Adaption of the ex vivo mycobacterial growth inhibition assay for use with murine lung cells.

In the absence of a correlate(s) of protection against human tuberculosis and a validated animal model of the disease, tools to facilitate vaccine development must be identified. We present an optimised ex vivo mycobacterial growth inhibition assay (MGIA) to assess the ability of host cells within the lung to inhibit mycobacterial growth, including Bacille Calmette-Guérin (BCG) and Mycobacterium tuberculosis (MTB) Erdman. Growth of BCG was reduced by 0.39, 0.96 and 0.73 log10 CFU following subcutaneous (s.c.) BCG, intranasal (i.n.) BCG, or BCG s.c. + mucosal boost, respectively, versus naïve mice. Comparatively, a 0.49 (s.c.), 0.60 (i.n.) and 0.81 (s.c. + mucosal boost) log10 reduction in MTB CFU was found. A BCG growth inhibitor, 2-thiophenecarboxylic acid hydrazide (TCH), was used to prevent quantification of residual BCG from i.n. immunisation and allow accurate MTB quantification. Using TCH, a further 0.58 log10 reduction in MTB CFU was revealed in the i.n. group. In combination with existing methods, the ex vivo lung MGIA may represent an important tool for analysis of vaccine efficacy and the immune mechanisms associated with vaccination in the organ primarily affected by MTB disease

Mucosal delivery of tuberculosis vaccines: a review of current approaches and challenges.

Introduction: Tuberculosis (TB) remains a major health threat and it is now clear that the current vaccine, BCG, is unable to arrest the global TB epidemic. A new vaccine is needed to either replace or boost BCG so that a better level of protection could be achieved. The route of entry of Mycobacterium tuberculosis, the causative organism, is via inhalation making TB primarily a respiratory disease. There is therefore good reason to hypothesize that a mucosally delivered vaccine against TB could be more effective than one delivered via the systemic route.Areas covered: This review summarizes the progress that has been made in the area of TB mucosal vaccines in the last few years. It highlights some of the strengths and shortcomings of the published evidence and aims to discuss immunological and practical considerations in the development of mucosal vaccines.Expert opinion: There is a growing body of evidence that the mucosal approach to vaccination against TB is feasible and should be pursued. However, further key studies are necessary to both improve our understanding of the protective immune mechanisms operating in the mucosa and the technical aspects of aerosolized delivery, before such a vaccine could become a feasible, deployable strategy

Influence of husbandry procedures on mouse locomotor activity

The currently used raL models of obesity and diabetes are derived from either Zucker or from Koletsky rats. Recently, we identified a spontaneous obese rat from out Wistar colony which is maintained as an inbred stock for the past 75 years. Initially, one of the male progeny in a litter was observed to have abnormal body weight for its age. The parents of this ral were identified, the progeny selectively bred, and a colony has been developed. This is designated as WNIN-0b. The colony is maintained by mating heterozygous animals (+/ob), as the homozygous (ob/ob) were found to be infertile. The trait is carried as an autosomal recessive mutation and the colony is currenfly in F7 generation.Obesity is visible in these mutants around 35 days of age. They are hyperphagic and reach a body weight of 500—600 g by 105 days of age. “Kinky” tail is characteristic of this mutant and this is visible around 50-60 days. Sexual maturity is delayed in female obese mutants, as judged by the day of vaginal opening. The animals are cuglyccmic and show hyperinsulinaemia, hypertriglyceridaemia, arid hypercholesterolemia. Another mutant showing hyperglycemia is also obtained fromthe obese colony. Unlike earlier models which are essentially derived from a randomAbred stock, this is the first report of a rat obese model, developed spontaneously from an inbred strain

Serum proteomics of active tuberculosis patients and contacts reveals unique processes activated during Mycobacterium tuberculosis infection.

Tuberculosis (TB) is the most lethal infection among infectious diseases. The specific aim of this study was to establish panels of serum protein biomarkers representative of active TB patients and their household contacts who were either infected (LTBI) or uninfected (EMI-TB Discovery Cohort, Pontevedra Region, Spain). A TMT (Tamdem mass tags) 10plex-based quantitative proteomics study was performed in quintuplicate containing a total of 15 individual serum samples per group. Peptides were analyzed in an LC-Orbitrap Elite platform, and raw data were processed using Proteome Discoverer 2.1. A total of 418 proteins were quantified. The specific protein signature of active TB patients was characterized by an accumulation of proteins related to complement activation, inflammation and modulation of immune response and also by a decrease of a small subset of proteins, including apolipoprotein A and serotransferrin, indicating the importance of lipid transport and iron assimilation in the progression of the disease. This signature was verified by the targeted measurement of selected candidates in a second cohort (EMI-TB Verification Cohort, Maputo Region, Mozambique) by ELISA and nephelometry techniques. These findings will aid our understanding of the complex metabolic processes associated with TB progression from LTBI to active disease

Bacillus Calmette-Guérin Induces PD-L1 Expression on Antigen-Presenting Cells via Autocrine and Paracrine Interleukin-STAT3 Circuits

Bacillus Calmette-Guérin (BCG) is the only licensed vaccine for tuberculosis (TB), and is also used as an immunotherapy for bladder cancer and other malignancies due to its immunostimulatory properties. Mycobacteria spp., however, are well known for their numerous immune evasion mechanisms that limit the true potential of their therapeutic use. One such major mechanism is the induction of programmed death ligand-1 (PD-L1), which mitigates adaptive immune responses. Here, we sought to unravel the molecular pathways behind PD-L1 up-regulation on antigen-presenting cells (APCs) by BCG. We found that infection of APCs with BCG induced PD-L1 up-regulation, but that this did not depend on direct infection, suggesting a soluble mediator for this effect. BCG induced potent quantities of IL-6 and IL-10, and the downstream transcription factor STAT3 was hyper-phosphorylated. Intracellular analyses revealed that levels of PD-L1 molecules were associated with the STAT3 phosphorylation state, suggesting a causal link. Neutralisation of the IL-6 or IL-10 cytokine receptors dampened STAT3 phosphorylation and BCG-mediated up-regulation of PD-L1 on APCs. Pharmacological inhibition of STAT3 achieved the same effect, confirming an autocrine-paracrine cytokine loop as a mechanism for BCG-mediated up-regulation of PD-L1. Finally, an in vivo immunisation model showed that BCG vaccination under PD-L1 blockade could enhance antigen-specific memory CD4 T-cell responses. These novel findings could lead to refinement of BCG as both a vaccine for infectious disease and as a cancer immunotherapy

Identifying key denning habitat to conserve brown bear (Ursus arctos) in Croatia

CONTEXT: The preservation of denning habitat is paramount to the recovery of threatened bear populations because of the effect that den site disturbance can have on cub mortality. Understanding habitat suitability for denning can allow management efforts to be directed towards the regions where conservation interventions would be most effective. AIM: We sought to identify the environmental and anthropogenic habitat variables associated with the presence of Eurasian brown bear (Ursus arctos) den sites in Croatia. Based on these associations, in order to inform future conservation decisions, we also sought to identify regions of high suitability for denning across Croatia. METHODS: Using the locations of 91 dens inhabited by bears between 1982 and 2011, we opted for the presence-only modelling option in software Maxent to determine the most important predictors of den presence, and thus predict the distribution of high-value denning habitat across Croatia. KEY RESULTS: We found that structural elements were the most important predictors, with ruggedness and elevation both relating positively to den presence. However, distance to nearest settlement was also positively associated with den presence. CONCLUSION: We determine that there is considerable denning habitat value in areas with high and rugged terrain as well as areas with limited human activity. We suspect that high and rugged terrain contains a greater concentration of the karstic formations used for denning than lower-lying regions. IMPLICATIONS: Our study presents the first habitat suitability model for brown bears in Croatia, and identifies core areas suitable for denning both within and outside the species’ current range. As such, it provides useful evidence for conservation decision making and the development of scientifically-based management plans. Our results also support the need for finer spatial scale studies that can reveal specific denning preferences of subpopulations

Quality of clothing fabrics in terms of their comfort properties

Quality of various clothing woven fabrics with respect to their comfort properties, such as electro-physical properties, air permeability, and compression properties has been studied. Fabrics are produced from cotton and cotton/polyester fibre blends in plain, twill, satin and basket weave. Results show that cotton fabrics have lower values of the volume resistivity, air permeability and compressive resilience but higher values of effective relative dielectric permeability and compressibility as compared to fabrics that have been produced from cotton/PES fibre blends. Regression analysis shows a strong linear correlative relationship between the air permeability and the porosity of the woven fabrics with very high coefficient of linear correlation (0.9807). It is also observed that comfort properties are determined by the structure of woven fabrics (raw material composition, type of weave) as well as by the fabrics surface condition. Findings of the studies have been used for estimating the quality of woven fabrics in terms of their comfort properties by the application of ranking method. It is concluded that the group of cotton fabrics exhibits better quality of comfort as compared to the group of cotton/PES blend fabrics.

Comparative efficacy of tandem autologous versus autologous followed by allogeneic hematopoietic cell transplantation in patients with newly diagnosed multiple myeloma: a systematic review and meta-analysis of randomized controlled trials

Background Despite advances in understanding of clinical, genetic, and molecular aspects of multiple myeloma (MM) and availability of more effective therapies, MM remains incurable. The autologous-allogeneic (auto-allo) hematopoietic cell transplantation (HCT) strategy is based on combining cytoreduction from high-dose (chemo- or chemoradio)-therapy with adoptive immunotherapy. However, conflicting results have been reported when an auto-allo HCT approach is compared to tandem autologous (auto-auto) HCT. A previously published meta-analysis has been reported; however, it suffers from serious methodological flaws. Methods A systematic search identified 152 publications, of which five studies (enrolling 1538 patients) met inclusion criteria. All studies eligible for inclusion utilized biologic randomization. Results Assessing response rates by achievement of at least a very good partial response did not differ among the treatment arms [risk ratio (RR) (95% CI) = 0.97 (0.87-1.09), p = 0.66]; but complete remission was higher in the auto-allo HCT arm [RR = 1.65 (1.25-2.19), p = 0.0005]. Event-free survival did not differ between auto-allo HCT group versus auto-auto HCT group using per-protocol analysis [hazard ratio (HR) = 0.78 (0.58-1.05)), p = 0.11] or using intention-to-treat analysis [HR = 0.83 (0.60-1.15), p = 0.26]. Overall survival (OS) did not differ among these treatment arms whether analyzed on per-protocol [HR = 0.88 (0.33-2.35), p = 0.79], or by intention-to-treat [HR = 0.80 (0.48-1.32), p = 0.39] analysis. Non-relapse mortality (NRM) was significantly worse with auto-allo HCT [RR (95%CI) = 3.55 (2.17-5.80), p \u3c 0.00001]. Conclusion Despite higher complete remission rates, there is no improvement in OS with auto-allo HCT; but this approach results in higher NRM in patients with newly diagnosed MM. At present, totality of evidence suggests that an auto-allo HCT approach for patients with newly diagnosed myeloma should not be offered outside the setting of a clinical trial

NON-INVASIVE BLOOD PRESSURE AND OTHER PHYSIOLOGICAL DATA IN CHEMICALLY IMMOBILIZED BROWN BEARS (URSUS ARCTOS)

Free-ranging brown bears (Ursus arctos) were snared and subsequently darted with a combination of xylazine-ketamine in Croatia (n = 5) or darted from a helicopter with a combination of medetomidine-tiletamine-zolazepam in Scandinavia (n = 20). Three adults and one yearling (1 year old) bear were captured in Croatia, with one adult being captured twice. The Scandinavian bears were divided into Group A (yearlings, n = 7) and Group B (subadults, n = 2 and adults, n = 11). The exertion time (time from activation of the trap or from the start of the helicopter chase to recumbency) and the induction time (time from darting to recumbency) were recorded. The rectal temperature (Tr) was measured as soon as possible after induction and then monitored at frequent intervals (varied between individuals) in immobilized bears. Blood pressure (BP) was measured with a non-invasive method (Korotkoff's technique) every 5 minutes. The heart rate (HR), respiratory rate (RR), and arterial haemoglobin oxygen saturation (SpO2) were recorded every 5 minutes. Reliability of the BP monitoring technique, trends of variation of the physiological variables, and the factors related to the capture were assessed. Both exertion and induction times were longer in Croatian bears than in Scandinavian bears. In Croatian bears, the Tr was either constant or slightly decreasing, with hyperthermia recorded in two individuals (Tr > 39.0° C). In Scandinavian bears, 17 of 20 individuals developed an initial hyperthermia. Four of five bears in Croatia and 17 of 20 bears in Scandinavia showed a decreasing trend in systolic and mean BP over time. According to the Korotkoff method, all bears were hypertensive (mean BP > 130 mmHg) with varying severity, and the systolic pressure was significantly lower in yearlings when compared to subadults and adults. Yearlings had significantly (p < 0.05) higher HR than subadults and adults, however there was no significant differences in RR, SpO2, and Tr between the age groups. All Croatian bears and 13 of 20 Scandinavian bears were moderately to severely hypoxemic (SpO2 < 90%). Further studies with simultaneous invasive and non-invasive (Korotkoff) BP monitoring techniques are required to confirm the accuracy of methods used in this study. The data presented here provides evidence of the physiological impact of different capture methods and chemical immobilization of brown bears in Croatia and Scandinavia