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Tuberculosis (TB) remains one of the deadliest infectious diseases worldwide, posing great social and economic burden to affected countries. Novel vaccine approaches are needed to increase protective immunity against the causative agent

Data Availability Statement: The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Copyright © 2022 Bitencourt, Peralta-Álvarez, Wilkie, Jacobs, Wright, Salman Almujri, Li, Harris, Smith, Elias, White, Satti, Sharpe, O’Shea, McShane and Tanner. Tuberculosis (TB) is a major global health problem and the only currently-licensed vaccine, BCG, is inadequate. Many TB vaccine candidates are designed to be given as a boost to BCG; an understanding of the BCG-induced immune response is therefore critical, and the opportunity to relate this to circumstances where BCG does confer protection may direct the design of more efficacious vaccines. While the T cell response to BCG vaccination has been well-characterized, there is a paucity of literature on the humoral response. We demonstrate BCG vaccine-mediated induction of specific antibodies in different human populations and macaque species which represent important preclinical models for TB vaccine development. We observe a strong correlation between antibody titers in serum versus plasma with modestly higher titers in serum. We also report for the first time the rapid and transient induction of antibody-secreting plasmablasts following BCG vaccination, together with a robust and durable memory B cell response in humans. Finally, we demonstrate a functional role for BCG vaccine-induced specific antibodies in opsonizing mycobacteria and enhancing macrophage phagocytosis in vitro, which may contribute to the BCG vaccine-mediated control of mycobacterial growth observed. Taken together, our findings indicate that the humoral immune response in the context of BCG vaccination merits further attention to determine whether TB vaccine candidates could benefit from the induction of humoral as well as cellular immunity. This work was funded in part by a small grant awarded to RT from the Royal Society of Tropical Medicine and Hygiene (RSTMH); the European Research Infrastructures for Poverty Related Diseases (EURIPRED), an EC seventh framework program (grant number 312661); TBVAC2020 (grant number 643381); and the Wellcome Trust (HMcS is a Wellcome Trust Investigator, grant code WT 206331/Z/17/Z). Human Study 1 was funded by the Bill & Melinda Gates Foundation (grant number OPP1112389) and human Study 2 was funded by a grant awarded to MO’S from the Wellcome Trust (grant number 103420/Z/13/Z). For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. This work was also supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Center (BRC).

The tuberculosis vaccine, Bacille Calmette-Guerin (BCG), also affords protection against non-tuberculous diseases attributable to heterologous immune mechanisms such as trained innate immunity, activation of non-conventional T-cells, and cross-reactive adaptive immunity. Aerosol vaccine delivery can target immune responses toward the primary site of infection for a respiratory pathogen. Therefore, we hypothesised that aerosol delivery of BCG would enhance cross-protective action against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and be a deployable intervention against coronavirus disease 2019 (COVID-19). Immune parameters were monitored in vaccinated and unvaccinated rhesus macaques for 28 days following aerosol BCG vaccination. High-dose SARS-CoV-2 challenge was applied by intranasal and intrabronchial instillation and animals culled 6-8 days later for assessment of viral, disease, and immunological parameters. Mycobacteria-specific cell-mediated immune responses were detected following aerosol BCG vaccination, but SARS-CoV-2-specific cellular- and antibody-mediated immunity was only measured following challenge. Early secretion of cytokine and chemokine markers associated with the innate cellular and adaptive antiviral immune response was detected following SARS-CoV-2 challenge in vaccinated animals, at concentrations that exceeded titres measured in unvaccinated macaques. Classical CD14+ monocytes and Vδ2 γδ T-cells quantified by whole-blood immunophenotyping increased rapidly in vaccinated animals following SARS-CoV-2 challenge, indicating a priming of innate immune cells and non-conventional T-cell populations. However, viral RNA quantified in nasal and pharyngeal swabs, bronchoalveolar lavage (BAL), and tissue samples collected at necropsy was equivalent in vaccinated and unvaccinated animals, and in-life CT imaging and histopathology scoring applied to pulmonary tissue sections indicated that the disease induced by SARS-CoV-2 challenge was comparable between vaccinated and unvaccinated groups. Hence, aerosol BCG vaccination did not induce, or enhance the induction of, SARS-CoV-2 cross-reactive adaptive cellular or humoral immunity, although an influence of BCG vaccination on the subsequent immune response to SARS-CoV-2 challenge was apparent in immune signatures indicative of trained innate immune mechanisms and primed unconventional T-cell populations. Nevertheless, aerosol BCG vaccination did not enhance the initial clearance of virus, nor reduce the occurrence of early disease pathology after high dose SARS-CoV-2 challenge. However, the heterologous immune mechanisms primed by BCG vaccination could contribute to the moderation of COVID-19 disease severity in more susceptible species following natural infection.

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Misfolded amyloid peptides are neurotoxic molecules associated with Alzheimer's disease. The Aβ

Studies demonstrate that small molecule targeting of atypical protein kinase C (aPKC) may provide an effective means to control vascular permeability, prevent edema, and reduce inflammation providing novel and important alternatives to anti-VEGF therapies for certain blinding eye diseases. Based on a literature tricyclic thieno[2,3-d]pyrimidine lead (1), an ATP-competitive inhibitor of the aPKC iota (ι) and aPKC zeta (ζ) isoforms, we have synthesized a small series of compounds in 1-2 steps from a readily available chloro intermediate. A single pyridine congener was also made using 2D NMR to assign regiochemistry. Within the parent pyrimidine series, a range of potencies was observed against aPKCζ whereas the pyridine congener was inactive. Selected compounds were also tested for their effect toward VEGF-induced permeability in BREC cells. The most potent of these (7l) was further assayed against the aPKCι isoform and showed a favorable selectivity profile against a panel of 31 kinases, including kinases from the AGC superfamily, with a focus on PKC isoforms and kinases previously shown to affect permeability. Further testing of 7l in a luciferase assay in HEK293 cells showed an ability to prevent TNF-α induced NFκB activation while not having any effect on cell survival. Intravitreal administration of 7l to the eye yielded a complete reduction in permeability in a test to determine whether the compound could block VEGF- and TNFα-induced permeability across the retinal vasculature in a rat model. The compound in mice displayed good microsomal stability and in plasma moderate exposure (AUC and Cmax), low clearance, a long half-life and high oral bioavailability. With IV dosing, higher levels were observed in the brain and eye relative to plasma, with highest levels in the eye by either IV or PO dosing. With a slow oral absorption profile, 7l accumulates in the eye to maintain a high concentration after dosing with higher levels than in plasma. Compound 7l may represent a class of aPKC inhibitors for further investigation.

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The NPY5 receptor binding and pharmacokinetic properties of a novel series of cis-1-oxo-heterocyclyl-4-amido-cyclohexane derivatives are described.

Introduction into the human body makes most nanoparticle systems susceptible to aggregation via nonspecific protein binding. Here, we developed a peptide-capped gold nanoparticle platform that withstands aggregation in undiluted human serum at 37 °C for 24 h. This biocompatible and natural system is based on mimicking human proteins which are enriched in negatively charged glutamic acid and positively charged lysine residues on their surface. The multifunctional EKEKEKE-PPPPC-Am peptide sequence consists of a stealth glutamic acid/lysine portion combined with a surface anchoring linker containing four prolines and a cysteine. Particle stability was measured via optical spectroscopy and dynamic light scattering in single protein, high salt, and undiluted human serum solutions. In vitro cell experiments demonstrate EKEKEKE-PPPPC-Am capped gold nanoparticles effectively minimize nonspecific cell uptake by nonphagocytic bovine aortic endothelial cells and phagocytic murine macrophage RAW 264.7 cells. Cytotoxicity studies show that peptide-capped gold nanoparticles do not affect cell viability. Finally, the peptide EKEKEKE-PPPPC-Am was extended with cyclic RGD to demonstrate specific cell targeting and stealth without using poly(ethylene glycol). Adding the functional peptide via peptide sequence extension avoids complex conjugation chemistries that are used for connection to synthetic materials. Inductively coupled plasma mass spectroscopy results indicate high aortic bovine endothelial cell uptake of c[RGDfE(SGG-KEKEKE-PPPPC-Am)] capped gold nanoparticles and low uptake of the control scrambled sequence c[RDGfE(SGG-KEKEKE-PPPPC-Am)] capped gold nanoparticles.

A novel series of aryl azetidinyl oxadiazoles are identified as mGluR5 positive allosteric modulators (PAMs) with improved physico-chemical properties. N-substituted cyclohexyl and exo-norbornyl carboxamides, and carbamate analogs of azetidines are moderate to potent mGluR5 PAMs. The aryl, lower alkyl carboxamides analogs and sulfonamide analogs of azetidines are moderate mGluR5 negative allosteric modulators (NAMs). In the aryl oxadiazole moiety, substituents such as fluoro, chloro and methyl are well tolerated at the meta position while para substituents led to either inactive compounds or NAMs. A tight pharmacophore and subtle 'PAM to NAM switching' with close analogs makes the optimization of the series extremely challenging.

A novel series of N-aryl pyrrolidinonyl oxadiazoles were identified as mGluR5 positive allosteric modulators (PAMs). Optimization of the initial lead compound 6a led to the identification of the 12c (-) enantiomer as a potent compound with acceptable in vitro clearance, CYP, hERG and PK properties. Para substituted N-aryl pyrrolidinonyl oxadiazoles are mGluR5 PAMs while the meta and ortho substituted N-aryl pyrrolidinonyl oxadiazoles are negative allosteric modulators (NAMs). Para fluoro substitution on the N-aryl group and meta chloro or methyl substituents on the aryl oxadiazole moiety are optimal for mGluR5 PAM efficacy. The existence of an exquisitely sensitive 'PAM to NAM switch' within this chemotype making it challenging for simultaneous optimization of potency and drug-like properties.

This study analyses changes in the bony architecture of the femoral head and neck in osteoarthritis. Five osteoarthritic hips were obtained from female patients undergoing total hip replacement and were compared to non-arthritic control specimens from the dissecting room. Analysis was carried out on a coronal bony slice of the femoral head and on a transverse section of the upper femoral neck. The results indicated that in the region of the compressive trabeculae the intertrabecular areas were generally smaller in the osteoarthritic specimens and that the inferior part of the cortex of the femoral neck had thickened. Our data thus quantified changes consistent with the thickening of compressive trabeculae reported by others and with thickening of the region of the femoral neck on which they converge. We found less trabecular bone in non-weight-bearing regions of the lower femoral head and in the medulla of the upper neck in the osteoarthritic specimens consistent with loss of tensile trabeculae. Scanning electron microscopy suggested that bone resorption activity was higher in the subchondral and non-weight-bearing regions of the osteoarthritic femoral heads than in the controls. Clin. Anat. 17:378–391, 2004. © 2004 Wiley-Liss, Inc.

Game context is widely accepted to influence performance but most data available is ‘categorical’ and addresses performance rather than activity. This study assessed direct effects of opposition and team ranking in field hockey to establish influences on activity. One hundred and eight (n = 108) female field hockey players (age 16–39 years) participated, giving 186 competition and 48 training analyses. Team average distance (mean ± SD) observed in a mid-ranked team during competition ranged from 5949 ± 611 to 7719 ± 257 m demonstrating an opposition rank effect (Pearson’s r = .71; adjusted R2 = .42). However, multiple linear regression analysis demonstrated a stronger relationship in lower ranked teams (Pearson’s r = .99; adjusted R2 = .97 through 3–9). In contrast, no team rank effect was observed during randomly monitored competition where team average distance ranged from 5177 ± 444 to 7316 ± 241 m (Pearson’s r = .15 and adjusted R2 = .12). In training, however, a team rank effect was observed in distance-related performance indicators where team average distance during small-sided games ranged from 5877 ± 188 to 3551 ± 193 m drill (per 70 min) with Pearson’s r = .95 and adjusted R2 = .87. The presence of contextual effects has significant practical implications for team sports where the training load assumed from competition may be overestimated.

This study assessed the relative demands of elite field hockey training and competition to determine whether familiar exercise prescription strategies provide an appropriate training stimulus. Sixteen elite male field hockey players (age, 25 ± 4 years; body mass, 70.9 ± 6.6 kg; and maximal oxygen consumption, 61.0 ± 2.1 ml·kg·min [mean ± SD]) participated in the study. Seventy-five elite level competition and 37 training analyses from 8 games and 4 training sessions were obtained. Training duration was longer than competition and covered a greater total distance (109 ± 2.5 vs. 74 ± 0.3 minutes and 7318 ± 221 vs. 5868 ± 75 m; p < 0.001 in both). The distance covered sprinting and running at high intensity was not different between training and competition (114 ± 6 vs. 116 ± 9 m when sprinting and 457 ± 6 vs. 448 ± 7 m for high-intensity running). More high-intensity accelerations were performed during training than in competition (37 ± 3 vs. 20 ± 2). Despite having lower predicted aerobic capacity and covering less distance in competition than in some previous studies, these data support the suggestion that it is high-intensity activity that differentiates international level competition and further suggests that international players can replicate the intensity of competition during small-sided games.

The pH dependence of matrix metalloproteinase (MMP) catalysis is described by a broad bell-shaped curve, indicating the involvement of two unspecified ionizable groups in proteolysis. Stromelysin-1 has a third pK(a) near 6, resulting in a uniquely sharp acidic catalytic optimum, which has recently been attributed to His(224). This suggests the presence of a critical, but unidentified, S1' substructure. Integrating biochemical characterizations of inhibitor-enzyme interactions with active site topography from corresponding crystal structures, we isolated contributions to the pH dependence of catalysis and inhibition of active site residues Glu(202) and His(224). The acidic pK(a) 5.6 is attributed to the Glu(202).zinc.H(2)O complex, consistent with a role for the invariant active site Glu as a general base in MMP catalysis. The His(224)-dependent substructure is identified as a tripeptide (Pro(221)-Leu(222)-Tyr(223)) that forms the substrate cleft lower wall. Substrate binding induces a beta-conformation in this sequence, which extends and anchors the larger beta-sheet of the enzyme. substrate complex and appears to be essential for productive substrate binding. Because the PXY tripeptide is strictly conserved among MMPs, this "beta-anchor" may represent a common motif required for macromolecular substrate hydrolysis. The striking acidic profile of stromelysin-1 defined by the combined ionization of Glu(202) and His(224) allows the design of highly selective inhibitors.

Effective inhibitors of matrix metalloproteinases (MMPs), a family of connective tissue-degrading enzymes, could be useful for the treatment of diseases such as cancer, multiple sclerosis, and arthritis. Many of the known MMP inhibitors are derived from peptide substrates, with high potency in vitro but little selectivity among MMPs and poor bioavailability. We have discovered nonpeptidic MMP inhibitors with improved properties, and report here the crystal structures of human stromelysin-1 catalytic domain (SCD) complexed with four of these inhibitors. The structures were determined and refined at resolutions ranging from 1.64 to 2.0 A. Each inhibitor binds in the active site of SCD such that a bulky diphenyl piperidine moiety penetrates a deep, predominantly hydrophobic S'1 pocket. The active site structure of the SCD is similar in all four inhibitor complexes, but differs substantially from the peptide hydroxamate complex, which has a smaller side chain bound in the S'1 pocket. The largest differences occur in the loop forming the "top" of this pocket. The occupation of these nonpeptidic inhibitors in the S'1 pocket provides a structural basis to explain their selectivity among MMPs. An analysis of the unique binding mode predicts structural modifications to design improved MMP inhibitors.

A new series of potent fused thiazole mGlu5 receptor positive allosteric modulators (PAMs) (10, 11 and 27-31) are disclosed and details of the SAR and optimization are described. Optimization of alkynyl thiazole 9 (Lu AF11205) led to the identification of potent fused thiazole analogs 10b, 27a, 28j and 31d. In general, substituted cycloalkyl, aryl and heteroaryl carboxamides, and carbamate analogs are mGlu5PAMs, whereas smaller alkyl carboxamide, sulfonamide and sulfamide analogs tend to be mGlu5 negative allosteric modulators (NAMs).

Purpose:The current study assessed the impact of full-game (FG) and time-on-pitch (TOP) procedures for global-positioning-system (GPS) analysis on the commonly used markers of physical performance in elite field hockey.Methods:Sixteen international male field hockey players, age 19–30, were studied (yielding 73 player analyses over 8 games). Physical activity was recorded using a 5-Hz GPS.Results:Distance covered, player load, maximum velocity, high-acceleration efforts, and distance covered at specified speed zones were all agreeable for both analysis procedures (P > .05). However, percentage time spent in 0–6 km/h was higher for FG (ES: –21% to –16%; P < .001), whereas the percentage time in all other speed zones (1.67–3.06 m/s, 3.06–4.17 m/s, 4.17–5.28 m/s, and > 6.39 m/s) and relative distance (m/min) were higher for TOP (ES: 8–10%, 2–7%, 2–3%, 1–1%, 0–1%, respectively; P < .001).Conclusions:These data demonstrate that GPS analysis procedures should be appropriate for the nature of the sport being studied. In field hockey, TOP and FG analysis procedures are comparable for distance-related variables but significantly different for time-dependent factors. Using inappropriate analysis procedures can alter the perceived physiological demand of elite field hockey because of “rolling” substitutions. Inaccurate perception of physiological demand could negatively influence training prescription (for both intensity and volume).

A series of 6-alkoxyisoindolin-1-ones with a magic shotgun pharmacological profile are presented as potential antipsychotics. The in vitro pharmacological profile includes D2 partial agonism (30–55%), 5-HT1A partial agonism (60–90%), and 5-HT2A antagonism. Selected compounds in this series displayed good in vivo activity and potency.

A new surface chemistry presenting an abundance of functional groups for ligand immobilization in an ultra-low fouling background all in one material for nanoparticles was introduced. This surface platform, as demonstrated by zwitterionic poly(carboxybetaine acrylamide) (polyCBAA) coated nanoparticles, is not only ultra stable in undiluted human blood serum, but also can be conjugated to biomolecules conveniently and effectively. Thus, this surface chemistry is ideal to create multi-functional nanoparticles for targeted delivery and diagnostics. In addition, this work clearly shows that 10% blood serum commonly used to evaluate the stability of nanoparticles is insufficient and a new evaluation criterion with undiluted blood serum is recommended.

The presence of plasma cholesteryl ester transfer protein (CETP) activity may be atherogenic, and, therefore, strategies to inhibit its activity or production may result in a beneficial effect on lipoprotein profiles and the disease process. The current report describes 4-phenyl-5-tridecyl-4H-1,2,4- triazole-3-thiol (PD 140195), a novel CETP inhibitor. The concentration-dependent inhibition of CETP by PD 140195 and the inhibitory monoclonal antibody (Mab) TP2 is demonstrated in a variety of in vitro assay systems. Molecular models of PD 140195 suggest a spatial mimicry of the cholesteryl ester structure. Despite the structural similarity, kinetic studies with a fluorescent cholesteryl ester analog suggest that the inhibition of transfer is not competitive. PD 140195 also selectively inhibited cholesteryl ester but not triglyceride transfer, while the Mab TP2 blocked CETP transfer of both. Studies were carried out to determine whether CETP inhibition observed in vitro could also be demonstrated in vivo. When PD 140195 was intravenously infused to anesthetized rabbits (up to 20 mg/kg), only transient CETP inhibition was observed. In vitro reconstitution studies in the presence of bovine serum albumin resulted in marked reduction of PD 140195 inhibitory activity. Thus, the low activity of PD 140195 in whole plasma probably results from binding to other plasma proteins.

In the present report we describe a simple and practical method to assess CETP activity in a defined system by use of microemulsions containing a fluorescent cholesteryl ester analog. The microemulsions are stable, simple to prepare, and can be made to defined composition. Initial transfer rates are easily determined by monitoring changes in fluorescence. We have used the fluorescent cholesteryl ester analog, cholesteryl 4,4-difluoro-5,7-dimethyl-4-boro-3 alpha, 4 alpha-diaza-3-indacenedodecanoate (BODIPY-CE), to demonstrate the utility of this assay. The assay takes advantage of the concentration-dependent self-quenching of BODIPY-CE, when this analog is incorporated into microemulsions. We have used this new assay to demonstrate fluorescent lipid transfer facilitated by rabbit and human d > 1.21 g/ml plasma fraction and recombinant human CETP. A known inhibitory monoclonal antibody (Mab) to human CETP blocked BODIPY-CE transfer in a dose-dependent manner. We have also used BODIPY-CE microemulsions to measure CETP activity in whole plasma.

Hypercholesterolemia is one of the few independent risk factors definitively linked to increased morbidity and mortality due to myocardial infarction. One possible therapy of current interest is the prevention of the absorption of dietary cholesterol by inhibiting the enzyme, acyl-CoA: cholesterol acyltransferase (ACAT), which catalyses the intracellular formation of cholesterol esters. Evidence is now accumulating that suggests that ACAT inhibition may not only lower plasma cholesterol levels, but may also have a direct effect at the artery wall, where ACAT has been shown to be responsible for the accumulation of cholesterol esters in arterial lesions. Drago Sliskovic and Andrew White discuss the importance of ACAT in the lipid transport system and the consequences of its inhibition in a variety of tissues, with emphasis on both lipid-lowering and anti-atherosclerotic effects.