Early pneumococcal clearance in mice induced by systemic immunization with recombinant BCG PspA-PdT prime and protein boost correlates with cellular and humoral immune response in bronchoalveolar fluids (BALF)

© 2019 The Author(s) An effective immunological response in the lungs during a pneumococcal infection is a key factor to the bacteria clearance and prevention of sepsis. In order to develop broad-range pneumococcal vaccines several pneumococcal proteins and strong adjuvants have been investigated. Previously, we constructed a recombinant BCG (rBCG) strain expressing a fragment of PspA (Pneumococcal surface protein A) fused to PdT (detoxified form of pneumolysin). Immunization of mice with a priming dose of rBCG PspA-PdT followed by a booster dose of rPspA-PdT fused protein induced a high antibody response in the serum and protected mice against lethal challenge. Here, we investigated the humoral and cellular immune response in the Bronchoalveolar lavage fluid (BALF). Immunization of mice with rBCG PspA-PdT / rPspA-PdT induced rapid clearance of bacteria after challenge, an early control of the cellular influx and reduced inflammatory cytokine levels in the BALF. In addition, rBCG PspA-PdT / rPspA-PdT induced higher lymphocyte recruitment to the lungs at 48 h, showing an increased percentage of CD4+ T cells. Furthermore, BALF samples from mice immunized with rBCG PspA-PdT / PspA-PdT showed high binding of IgG2c and enhanced complement deposition on the pneumococcal surface; antibody binding was specific to PspA as no binding was observed to a PspA-knockout strain. Taken together, our results show that the immunization with rBCG PspA-PdT / rPspA-PdT induces humoral and cellular immune responses in the lungs, promotes an early clearance of pneumococci and protects against the systemic dissemination of pneumococci

A bivalent recombinant mycobacterium bovis BCG expressing the S1 subunit of the pertussis toxin induces a polyfunctional CD4 ⁺ T cell immune response

© 2019 Alex I. Kanno et al. Background. A recombinant BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (rBCG-S1PT), previously constructed by our research group, demonstrated the ability to develop high protection in mouse models of pertussis challenge which correlated with the induction of a Th1 immune response pattern. The Th1 immune response induced by rBCG-S1PT treatment was also confirmed in the murine orthotopic bladder cancer model, in which the intravesical instillation of rBCG-S1PT resulted in an improved antitumor effect. Based on these observations, we hypothesize that the reengineering of the S1PT expression in BCG could increase the efficiency of the protective Th1 immune response in order to develop a new alternative of immunotherapy in bladder cancer treatment. Objectives. To construct rBCG strains expressing S1PT from extrachromosomal (rBCG-S1PT) and integrative vectors (rBCG-Sli), or their combination, generating the bivalent strain (rBCG-S1+S1i), and to evaluate the respective immunogenicity of rBCG strains in mice. Methods. Mycobacterial plasmids were constructed by cloning the s1pt gene under integrative and extrachromosomal vectors and used to transform BCG, individually or in combination. Antigen expression and localization were confirmed by Western blot. Mice were immunized with wild-type BCG or the rBCG strains, and cytokines quantification and flow cytometry analysis were performed in splenocytes culture stimulated with mycobacterial-specific proteins. Findings. S1PT expression was confirmed in all rBCG strains. The extrachromosomal vector directs S1PT to the cell wall-associated fraction, while the integrative vector directs its expression mainly to the intracellular fraction. Higher levels of IFN-γ were observed in the splenocytes culture from the group immunized with rBCG-S1i in comparison to BCG or rBCG-S1PT. rBCG-S1+S1i showed higher levels of CD4 + IFN-γ + and double-positive CD4 + IFN-γ + TNF-α + T cells. Conclusions. rBCG-S1+S1i was able to express the two forms of S1PT and elicited higher induction of polyfunctional CD4 + T cells, indicating enhanced immunogenicity and suggesting its use as immunotherapy for bladder cancer

New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters.

The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response

A Combination of Recombinant Mycobacterium bovis BCG Strains Expressing Pneumococcal Proteins Induces Cellular and Humoral Immune Responses and Protects against Pneumococcal Colonization and Sepsis.

Pneumococcal diseases remain a substantial cause of mortality in young children in developing countries. The development of potentially serotype-transcending vaccines has been extensively studied; ideally, such a vaccine should include antigens that are able to induce protection against colonization (likely mediated by interleukin-17A [IL-17A]) and invasive disease (likely mediated by antibody). The use of strong adjuvants or alternative delivery systems that are able to improve the immunological response of recombinant proteins has been proposed but poses potential safety and practical concerns in children. We have previously constructed a recombinant Mycobacterium bovis BCG strain expressing a pneumococcal surface protein A (PspA)-PdT fusion protein (rBCG PspA-PdT) that was able to induce an effective immune response and protection against sepsis in a prime-boost strategy. Here, we constructed two new rBCG strains expressing the pneumococcal proteins SP 0148 and SP 2108, which confer IL-17A-dependent protection against pneumococcal colonization in mouse models. Immunization of mice with rBCG 0148 or rBCG 2108 in a prime-boost strategy induced IL-17A and gamma interferon (IFN-γ) production. The combination of these rBCG strains with rBCG PspA-PdT (rBCG Mix), followed by a booster dose of the combined recombinant proteins (rMix) induced an IL-17A response against SP 0148 and SP 2108 and a humoral response characterized by increased levels of IgG2c against PspA and functional antibodies against pneumolysin. Furthermore, immunization with the rBCG Mix prime/rMix booster (rBCG Mix/rMix) provides protection against pneumococcal colonization and sepsis. These results suggest the use of combined rBCG strains as a potentially serotype-transcending pneumococcal vaccine in a prime-boost strategy, which could provide protection against pneumococcal colonization and sepsis

Antioxidant activities of sulfated polysaccharides from brown and red seaweeds

The in vitro antioxidant activities of the following six sulfated polysaccharides were investigated: iota, kappa and lambda carrageenans, which are widely used in the food industry, fucoidan (homofucan) from the edible seaweed Fucus vesiculosus and fucans (heterofucans) F0.5 and F1.1 from the seaweed Padina gymnospora. With respect to the inhibition of superoxide radical formation, fucoidan had an IC50 (the half maximal inhibitory concentration) of 0.058 mg·mL−1, while the IC50 for the kappa, iota and lambda carrageenans were 0.112, 0.332 and 0.046 mg·mL−1, respectively. All of the samples had an inhibitory effect on the formation of hydroxyl radicals. The results of peroxidation tests showed that fucoidan had an IC50 of 1.250 mg·mL−1 and that the kappa, iota and lambda carrageenans had an IC50 of 2.753 and 2.338 and 0.323 mg·mL−1, respectively. Fucan fractions showed low antioxidant activity relative to fucoidan. These results clearly indicate the beneficial effect of algal polysaccharides as antioxidants

The Effect of Exercise Training on Diastolic and Systolic Function After Acute Myocardial Infarction: A Randomized Study

After acute myocardial infarction (AMI), diastolic dysfunction is frequent and an important determinant of adverse outcome. However, few interventions have proven to be effective in improving diastolic function. We aimed to determine the effect of exercise training on diastolic and systolic function after AMI.One month after AMI, 188 patients were prospectively randomized (1:1) to an 8-week supervised program of endurance and resistance exercise training (n = 86; 55.9 ± 10.8 years) versus standard of care (n = 89; 55.4 ± 10.3 years). All patients were submitted to detailed echocardiography and cardiopulmonary exercise test, at baseline and immediately after the study. Diastolic function was evaluated by the determination of tissue-Doppler derived early diastolic velocities (E' velocity at the septal and lateral sides of mitral annulus) and by the E/E' (ratio between the E wave velocity from mitral inflow and the E' velocity) as recommended in the consensus document for diastolic function assessment.At the end of the study, there was no significant change in E' septal velocity or E/E' septal ratio in the exercise group. We observed a small, although nonsignificant, improvement in E' lateral (mean change 0.1 ± 2.0 cm/s; P = 0.40) and E/E' lateral ratio (mean change of -0.3 ± 2.5; P = 0.24), while patients in the control group had a nonsignificant reduction in E' lateral (mean change -0.4 ± 1.9 cm/s; P = 0.09) and an increase in E/E' lateral ratio (mean change + 0.3 ± 3.3; P = 0.34). No relevant changes occurred in other diastolic parameters. The exercise-training program also did not improve systolic function (either tissue Doppler systolic velocities or ejection fraction).Exercise capacity improved only in the exercise-training group, with an increase of 1.6 mL/kg/min in pVO2 (P = 0.001) and of 1.9 mL/kg/min in VO2 at anaerobic threshold (P < 0.001).After AMI, an 8-week endurance plus resistance exercise-training program did not significantly improve diastolic or systolic function, although it was associated with an improvement in exercise capacity parameters

Bioactive Hydrogel Marbles

Liquid marbles represented a signifcant advance in the manipulation of fuids as they used particle flms to confne liquid drops, creating a robust and durable soft solid. We exploit this technology to engineering a bioactive hydrogel marble (BHM). Specifcally, pristine bioactive glass nanoparticles were chemically tuned to produce biocompatible hydrophobic bioactive glass nanoparticles (H-BGNPs) that shielded a gelatin-based bead. The designed BHM shell promoted the growth of a bone-like apatite layer upon immersion in a physiological environment. The fabrication process allowed the efcient incorporation of drugs and cells into the engineered structure. The BHM provided a simultaneously controlled release of distinct encapsulated therapeutic model molecules. Moreover, the BHM sustained cell encapsulation in a 3D environment as demonstrated by an excellent in vitro stability and cytocompatibility. The engineered structures also showed potential to regulate a pre-osteoblastic cell line into osteogenic commitment. Overall, these hierarchical nanostructured and functional marbles revealed a high potential for future applications in bone tissue engineering.Portuguese Foundation for Science and Technology − FCT (Grant Nos SFRH/BD/73174/2010 and SFRH/BD/73172/2010, respectively), from the program POPH/FSE from QREN. The authors would like to acknowledge the support of the European Research Council grant agreement ERC-2014-ADG-669858 for project ATLASinfo:eu-repo/semantics/publishedVersio

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration