Association of Secondhand Smoke Exposure with Pediatric Invasive Bacterial Disease and Bacterial Carriage: A Systematic Review and Meta-analysis

Background: A number of epidemiologic studies have observed an association between secondhand smoke (SHS) exposure and pediatric invasive bacterial disease (IBD) but the evidence has not been systematically reviewed. We carried out a systematic review and meta-analysis of SHS exposure and two outcomes, IBD and pharyngeal carriage of bacteria, for Neisseria meningitidis (N. meningitidis), Haemophilus influenzae type B (Hib), and Streptococcus pneumoniae (S. pneumoniae). Methods and Findings: Two independent reviewers searched Medline, EMBASE, and selected other databases, and screened articles for inclusion and exclusion criteria. We identified 30 case-control studies on SHS and IBD, and 12 crosssectional studies on SHS and bacterial carriage. Weighted summary odd ratios (ORs) were calculated for each outcome and for studies with specific design and quality characteristics. Tests for heterogeneity and publication bias were performed. Compared with those unexposed to SHS, summary OR for SHS exposure was 2.02 (95% confidence interval [CI] 1.52–2.69) for invasive meningococcal disease, 1.21 (95% CI 0.69–2.14) for invasive pneumococcal disease, and 1.22 (95% CI 0.93–1.62) for invasive Hib disease. For pharyngeal carriage, summary OR was 1.68 (95% CI, 1.19–2.36) for N. meningitidis, 1.66 (95% CI 1.33–2.07) for S. pneumoniae, and 0.96 (95% CI 0.48–1.95) for Hib. The association between SHS exposure and invasive meningococcal and Hib diseases was consistent regardless of outcome definitions, age groups, study designs, and publication year. The effect estimates were larger in studies among children younger than 6 years of age for all three IBDs, and in studies with the more rigorous laboratory-confirmed diagnosis for invasive meningococcal disease (summary OR 3.24; 95% CI 1.72–6.13). Conclusions: When considered together with evidence from direct smoking and biological mechanisms, our systematic review and meta-analysis indicates that SHS exposure may be associated with invasive meningococcal disease. The epidemiologic evidence is currently insufficient to show an association between SHS and invasive Hib disease or pneumococcal disease. Because the burden of IBD is highest in developing countries where SHS is increasing, there is a need for high-quality studies to confirm these results, and for interventions to reduce exposure of children to SHS

Multidimensional Methods for the Formulation of Bipharmaceuticals and Vaccines

Determining and preserving the higher order structural integrity and conformational stability of proteins, plasmid DNA and macromolecular complexes such as viruses, virus-like particles and adjuvanted antigens is often a significant barrier to the successful stabilization and formulation of biopharmaceutical drugs and vaccines. These properties typically must be investigated with multiple lower resolution experimental methods, since each technique monitors only a narrow aspect of the overall conformational state of a macromolecular system. This review describes the use of empirical phase diagrams (EPDs) to combine large amounts of data from multiple high-throughput instruments and construct a map of a target macromolecule's physical state as a function of temperature, solvent conditions, and other stress variables. We present a tutorial on the mathematical methodology, an overview of some of the experimental methods typically used, and examples of some of the previous major formulation applications. We also explore novel applications of EPDs including potential new mathematical approaches as well as possible new biopharmaceutical applications such as analytical comparability, chemical stability, and protein dynamics

An Improved Methodology for Multidimensional High- Throughput Preformulation Characterization of Protein Conformational Stability

The Empirical Phase Diagram (EPD) technique is a vector-based multidimensional analysis method for summarizing large data sets from a variety of biophysical techniques. It can be used to provide comprehensive preformulation characterization of a macromolecule’s higher-order structural integrity and conformational stability. In its most common mode, it represents a type of stimulus-response diagram using environmental variables such as temperature, pH, and ionic strength as the stimulus, with alterations in macromolecular structure being the response. Until now EPD analysis has not been available in a high throughput mode because of the large number of experimental techniques and environmental stressor/stabilizer variables typically employed. A new instrument has been developed that combines circular dichroism, UV-absorbance, fluorescence spectroscopy and light scattering in a single unit with a 6-position temperature controlled cuvette turret. Using this multifunctional instrument and a new software system we have generated EPDs for four model proteins. Results confirm the reproducibility of the apparent phase boundaries and protein behavior within the boundaries. This new approach permits two EPDs to be generated per day using only 0.5 mg of protein per EPD. Thus, the new methodology generates reproducible EPDs in high-throughput mode, and represents the next step in making such determinations more routine

Structure-Function Analysis of Invasion Plasmid Antigen C (IpaC) from Shigella flexneri

Shigella flexneri causes a self-limiting gastroenteritis in humans, characterized by severe localized inflammation and ulceration of the colonic mucosa. Shigellosis most often targets young children in underdeveloped countries. Invasion plasmid antigen C (IpaC) has been identified as the primary effector protein for Shigella invasion of epithelial cells. Although an initial model of IpaC function has been developed, no detailed structural information is available that could assist in a better understanding of the molecular basis for its interactions with the host cytoskeleton and phospholipid membrane. We have therefore initiated structural studies of IpaC, IpaC I′, (residues 101–363 deleted), and IpaC ΔH (residues 63–170 deleted). The secondary and tertiary structure of the protein was examined as a function of temperature, employing circular dichroism and high resolution derivative absorbance techniques. ANS (8-anilino-1-napthalene sulfonic acid) was used to probe the exposure of the hydrophobic surfaces under different conditions. The interaction of IpaC and these mutants with a liposome model (liposomes with entrapped fluorescein) was also examined. Domain III (residues 261–363) was studied using linker-scanning mutagenesis. It was shown that domain III contains periodic, sequence-dependent activity, suggesting helical structure in this section of the protein. In addition to these structural studies, investigation into the actin nucleation properties of IpaC was conducted, and actin nucleation by IpaC and some of the mutants was exhibited. Structure-function relationships of IpaC are discussed

Synthetic Cationic Autoantigen Mimics Glatiramer Acetate Persistence at the Site of Injection and Is Efficacious Against Experimental Autoimmune Encephalomyelitis

A synthetic peptide, K-PLP, consisting of 11-unit poly-lysine (K11) linked via polyethylene glycol (PEG) to proteolipid protein epitope (PLP) was synthesized, characterized, and evaluated for efficacy in ameliorating experimental autoimmune encephalomyelitis (EAE) induced by PLP. K-PLP was designed to mimic the cationic nature of the relapsing-remitting multiple sclerosis treatment, glatiramer acetate (GA). With a pI of ~10, GA is able to form visible aggregates at the site of injection via electrostatic interactions with the anionic extracellular matrix. Aggregation further facilitates the retention of GA at the site of injection and draining lymph nodes, which may contribute to its mechanism of action. K-PLP with a pI of ~11, was found to form visible aggregates in the presence of glycosaminoglycans and persist at the injection site and draining lymph nodes in vivo, similar to GA. Additionally, EAE mice treated with K-PLP showed significant inhibition of clinical symptoms compared to free poly-lysine and to PLP, which are the components of K-PLP. The ability of the poly-lysine motif to retain PLP at the injection site, which increased the local exposure of PLP to immune cells may be an important factor affecting drug efficacy

The role of covalent dimerization on the physical and chemical stability of the EC1 domain of human E-cadherin

The objective of this work was to evaluate the solution stability of the EC1 domain of E-cadherin under various conditions. The EC1 domain was incubated at various temperatures (4, 37, and 70 °C) and pH values (3.0, 7.0, and 9.0). At pH 9.0 and 37 or 70 °C, a significant loss of EC1 was observed due to precipitation and a hydrolysis reaction. The degradation was suppressed upon addition of DTT, suggesting that the formation of EC1 dimer facilitated the EC1 degradation. At 4 °C and various pH values, the EC1 secondary and tertiary showed changes upon incubation up to 28 days, and DTT prevented any structural changes upon 28 days of incubation. Molecular dynamics simulations indicated that the dimer of EC1 has higher mobility than does the monomer; this higher mobility of the EC1 dimer may contribute to instability of the EC1 domain

Multicentre validation of the bedside paediatric early warning system score: a severity of illness score to detect evolving critical illness in hospitalised children

Abstract Introduction The timely provision of critical care to hospitalised patients at risk for cardiopulmonary arrest is contingent upon identification and referral by frontline providers. Current approaches require improvement. In a single-centre study, we developed the Bedside Paediatric Early Warning System (Bedside PEWS) score to identify patients at risk. The objective of this study was to validate the Bedside PEWS score in a large patient population at multiple hospitals. Methods We performed an international, multicentre, case-control study of children admitted to hospital inpatient units with no limitations on care. Case patients had experienced a clinical deterioration event involving either an immediate call to a resuscitation team or urgent admission to a paediatric intensive care unit. Control patients had no events. The scores ranged from 0 to 26 and were assessed in the 24 hours prior to the clinical deterioration event. Score performance was assessed using the area under the receiver operating characteristic (AUCROC) curve by comparison with the retrospective rating of nurses and the temporal progression of scores in case patients. Results A total of 2,074 patients were evaluated at 4 participating hospitals. The median (interquartile range) maximum Bedside PEWS scores for the 12 hours ending 1 hour before the clinical deterioration event were 8 (5 to 12) in case patients and 2 (1 to 4) in control patients (P < 0.0001). The AUCROC curve (95% confidence interval) was 0.87 (0.85 to 0.89). In case patients, mean scores were 5.3 at 20 to 24 hours and 8.4 at 0 to 4 hours before the event (P < 0.0001). The AUCROC curve (95% CI) of the retrospective nurse ratings was 0.83 (0.81 to 0.86). This was significantly lower than that of the Bedside PEWS score (P < 0.0001). Conclusions The Bedside PEWS score identified children at risk for cardiopulmonary arrest. Scores were elevated and continued to increase in the 24 hours before the clinical deterioration event. Prospective clinical evaluation is needed to determine whether this score will improve the quality of care and patient outcomes

Soluble Antigen Arrays for Selective Desensitization of Insulin-Reactive B Cells

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Molecular Pharmaceutics, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.molpharmaceut.8b01250.Autoimmune diseases are believed to be highly dependent on loss of immune tolerance to self-antigens. Currently, no treatments have been successful clinically in inducing autoantigen-specific tolerance, including efforts to utilize antigen-specific immunotherapy (ASIT) to selectively correct the aberrant autoimmunity. Soluble antigen arrays (SAgAs) represent a novel autoantigen delivery system composed of a linear polymer, hyaluronic acid (HA), displaying multiple copies of conjugated autoantigen. We have previously reported that Soluble Antigen Arrays proteolipid protein (SAgAPLP) induced tolerance to a specific multiple sclerosis (MS) autoantigen, proteolipid peptide (PLP). Utilizing SAgA technology, we have developed a new ASIT as a possible type 1 diabetes (T1D) therapeutic by conjugating human insulin to HA, known as Soluble Antigen Array Insulin (SAgAIns). Three types were synthesized: low valency lvSAgAIns (2 insulins per HA), medium valency mvSAgAIns (4 insulins per HA) and, high valency hvSAgAIns (9 insulins per HA) to determine if valency differentially modulates the ex vivo activity of insulin-binding B cells (IBCs). Extensive biophysical characterization was performed for the SAgA molecules. SAgAIns molecules were successfully used to affect the biologic activity of IBCs by inducing desensitization of the B cell antigen receptors (BCR). SAgAIns bound specifically to insulin-reactive B cells without blocking epitopes recognized by antibodies against the Fc regions of membrane immunoglobulin or CD79 transducer components of the BCR. Pre-incubation of IBCs (125Tg) with SAgAIns, but not HA alone, rendered the IBCs refractory to re-stimulation. SAgAIns induced a decrease in BCR expression and IP3R-mediated intracellular calcium release. Surprisingly, SAgAIns binding to BCR on the surface of IBCs induced the observed effects at both high and low SAgAIns valency. Future studies aim to test the effects of SAgAIns on disease progression in the VH125.NOD mouse model of T1D.NIH T32 GM00854

Impact of Detergent on the Biophysical Properties and Immune Response of the IpaDB Fusion Protein, a Candidate Subunit Vaccine against Shigella spp.

Shigella spp. are causative agents of bacillary dysentery, a human illness with high global morbidity levels, particularly among elderly and infant populations. Shigella infects via the fecal-oral route, and its virulence is dependent upon a type III secretion system (T3SS). Two components of the exposed needle tip complex of the Shigella T3SS, invasion plasmid antigen D (IpaD) and IpaB, have been identified as broadly protective antigens in the mouse lethal pneumonia model. A recombinant fusion protein (DB fusion) was created by joining the coding sequences of IpaD and IpaB. The DB fusion is coexpressed with IpaB\u27s cognate chaperone, IpgC, for proper recombinant expression. The chaperone can then be removed by using the mild detergents octyl oligooxyethelene (OPOE) or N,N-dimethyldodecylamine N-oxide (LDAO). The DB fusion in OPOE or LDAO was used for biophysical characterization and subsequent construction of an empirical phase diagram (EPD). The EPD showed that the DB fusion in OPOE is most stable at neutral pH below 55°C. In contrast, the DB fusion in LDAO exhibited remarkable thermal plasticity, since this detergent prevents the loss of secondary and tertiary structures after thermal unfolding at 90°C, as well as preventing thermally induced aggregation. Moreover, the DB fusion in LDAO induced higher interleukin-17 secretion and provided a higher protective efficacy in a mouse challenge model than did the DB fusion in OPOE. These data indicate that LDAO might introduce plasticity to the protein, promoting thermal resilience and enhanced protective efficacy, which may be important in its use as a subunit vaccine

The novel mu-opioid antagonist, GSK1521498, reduces ethanol consumption in C57BL/6J mice.

RATIONALE Using the drinking-in-the-dark (DID) model, we compared the effects of a novel mu-opioid receptor antagonist, GSK1521498, with naltrexone, a licensed treatment of alcohol dependence, on ethanol consumption in mice. OBJECTIVE We test the ability of GSK1521498 to reduce alcohol consumption and compare its intrinsic efficacy to that of naltrexone by comparing the two drugs at doses matched for equivalent receptor occupancy. METHODS Thirty-six C57BL/6J mice were tested in a DID procedure. In 2-day cycles, animals experienced one baseline, injection-free session, and one test session when they received two injections, one of test drug and one placebo. All animals received GSK1521498 (0, 0.1, 1 and 3 mg/kg, i.p., 30 min pre-treatment) and naltrexone (0, 0.1, 1 and 3 mg/kg, s.c. 10 min pre-treatment) in a cross-over design. Receptor occupancies following the same doses were determined ex vivo in separate groups by autoradiography, using [3H]DAMGO. Binding in the region of interest was measured integrally by computer-assisted microdensitometry and corrected for non-specific binding. RESULTS Both GSK1521498 and naltrexone dose-dependently decreased ethanol consumption. When drug doses were matched for 70-75 % receptor occupancy, GSK1521498 3 mg/kg, i.p., caused a 2.5-fold greater reduction in alcohol consumption than naltrexone 0.1 mg/kg, s.c. Both GSK1521498 and naltrexone significantly reduced sucrose consumption at a dose of 1 mg/kg but not 0.1 mg/kg. In a test of conditioned taste aversion, GSK1521498 (3 mg/kg) reduced sucrose consumption 24 h following exposure to a conditioning injection. CONCLUSIONS Both opioid receptor antagonists reduced alcohol consumption but GK1521498 has higher intrinsic efficacy than naltrexone