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

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

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

Plant-expressed Fc-fusion protein tetravalent dengue vaccine with inherent adjuvant properties.

Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi-Pasteur Denvaxia vaccine does not protect children under the age of nine and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant-expression to produce a humanised and highly immunogenic Poly-Immunoglobulin G Scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralising antibodies and evidence of cell-mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen-specific CD4+ and CD8+ T cell proliferation, IFN-γ and antibody production. The purified polymeric fraction of dengue PIGS (D-PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low affinity Fcγ receptors on antigen-presenting cells. These results show that the plant-expressed D-PIGS have the potential for translation towards a safe and easily scalable single antigen based tetravalent dengue vaccine. This article is protected by copyright. All rights reserved

PPAR agonists as add-on treatment with metformin in management of type 2 diabetes:a systematic review and meta-analysis

The combination of metformin and the peroxisome proliferator-activated receptors (PPAR) agonists offers a promising avenue for managing type 2 diabetes (T2D) through their potential complementary mechanisms of action. The results from randomized controlled trials (RCT) assessing the efficacy of PPAR agonists plus metformin versus metformin alone in T2D are inconsistent, which prompted the conduct of the systematic review and meta-analysis. We searched MEDLINE and EMBASE from inception (1966) to March 2023 to identify all RCTs comparing any PPAR agonists plus metformin versus metformin alone in T2D. Categorical variables were summarized as relative risk along with 95% confidence interval (CI). Twenty RCTs enrolling a total of 6058 patients met the inclusion criteria. The certainty of evidence ranged from moderate to very low. Pooled results show that using PPAR agonist plus metformin, as compared to metformin alone, results in lower concentrations of fasting glucose [MD = - 22.07 mg/dl (95% CI - 27.17, - 16.97), HbA1c [MD = - 0.53% (95% CI - 0.67, - 0.38)], HOMA-IR [MD = - 1.26 (95% CI - 2.16, - 0.37)], and fasting insulin [MD = - 19.83 pmol/L (95% CI - 29.54, - 10.13)] without significant increase in any adverse events. Thus, synthesized evidence from RCTs demonstrates the beneficial effects of PPAR agonist add-on treatment versus metformin alone in T2D patients. In particular, novel dual PPARα/γ agonist (tesaglitazar) demonstrate efficacy in improving glycaemic and lipid concentrations, so further RCTs should be performed to elucidate the long-term outcomes and safety profile of these novel combined and personalized therapeutic strategies in the management of T2D.PROSPERO registration no. CRD42023412603.</p

Structured decision-making drives guidelines panels’ recommendations ‘for’ but not ‘against’ health interventions

Background: The determinants of guideline panels’ recommendations remain uncertain. Objective: To investigate factors considered by members of 8 panels convened by the American Society of Hematology (ASH) to develop guidelines using GRADE system. Study Design and Setting: web-based survey of the participants in the ASH guidelines panels. Analysis: two level hierarchical, random-effect, multivariable regression analysis to explore the relation between GRADE and non-GRADE factors and strength of recommendations (SOR). Results: In the primary analysis, certainty in evidence [OR=1.83; (95CI% 1.45 to 2.31)], balance of benefits and harms [OR=1.49 (95CI% 1.30 to 1.69)] and variability in patients’ values and preferences [OR=1.47 (95CI% 1.15 to 1.88)] proved the strongest predictors of SOR. In a secondary analysis, certainty of evidence was associated with a strong recommendation [OR=3.60 (95% CI 2.16 to 6.00)] when panel members recommended “for” interventions but not when they made recommendations “against” [OR=0.98 (95%CI: 0.57 to 1.8)] consistent with “yes” bias. Agreement between individual members and the group in rating SOR varied (kappa ranged from -0.01 to 0.64). Conclusion: GRADE’s conceptual framework proved, in general, highly associated with SOR. Failure of certainty of evidence to be associated with SOR against an intervention, suggest the need for improvements in the process

Immune-Complex Mimics as a Molecular Platform for Adjuvant-Free Vaccine Delivery

Protein-based vaccine development faces the difficult challenge of finding robust yet non-toxic adjuvants suitable for humans. Here, using a molecular engineering approach, we have developed a molecular platform for generating self-adjuvanting immunogens that do not depend on exogenous adjuvants for induction of immune responses. These are based on the concept of Immune Complex Mimics (ICM), structures that are formed between an oligomeric antigen and a monoclonal antibody (mAb) to that antigen. In this way, the roles of antigens and antibodies within the structure of immune complexes are reversed, so that a single monoclonal antibody, rather than polyclonal sera or expensive mAb cocktails can be used. We tested this approach in the context of Mycobacterium tuberculosis (MTB) infection by linking the highly immunogenic and potentially protective Ag85B with the oligomeric Acr (alpha crystallin, HspX) antigen. When combined with an anti-Acr monoclonal antibody, the fusion protein formed ICM which bound to C1q component of the complement system and were readily taken up by antigen-presenting cells in vitro. ICM induced a strong Th1/Th2 mixed type antibody response, which was comparable to cholera toxin adjuvanted antigen, but only moderate levels of T cell proliferation and IFN-γ secretion. Unfortunately, the systemic administration of ICM did not confer statistically significant protection against intranasal MTB challenge, although a small BCG-boosting effect was observed. We conclude that ICM are capable of inducing strong humoral responses to incorporated antigens and may be a suitable vaccination approach for pathogens other than MTB, where antibody-based immunity may play a more protective role

Systemic and Oral Immunogenicity of Porcine Epidemic Diarrhea Virus Antigen Fused to Poly-Fc of Immunoglobulin G and Expressed in ΔXT/FT Nicotiana benthamiana Plants.

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family has become increasingly probelmatic in the pig farming industry. Currently, there are no effective, globally applicable vaccines against PEDV. Here, we tested a recombinant PEDV vaccine candidate based on the expression of the core neutralising epitope (COE) of PEDV conjugated to polymeric immunoglobulin G scaffold (PIGS) in glycoengineered Nicotiana benthamiana plants. The biological activity of COE-PIGS was demonstrated by binding to C1q component of the complement system, as well as the surface of antigen-presenting cells (APCs) in vitro. The recombinant COE-PIGS induced humoral and cellular immune responses specific for PEDV after both systemic and mucosal vaccination. Altogether, the data indicated that PEDV antigen fusion to poly-Fc could be a promising vaccine platform against respiratory PEDV infection

The ansamycin antibiotic, rifamycin SV, inhibits BCL6 transcriptional repression and forms a complex with the BCL6-BTB/POZ domain

BCL6 is a transcriptional repressor that is over-expressed due to chromosomal translocations, or other abnormalities, in ~40% of diffuse large B-cell lymphoma. BCL6 interacts with co-repressor, SMRT, and this is essential for its role in lymphomas. Peptide or small molecule inhibitors, which prevent the association of SMRT with BCL6, inhibit transcriptional repression and cause apoptosis of lymphoma cells in vitro and in vivo. In order to discover compounds, which have the potential to be developed into BCL6 inhibitors, we screened a natural product library. The ansamycin antibiotic, rifamycin SV, inhibited BCL6 transcriptional repression and NMR spectroscopy confirmed a direct interaction between rifamycin SV and BCL6. To further determine the characteristics of compounds binding to BCL6-POZ we analyzed four other members of this family and showed that rifabutin, bound most strongly. An X-ray crystal structure of the rifabutin-BCL6 complex revealed that rifabutin occupies a partly non-polar pocket making interactions with tyrosine58, asparagine21 and arginine24 of the BCL6-POZ domain. Importantly these residues are also important for the interaction of BLC6 with SMRT. This work demonstrates a unique approach to developing a structure activity relationship for a compound that will form the basis of a therapeutically useful BCL6 inhibitor