Lionex (Germany)

Background Military personnel engaged in intense activities must consume enough quality protein in their diet to maintain protein balance and promote recovery. Plant-based proteins are considered lower-quality protein sources than animal proteins due to their lower amount of essential amino acids (EAA) and only containing trace amounts of creatine. The purpose of this study was to determine whether ingestion of military-style meals-ready-to-eat (MREs) containing plant-based (Plant) or pork-based (Pork) sources or protein would affect recovery after the Army Combat Fitness Test (ACFT).Methods Twenty-three (n = 23) Corps of Cadets members participated in a randomized, double-blind, placebo-controlled, and crossover-designed study where they consumed Plant or Pork protein containing MREs. On testing days, participants completed a battery of cognitive function tests (i.e. the trail making test [TMT], Profile of Mood States [POMs], and psychomotor vigilance task test [PVT]) before consuming a pre-exercise meal. After four hours, participants performed the ACFT. Participants were then fed 3 MREs daily containing ≈ 1,250 kcals, 145 g carbohydrate, 45 g protein, 60 g fat, providing 1.7 g/kg/d of protein. Plant-based MREs contained 15.2 g/d (0.20 g/kg/d) of EAA and 0.215 g/d (0.003 g/kg/d of creatine) compared to 22.9 g/d (0.31 g/kg/d) of EAA and 1.82 g/d (0.024 g/kg/d) of creatine in the pork-based MREs. Participants returned to the lab in a fasted condition at 0600 after 24, 48, and 72 hours of recovery while being fed 3 MREs daily and having these markers of cognition assessed. Data were analyzed using general linear model statistics and percentage changes from baseline with 95% confidence intervals.Results Multivariate analysis revealed a time (p < 0.001, ηp2 = 0.281) with no treatment ×time effect (p = 0.330, ηp2 = 0.026) for the TMT, wherein the univariate analysis indicated improvements in the time required to complete TMT tasks A (p < 0.001, ηp2 = 0.386) and B (p < 0.001, ηp2 = 0.214) tests, with no interaction effects observed between treatments (p > 0.05). The percentage change analysis (Figure 1) displays the improvements for time-to-completion of the TMT tasks. Regarding the POMs results, multivariate analysis revealed a time (p < 0.001, ηp2 = 0.128) with no treatment ×time interaction effect (p = 0.927, ηp2 = 0.026). Univariate analysis revealed a decrease over time in vigor ratings (p < 0.001, ηp2 = 0.225) and total mood disturbance score (TMDS, p < 0.001, ηp2 = 0.196) with no treatment ×time interaction effect observed (p > 0.05). Percentage change analysis revealed that depression ratings tended to be lower after 48 hours in the Pork diet (−54 %[−115, 7], p = 0.086) than the Plant diet (p = 0.08), but no differences were found between diets for the TMDS. Lastly, the multivariate analysis revealed no time (p = 0.472, ηp2 = 0.022) with a treatment ×time effect (p = 0.038, ηp2 = 0.044). Univariate analysis revealed an interaction effect in the correct response reaction time (p = 0.002, ηp2 = 0.111), the percentage change analysis showing reaction time for correct responses tended to be slower with pork diet.[Figure: see text]Conclusion Consuming Pork containing MREs for three days following intense military-style training may lessen perceptions of depression but does not appear to improve measures of cognitive function parameters when compared to the Plant-based MREs. While it may be prudent to consume MREs with high-quality protein sources, short-term ingestion of MREs containing protein with higher EAAs and creatine has limited effects on mood states and cognitive function.Registered clinical trial #ISRCTN47322504

Mycobacterium tuberculosis (M.tb) infections remain one of the most significant causes of mortality worldwide. The current situation shows an emergence of new antibiotic-resistant strains making it difficult to control the tuberculosis (TB) disease. A large part of its success as a pathogen is due to its ability to persist for years or even decades without causing evident clinical manifestations. M.tb is highly successful in evading the host-defense by manipulating host-signalling pathways. Although macrophages are generally viewed as the key cell type involved in harboring M.tb, growing evidence shows that neutrophils also play a fundamental role. Both cells are known to act in multiple ways when encountering an invading pathogen, including phagocytosis, release of cytokines and chemokines, and oxidative burst. In addition, the formation of neutrophil extracellular traps (NETs) and macrophage extracellular traps (METs) has been described to contribute to M.tb infections. NETs/METs are extracellular DNA fibers with associated granule components, which are released upon activation of the cells by the pathogen or by pro-inflammatory mediators. On one hand, they can lead to a protective immune response by entrapment and killing of pathogens. However, on the other hand, they can also play a severe pathological role by inducing tissue damage. Extracellular traps (ETs) produced in the pulmonary alveoli can expand easily and expose tissue-damaging factors with detrimental effects. Since host-directed therapies offer a complementary strategy in TB, the knowledge of NET/MET formation is important for understanding potential protective versus detrimental pathways during innate immune signaling. In this review, we summarize the progress made in understanding the role of NETs/METs in the pathogenesis of TB.

Excessive lung inflammation and airway epithelial damage are hallmarks of human inflammatory lung diseases, such as cystic fibrosis (CF). Enhancement of innate immunity provides protection against pathogens while reducing lung-damaging inflammation. However, the mechanisms underlying innate immunity–mediated protection in the lung remain mysterious, in part because of the lack of appropriate animal models for these human diseases. TLR5 (Toll-like receptor 5) stimulation by its specific ligand, the bacterial protein flagellin, has been proposed to enhance protection against several respiratory infectious diseases, although other cellular events, such as calcium signaling, may also control the intensity of the innate immune response. Here, we investigated the molecular events prompted by stimulation with flagellin and its role in regulating innate immunity in the lung of the pig, which is anatomically and genetically more similar to humans than rodent models. We found that flagellin treatment modulated NF-κB signaling and intracellular calcium homeostasis in airway epithelial cells. Flagellin pretreatment reduced the NF-κB nuclear translocation and the expression of proinflammatory cytokines to a second flagellin stimulus as well as to Pseudomonas aeruginosa infection. Moreover, in vivo administration of flagellin decreased the severity of P. aeruginosa–induced pneumonia. Then we confirmed these beneficial effects of flagellin in a pathological model of CF by using ex vivo precision-cut lung slices from a CF pigz model. These results provide evidence that flagellin treatment contributes to a better regulation of the inflammatory response in inflammatory lung diseases such as CF.

Infections with antimicrobial-resistant (AMR) bacteria are globally on the rise. In the future, multi-resistant infections will become one of the major problems in global health care. In order to enable reserve antibiotics to retain their effect as long as possible, broad-spectrum antibiotics must be used sparingly. This can be achieved by a rapid microfluidic phenotypic antibiotic susceptibility test, which provides the information needed for a targeted antibiotic therapy in less time than conventional tests. Such microfluidic tests must cope with a low bacteria concentration. On-chip filtering of the samples to accumulate bacteria can shorten the test time. By means of fluorescence microscopy, we examined a novel nanogap filtration principle to hold back Escherichia coli and to perform cultivation experiments with and without antibiotics present. Microfluidic chips based on the nanogap flow principle showed to be useful for the concentration and cultivation of E. coli. With a concentration of 106 cells/mL, a specific growth rate of 0.013 min−1 and a doubling time of 53 min were achieved. In the presence of an antibiotic, no growth was observed. The results prove that this principle can, in future, be used in fast and marker-free antimicrobial susceptibility testing (AST).

MTBVAC is a live-attenuated Mycobacterium tuberculosis vaccine, currently under clinical development, that contains the major antigens ESAT6 and CFP10. These antigens are absent from the current tuberculosis vaccine, BCG. Here we compare the protection induced by BCG and MTBVAC in several mouse strains that naturally express different MHC haplotypes differentially recognizing ESAT6 and CFP10. MTBVAC induces improved protection in C3H mice, the only of the three tested strains reactive to both ESAT6 and CFP10. Deletion of both antigens in MTBVAC reduces its efficacy to BCG levels, supporting a link between greater efficacy and CFP10- and ESAT6-specific reactogenicity. In addition, MTBVAC (but not BCG) triggers a specific response in human vaccinees against ESAT6 and CFP10. Our results warrant further exploration of this response as potential biomarker of protection in MTBVAC clinical trials.

Infection of vascular endothelial cells (ECs) is assumed to contribute to dissemination of human cytomegalovirus (HCMV). Investigation of virus-host interactions in ECs such as human umbilical vein endothelial cells (HUVECs) is limited due to the low maximal passage numbers of these primary cells. We tested a conditionally immortalized EC line (HEC-LTT) and a permanent cell line (EA.hy926) for their susceptibility to HCMV infection. Both cell lines resembled HUVECs in that they allowed for entry and immediate early protein expression of highly endotheliotropic HCMV strains but not of poorly endotheliotropic strains, rendering them suitable for analysis of the viral entry mechanism in ECs. The late phase of viral replication and release, however, was supported by growth-controlled HEC-LTT cells but not by EA.hy926 cells. HEC-LTT cells support both the early and late phase of viral replication and release infectious progeny virus at titers comparable to primary HUVECs; thus, the HEC-LTT cell line is a cell culture model representing the full viral replicative cycle of HCMV in ECs. The implementation of permanent HEC-LTT and EA.hy926 cell lines in HCMV research will facilitate long-term approaches that are not feasible in primary HUVECs.

There is evidence that the same mycobacterial heat shock proteins (Mtb-HSPs), especially HSP16, the main marker of mycobacteria dormant stage, occur in sarcoid tissues and in circulated immune complexes and prompt the immune responses against the different genetic background, leading to the development of acute sarcoidosis (SA)/Löfgren syndrome, chronic SA, latent tuberculosis (TB), or active TB. In SA there is increased monocytes phagocytic activity, decreased clearance of antigens/immune complexes by monocytes, which are resistant to apoptosis, and decreased serum microbicidal/degradable nitrate⁄nitrite (NOx) concentration. Reduction in NOx may result from the reaction of NOx with superoxide with subsequent production of peroxynitrite (ONOO-). In this study, therefore, we evaluated NOx and ONOO- levels in supernatants of peripheral blood mononuclear cells cultures treated with Mtb-HSPs from 20 SA patients, 19 TB patients, and 21 healthy volunteers using Griess and rhodamine fluorescence methods. We found significantly greater NOx and ONOO- concentrations with/without Mtb-HSPs stimulation in SA and TB patients than in controls. However, there were significantly lower NOx and higher ONOO- levels after Mtb-HSPs induction in SA than TB patients. In summary, in contrast to active TB, increased ONOO- concentration may explain the low level of NOx with induction of M. tuberculosis genetic dormancy program via higher Mtb-HSP16 expression in SA.

BackgroundTuberculosis is the leading cause of death due to bacterial infections worldwide, mainly caused by Mycobacterium tuberculosis. The antigen 85 complex comprises a set of major secreted proteins of M. tuberculosis, which are potential biomarkers for diagnostic.ResultsIn this work, the first human single chain fragment variable (scFv) antibodies specific for the tuberculosis biomarker 85 B were selected by phage display from naïve antibody gene libraries (HAL7/8). Produced as scFv-Fc in mammalian cells, these antibodies were further characterized and analysed for specificity and applicability in different tuberculosis antigen detection assays. Sandwich detection of recombinant 85 B was successful in enzyme linked immunosorbent assay (ELISA), lateral flow immunoassay and immunoblot. Whereas detection of M. tuberculosis cell extracts and culture filtrates was only possible in direct ELISA and immunoblot assays. It was found that the conformation of 85 B, depending on sample treatment, influenced antigen detection.ConclusionsRecombinant antibodies, selected by phage display, may be applicable for 85 B detection in various assays. These antibodies are candidates for the development of future point of care tuberculosis diagnostic kits. Using 85 B as a biomarker, the antigen conformation influenced by sample treatment is important.

The diagnosis of childhood active tuberculosis (aTB) and latent Mycobacterium tuberculosis (M. tuberculosis) infection (LTBI) remains a challenge, and the replacement of tuberculin skin tests (TST) with commercialized gamma interferon (IFN-γ) release assays (IGRA) is not currently recommended. Two hundred sixty-six children between 1 month and 15 years of age, 214 of whom were at risk of recent M. tuberculosis infection and 51 who were included as controls, were prospectively enrolled in our study. According to the results of a clinical evaluation, TST, chest X ray, and microbiological assessment, each children was classified as noninfected, having LTBI, or having aTB. Long-incubation-time purified protein derivative (PPD), ESAT-6, and CFP-10 IGRA were performed and evaluated for their accuracy in correctly classifying the children. Whereas both TST and PPD IGRA were suboptimal for detecting aTB, combining the CFP-10 IGRA with a TST or with a PPD IGRA allowed us to detect all the children with aTB with a specificity of 96% for children who were positive for the CFP-10 IGRA. Moreover, the combination of the CFP-10 IGRA and PPD IGRA detected 96% of children who were eventually classified as having LTBI, but a strong IFN-γ response to CFP-10 (defined as >500 pg/ml) was highly suggestive of aTB, at least among the children who were <3 years old. The use of long-incubation-time CFP-10 IGRA and PPD IGRA should help clinicians to quickly identify aTB or LTBI in young children.

Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex group of bacteria. Vaccination of cattle might offer a long-term solution for controlling the disease and priority has been given to the development of a cattle vaccine against bTB. Identification of biomarkers in tuberculosis research remains elusive and the goal is to identify host correlates of protection. We hypothesized that by studying global gene expression we could identify in vitro predictors of protection that could help to facilitate vaccine development. Calves were vaccinated with BCG or with a heterologous BCG prime adenovirally vectored subunit boosting protocol. Protective efficacy was determined after M. bovis challenge. RNA was prepared from PPD-stimulated PBMC prepared from vaccinated-protected, vaccinated-unprotected and unvaccinated control cattle prior to M. bovis challenge and global gene expression determined by RNA-seq. 668 genes were differentially expressed in vaccinated-protected cattle compared with vaccinated-unprotected and unvaccinated control cattle. Cytokine-cytokine receptor interaction was the most significant pathway related to this dataset with IL-22 expression identified as the dominant surrogate of protection besides INF-γ. Finally, the expression of these candidate genes identified by RNA-seq was evaluated by RT-qPCR in an independent set of PBMC samples from BCG vaccinated and unvaccinated calves. This experiment confirmed the importance of IL-22 as predictor of vaccine efficacy.