2007; Macquet et al., 2007b), arabinans (Arsovski et al., 2009), mannans (Yu et al., 2014), and cellulose (Harpaz-Saad et al., 2011; Mendu et al., 2011; Sullivan et al., 2011), all of which influence the physical properties of the layers.Adherent mucilage has a distinct structure, which can be examined using cell wall dyes and antibodies. When treated with cellulose-specific dyes, densely stained rays extend from the top of each columella to the outer edge of the adherent layer, many cell lengths above the seed surface (Mendu et al., 2011; Sullivan et al., 2011). Cytological evidence indicates that cellulose, pectins, and mannans are components of the ray (Haughn and Western, 2012; Griffiths et al., 2014; North et al., 2014; Yu et al., 2014), although the exact manner in which they are assembled is unknown.Cellulose is abundant in mucilage rays and mediates adherence. Loss-of-function mutations in CELLULOSE SYNTHASE5 (CESA5) result in reduced cellulose levels and increased detachment of mucilage from the seed (Harpaz-Saad et al., 2011; Mendu et al., 2011; Sullivan et al., 2011; Griffiths et al., 2014). How a reduction in cellulose results in a loss of adherence is still unknown, but it likely involves interaction with other mucilage components such as pectin and arabinogalactan proteins (Griffiths et al., 2014). Since cesa5 mutants still have some cellulose in the rays of the adherent mucilage halo (Mendu et al., 2011; Sullivan et al., 2011), additional cellulose synthases must be involved in mucilage cellulose biosynthesis.The Arabidopsis genome encodes 10 different CESAs (Delmer, 1999; Richmond and Somerville, 2000). Multiple lines of evidence suggest that three different CESAs are required to form one active cellulose synthase complex (CSC; for review, see Somerville, 2006). CSCs are membrane-bound protein complexes that synthesize cellulose microfibrils in the apoplast (for review, see Somerville, 2006; Endler and Persson, 2011; Lei et al., 2012). CESA1, CESA3, and CESA6 are considered the core components of the primary wall CSC (Desprez et al., 2007; Persson et al., 2007). CESA2, CESA5, and CESA9 are partially redundant to CESA6 in primary wall biosynthesis, and genetic evidence suggests that each of these CESA polypeptides can form a functional CSC with CESA3 and CESA1 (Desprez et al., 2007; Persson et al., 2007). CESA10 is expressed in young plants, stems, floral tissue, and the base of rosette leaves (Beeckman et al., 2002; Doblin et al., 2002), but its function in cellulose biosynthesis is unclear. Other cesa mutant lines have been examined for altered mucilage phenotypes (cesa1, radially swollen1 [Burn et al., 2002; Sullivan et al., 2011], cesa2, cesa6, and cesa9 [Mendu et al., 2011]; CESA3, je5 [Sullivan et al., 2011] and cesa10-1 [Sullivan et al., 2011]); to date, only CESA5 has been shown to be required for cellulose biosynthesis during mucilage deposition.Two mutant alleles of CESA3, isoxaben resistant1-1 (ixr1-1) and ixr1-2, were isolated in a screen for resistance to the herbicide isoxaben (Scheible et al., 2001). Isoxaben inhibits the incorporation of Glc into the emerging cellulose polymer and is considered a potent and specific inhibitor of cellulose biosynthesis (Heim et al., 1990). Homozygous ixr1-1 and ixr1-2 lines show increased resistance to the herbicide, and the mutations causing this resistance were mapped to the genomic locus of CESA3 (Heim et al., 1990; Scheible et al., 2001). The ixr1-1 and ixr1-2 mutations cause amino acid substitutions near the C terminus of the CESA3 protein. ixr1-1 causes a Gly-to-Asn substitution (G998A) located in a transmembrane domain, while ixr1-2 contains a Thr-to-Ile substitution (T942I) in an apoplastic region of the protein between two transmembrane domains (Scheible et al., 2001). Recently, the ixr1-2 allele was shown to affect the velocity of CSCs in the plasma membrane, which consequently modifies cellulose crystallinity in the cell wall (Harris et al., 2012). It is not exactly clear how the ixr1-1 mutation affects cellulose biosynthesis. The effects of either of these mutations on seed coat mucilage have not been investigated.Since mucilage is composed primarily of pectins with smaller amounts of cellulose, seed coat epidermal cells represent an excellent system to study cellulose biosynthesis and interactions between cellulose and other wall components in muro. In this study, we investigated how cellulose is synthesized and deposited in seed coat epidermal cells. We show that at least three different CESA proteins are highly expressed in the seed coat epidermis during mucilage biosynthesis. These CESAs are oriented and move in a linear fashion around the cytoplasmic column of each cell in an identical pattern to cortical microtubules. In addition, we provide evidence that the adherent mucilage has a helical structure that expands and unwinds during extrusion to form the mucilage ray. We propose that during seed coat epidermal cell development, the biosynthesis of cellulose predetermines the structure of rays in the adherent mucilage layer.     

Mutual regulation between mitochondrial ATPase and respiratory chain activities

Compounds made from the reaction of fluorescamine with simple primary amines and with mycosamine-containing macrolide antibiotics (e.g., amphotericin B) are used to investigate possible interactions between ATPase and respiration enzymes in rat liver mitochondria. The following observations have been made. (1) The acyclic form of the benzyl amine-fluorescamine compound stimulates the ATPase-linked inorganic phosphate formation, and this stimulation is not affected by rotenone, antimycin A, and potassium cyanide. In contrast, the respiratory inhibitors are able to prevent the stimulation of ATPase activity that is caused by conventional uncouplers e.g., 2,4-dinitrophenol. (2) The acyclic form of the amphotericin B-fluorescamine compound has no effect on ATPase-linked inorganic phosphate formation rate. However, in the presence of the antibiotic-fluorescamine compounds, the respiratory inhibitors are no longer able to prevent the uncoupler-stimulated ATPase activity. (3) The amine-fluorescamine modifiers have no effect on rotenone-sensitive NADH-cytochrome c reductase, on succinate-cytochrome c reductase, and on cytochrome oxidase in submitochondrial particles. (4) The amine-fluorescamine modifiers decrease the rate of the ATP-driven NAD⁺ reduction by succinate in submitochondrial particles. (5) The amine-fluorescamine modifiers inhibit the stimulation of respiration that is caused by conventional uncouplers, although the modifiers have no effect on the kinetics of the proton influx induced by uncouplers. The data are consistent with the hypothesis that the ATPase-linked and respiration-linked proton pumps may interact directly with each other, and this step establishes the mutual regulation between ATPase and respiratory activities.     

B-cell maturation protein, which binds the tumor necrosis factor family members BAFF and APRIL, is dispensable for humoral immune responses

B-cell maturation protein (BCMA) is a member of the tumor necrosis factor (TNF) receptor family and is expressed in B lymphocytes. BCMA binds two TNF family members, BAFF and APRIL, that stimulate cellular proliferation. BAFF in particular has been shown to influence B-cell survival and activation, and transgenic mice overexpressing BAFF have a lupus-like autoimmune disorder. We have inactivated BCMA in the mouse germ line. BCMA(-/-) mice have normal B-cell development, and the life span of mutant B lymphocytes is comparable to that of wild-type B cells. The humoral immune responses of BCMA(-/-) mice to T-cell-independent antigens as well as high and low doses of T-cell-dependent antigens are also intact. In addition, mutant mice have normal splenic architecture, and germinal centers are formed during an ongoing immune response. These data suggest a functional redundancy of BCMA in B-cell physiology that is probably due to the presence of TACI, another TNF receptor family member that is expressed on B cells and that can also bind BAFF and APRIL.     

DHR3 is required for the prepupal-pupal transition and differentiation of adult structures during Drosophila metamorphosis.

Pulses of the steroid hormone ecdysone activate genetic regulatory hierarchies that coordinate the developmental changes associated with Drosophila metamorphosis. A high-titer ecdysone pulse at the end of larval development triggers puparium formation and induces expression of the DHR3 orphan nuclear receptor. Here we use both a heat-inducible DHR3 rescue construct and clonal analysis to define DHR3 functions during metamorphosis. Clonal analysis reveals requirements for DHR3 in the development of adult bristles, wings, and cuticle, and no apparent function in eye or leg development. DHR3 mutants rescued to the third larval instar also reveal essential functions during the onset of metamorphosis, leading to lethality during prepupal and early pupal stages. The phenotypes associated with these lethal phases are consistent with the effects of DHR3 mutations on ecdysone-regulated gene expression. Although DHR3 has been shown to be sufficient for early gene repression at puparium formation, it is not necessary for this response, indicating that other negative regulators may contribute to this pathway. In contrast, DHR3 is required for maximal expression of the midprepupal regulatory genes, EcR, E74B, and betaFTZ-1. Reductions in EcR and betaFTZ-F1 expression, in turn, lead to submaximal early gene induction in response to the prepupal ecdysone pulse and corresponding defects in adult head eversion and salivary gland cell death. These studies demonstrate that DHR3 is an essential regulator of the betaFTZ-F1 midprepupal competence factor, providing a functional link between the late larval and prepupal responses to ecdysone. Induction of DHR3 in early prepupae ensures that responses to the prepupal ecdysone pulse will be distinct from responses to the late larval pulse and thus that the animal progresses in an appropriate manner through the early stages of metamorphosis.