Delirium and the risk of developing dementia: a cohort study of 12 949 patients.

BACKGROUND: Delirium is an important risk factor for subsequent dementia. However, the field lacks large studies with long-term follow-up of delirium in subjects initially free of dementia to clearly establish clinical trajectories. METHODS: We undertook a retrospective cohort study of all patients over the age of 65 diagnosed with an episode of delirium who were initially dementia free at onset of delirium within National Health Service Greater Glasgow & Clyde between 1996 and 2020 using the Safe Haven database. We estimated the cumulative incidence of dementia accounting for the competing risk of death without a dementia diagnosis. We modelled the effects of age at delirium diagnosis, sex and socioeconomic deprivation on the cause-specific hazard of dementia via cox regression. RESULTS: 12 949 patients with an incident episode of delirium were included and followed up for an average of 741 days. The estimated cumulative incidence of dementia was 31% by 5 years. The estimated cumulative incidence of the competing risk of death without dementia was 49.2% by 5 years. The cause-specific hazard of dementia was increased with higher levels of deprivation and also with advancing age from 65, plateauing and decreasing from age 90. There did not appear to be a relationship with sex. CONCLUSIONS: Our study reinforces the link between delirium and future dementia in a large cohort of patients. It highlights the importance of early recognition of delirium and prevention where possible.

Effect of a Pluronic(®) P123 formulation on the nitric oxide-generating drug JS-K.

PURPOSE: O(2)-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] or JS-K is a nitric oxide-producing prodrug of the arylated diazeniumdiolate class with promising anti-tumor activity. JS-K has challenging solubility and stability properties. We aimed to characterize and compare Pluronic(®) P123-formulated JS-K (P123/JS-K) with free JS-K. METHODS: We determined micelle size, shape, and critical micelle concentration of Pluronic(®) P123. Efficacy was evaluated in vitro using HL-60 and U937 cells and in vivo in a xenograft in NOD/SCID IL2Rγ (null) mice using HL-60 cells. We compared JS-K and P123/JS-K stability in different media. We also compared plasma protein binding of JS-K and P123/JS-K. We determined the binding and Stern Volmer constants, and thermodynamic parameters. RESULTS: Spherical P123/JS-K micelles were smaller than blank P123. P123/JS-K formulation was more stable in buffered saline, whole blood, plasma and RPMI media as compared to free JS-K. P123 affected the protein binding properties of JS-K. In vitro it was as efficacious as JS-K alone when tested in HL-60 and U937 cells and in vivo greater tumor regression was observed for P123/JS-K treated NOD/SCID IL2Rγ (null) mice when compared to free JS-K-treated NOD/SCID IL2Rγ (null) mice. CONCLUSIONS: Pluronic(®) P123 solubilizes, stabilizes and affects the protein binding characteristics of JS-K. P123/JS-K showed more in vivo anti-tumor activity than free JS-K.

Co-formulation of the rF1V plague vaccine with depot-formulated cytokines enhances immunogenicity and efficacy to elicit protective responses against aerosol challenge in mice.

This study evaluated a depot-formulated cytokine-based adjuvant to improve the efficacy of the recombinant F1V (rF1V) plague vaccine and examined the protective response following aerosol challenge in a murine model. The results of this study showed that co-formulation of the Alhydrogel-adsorbed rF1V plague fusion vaccine with the depot-formulated cytokines recombinant human interleukin 2 (rhuIL-2) and/or recombinant murine granulocyte macrophage colony-stimulating factor (rmGM-CSF) significantly enhances immunogenicity and significant protection at lower antigen doses against a lethal aerosol challenge. These results provide additional support for the co-application of the depot-formulated IL-2 and/or GM-CSF cytokines to enhance vaccine efficacy.

The magnitude of the germinal center B cell and T follicular helper cell response predicts long-lasting antibody titers to plague vaccination.

The development of a safe and effective vaccine against Yersinia pestis, the causative organism for plague disease, remains an important global health priority. Studies have demonstrated effective immune-based protection against plague challenge that is induced by plague antigen subunit vaccination in an aqueous alhydrogel formulation; however, whether these candidate vaccines in this formulation and presentation, induce long-lasting immunological memory in the form of durable cellular and antibody recall responses has not been fully demonstrated. In this study, we analyzed germinal center T follicular helper and germinal center B cell responses following F1V and F1 + V plague subunit immunization of mice with vaccines formulated in various adjuvants. Our data demonstrate that recombinant plague protein immunization formulated with IL-2/GM-CSF cytokines bound to alhydrogel adjuvant drive an increase in the magnitude of the germinal center T follicular helper and germinal center B cell responses following primary immunization, compared to vaccines formulated with Alhydrogel adjuvant alone. In contrast, plague protein subunit immunization combined with CpG ODN bound to alhydrogel increased the magnitude and duration of the germinal center Tfh and B cell responses following booster immunization. Importantly, enhanced germinal center Tfh and B cell responses correlated with long-lasting and high F1V-specific antibody titers and more robust antibody recall responses to F1V re-exposure. These findings indicate that vaccine formulations that drive enhancement of the germinal center Tfh and B cell responses are critical for inducing durable plague-specific humoral immunity.

Validation of a Dendritic Cell and CD4+ T Cell Restimulation Assay Contributing to the Immunogenicity Risk Evaluation of Biotherapeutics.

Immunogenicity, defined as the ability to provoke an immune response, can be either wanted (i.e., vaccines) or unwanted. The latter refers to an immune response to protein or peptide therapeutics, characterized by the production of anti-drug antibodies, which may affect the efficacy and/or the safety profiles of these drugs. Consequently, evaluation of the risk of immunogenicity early in the development of biotherapeutics is of critical importance for defining their efficacy and safety profiles. Here, we describe and validate a fit-for-purpose FluoroSpot-based in vitro assay for the evaluation of drug-specific T cell responses. A panel of 24 biotherapeutics with a wide range of clinical anti-drug antibody response rates were tested in this assay. We demonstrated that using suitable cutoffs and donor cohort sizes, this assay could identify most of the compounds with high clinical immunogenicity rates (71% and 78% for sensitivity and specificity, respectively) while we characterized the main sources of assay variability. Overall, these data indicate that the dendritic cell and CD4+ T cell restimulation assay published herein could be a valuable tool to assess the risk of drug-specific T cell responses and contribute to the selection of clinical candidates in early development.

In-plane parallel scanning: a microarray technology for point-of-care testing.

A new microarray technology is described for rapid, inexpensive, multiplex diagnostics assays. Referred to as "in-plane parallel scanning" (IPPS), this technology replaces expensive laser scanning with a grid of 100-µm-wide waveguides embedded in the chip's substrate, enabling real-time quantification of molecular complex formation on the chip's surface. Compared to conventional microarray technology, IPPS has advantages of shorter assay time and lower instrument cost and complexity so that the platform can potentially be used in point-of-care (POC) settings. Two different chip formats are described: a low-density microarray with 10 sensing wells (IPPS-10) and a medium-density one with 100 sensing wells (IPPS-100). Performance was evaluated in two different proof-of-principle immunoassays: interleukin-1ß (IL-1ß) and Clostridium difficile toxin A. The two assays gave similar limits of detection of 0.67 and 0.94 pM, respectively. A saturation kinetics model described the sensor response with apparent dissociation constants of 511 pM for IL-1ß and 6.47 nM for C. difficile toxin A toxoid. The multiplexing capabilities of the IPPS technology were also demonstrated in a multiplex assay for both analytes on the same IPPS-10 chip. Based on these results, the IPPS technology holds promise for translating diagnostic microarrays into near-patient environments.

Optical system design for biosensors based on CCD detection.

The use of Charge Coupled Device (CCD) detectors as an integral part of a biosensing system has become widespread in recent years due to several advantages of this type of detection, such as the ability to image multiple zones on the sensor, the flexibility of defining the sensing configuration and the low-noise performance of the detectors. The specification of the CCD as well as the selection of the other components in this system--including the source and the filters--is driven by the particular transduction mechanism, but all parts must be matched. Particular attention must be paid to reducing the various noise components of the CCD to obtain the lowest detection level, and it is shown that cooling the CCD is often a wise choice.

Rapid throughput solubility screening method for BCS class II drugs in animal GI fluids and simulated human GI fluids using a 96-well format.

A rapid solubility-screening assay was developed with a focus on Biopharmaceutic Classification Scheme (BCS) class II drug solubility in animal and simulated human gastrointestinal (GI) fluids. The assay enables biologically promising drug leads to be evaluated for solubility limitations earlier in the drug development process, minimizes GI fluid needs, and produces in vitro solubility information with potential in vivo implications. A number of BCS II drugs were dissolved in DMSO at approximately 40 mM, and robotically distributed to a 96-well plate. The DMSO was evaporated and drugs were equilibrated with selected GI fluids, both fed and fasted states. After equilibration, precipitated wells were subjected to HPLC analysis. A spreadsheet calculated solubility automatically from HPLC output. Intra-day, inter-day, and inter-plate reproducibility were within 15% RSTD for the tested drugs with the primary source of variability being injection precision of our injector system. The reported solubility from screening assays was well correlated with literature data (r(2) = 0.80) with a slope of 0.86 and (r(2) = 0.99) with a slope of 0.89. This screening assay converts conventional solubility measurements to a 96-well format for increased throughput (>12 samples/h), reduces fluid needs, and minimizes drug consumption.

Rapid throughput screening of apparent KSP values for weakly basic drugs using 96-well format.

A rapid-throughput screening assay was developed to estimate the salt solubility parameter, K(SP), with a minimal quantity of drug. This assay allows for early evaluation of salt limited solubility with a large number of counter-ions and biologically promising drug leads. Drugs dissolved (typically 10 mM) in DMSO are robotically distributed to a 96-well plate. DMSO is evaporated, and drugs are equilibrated with various acids at different concentrations (typically <1 M) to yield final total drug concentrations around 2.5 mM. The plate is checked for precipitation. Filtrates from only those precipitated wells were subjected to rapid gradient HPLC analysis. An iterative procedure is employed to calculate all species concentrations based on mass and charge balance equations. The apparent K(SP) values assuming 1:1 stoichiometry are determined from counter-ion and ionized drug activities. A correlation coefficient >0.975 for eight drugs totaling 16 salts is reported. Intra-day and inter-day reproducibility was <10%. Conventional apparent K(SP) measurements were translated to 96-well format for increased throughput and minimal drug consumption (typically 10 mg) to evaluate at least eight different counter-ions. Although the current protocol estimates K(SP) from 10(-3) to 10(-7) M, the dynamic range of the assay could be expanded by adjusting drug and counter-ion concentrations.

Neuroimmune Biomarkers in Mental Illness.

Exploration of neuroimmune mechanisms is vital to the understanding of the pathogenesis and pathophysiology of mental disorders. Inflammatory and immune mechanisms are increasingly understood to underpin a number of neuropsychiatric disorders, with an ever-expanding evidence base drawn from basic science to large-scale epidemiological data. Unravelling of these mechanisms should lead to biomarker discovery and potential new avenues for therapeutics that modulate immunological mechanisms. Identification of neuroimmune biomarkers is vital to improving diagnosis, stratification and treatment of mental disorders. There is an urgent clinical need for new therapeutic approaches with poor treatment response and treatment resistance a major problem for many psychiatric disorders including depression and schizophrenia. Neurodegenerative psychiatric disorders such as Alzheimer's also have clear neuroimmune underpinnings and manifest an urgent clinical need for improvements in diagnosis and research towards transformative disease-modifying treatments. This chapter provides some background on the role of the neuroimmune system in mental illness, exploring the role for biomarkers, in addition to reviewing the current state of knowledge in this exciting field. We also reflect on the inherent challenges and methodological pitfalls faced by research in this field, including the complexity of conceptualising multidimensional mental disorders and the dynamic shifting sands of the immune system.

Three-dimensional structures of idiotypically related Fabs with intermediate and high affinity for fluorescein.

Multi-disciplinary studies of fluorescein-protein conjugates have led to the generation of a family of antibodies with common idiotypes and affinities for fluorescein ranging over five orders of magnitude. The high affinity 4-4-20 prototype traps the ligand in a highly complementary binding slot, which is lined by multiple aromatic side-chains. An antibody (9-40) of intermediate affinity belongs to the same idiotypic family as 4-4-20 and shares substantial amino acid identities within the VL and VH domains. To establish the structural basis for the affinity differences, we solved the crystal structure of the 9-40 Fab-fluorescein complex at a resolution of 2.3A. Similar to 4-4-20, 9-40 binds fluorescein in a tight aromatic slot with its xanthenonyl ring system accommodated by end-on insertion. However, the combined effects of the amino acid substitutions have resulted in reorganization of the binding site, with the HCDR3 loops showing the greatest differences in conformations. Access to the binding site of 9-40 is substantially more open, leaving the fluorescein's phenylcarboxylate moiety partially exposed to solvent. In addition to the usage of a different D (diversity) mini-gene encoding the HCDR3 loop, the decrease in fluorescein affinity in the 9-40 antibody family appears to be correlated with the substitution of histidine (9-40) for arginine (4-4-20) in position 34 of the antibody light chains.

Probing bis-ANS binding sites of different affinity on aggregated IgG by steady-state fluorescence, time-resolved fluorescence and isothermal titration calorimetry.

Fluorescent dyes, for example, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (bis-ANS) are extensively used to detect nonnative protein structures in therapeutic protein products, for example, during formulation development by monitoring the greatly enhanced dye fluorescence upon binding to nonnative species. Our aim was to characterize the level of heterogeneity of bis-ANS binding sites in a thermally stressed monoclonal antibody (IgG) formulation by steady-state fluorescence, time-resolved fluorescence and isothermal titration calorimetry (ITC), and to obtain apparent dissociation constants (Kd ) by data fitting. Because the methods differ in their underlying measurement principles, they provide different information on binding properties of bis-ANS to thermally stressed IgG. We found very heterogeneous bis-ANS binding sites on thermally stressed IgG, with apparent Kd values ranging from as low as 50 nM (time-resolved fluorescence) to 63 µM (ITC). Steady-state fluorescence and ITC gave insight into an overall binding affinity of a wide population of dye binding sites with micromolar Kd values. Time-resolved fluorescence was particularly sensitive to high-affinity binding sites with nanomolar Kd s. The heterogeneity of the bis-ANS binding sites reflects the complex, heterogeneous nature of the heat-stressed IgG used in this study. To probe such heterogeneity adequately, one should apply complementary analytical methods under various experimental conditions as presented in this paper. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:1294-1305, 2011.

A liposome permeability model for stratum corneum lipid bilayers based on commercial lipids.

The objective was to evaluate stratum corneum lipid liposomes (SCLLs) prepared from commercial lipids as a convenient model system for studying the mechanisms of chemical permeation enhancers. Liposomes prepared from extracted stratum corneum lipids (ESCLLs) were used as a control. Three different types of SCLLs were prepared by sonication or extrusion from mixtures of commercial ceramides, cholesterol, free fatty acids, and cholesterol 3-sulfate (SCLL-I-III; 55/25/15/5 weight ratio). Absolute mannitol permeabilities were 5- to 20-fold lower in SCLLs than in ESCLLs. 1-alkyl-2-pyrrolidone enhancers produced the same enhancement factor for mannitol efflux in sonicated SCLLs (SCLL-I) as reported previously for the ESCLLs. Enhancer-induced changes at graded depths were further monitored in SCLL-I vesicles using fluorescence spectroscopy with n-(9-anthroyloxy) fatty acid fluorescent probes. Lipid packing order, as determined from rotational correlation times derived from steady-state anisotropy and lifetime data, was found to be higher in SCLL-I than in ESCLLs. 1-alkyl-2-pyrrolidones were found to increase the fluidity of the bilayers to approximately the same extent at intermediate depths (C6-C9) as previously reported for the ESCLLs. The present results demonstrate that the sonicated SCLL model may be useful for studying the mechanisms of action of transdermal permeation enhancers.

Aliskiren, an oral renin inhibitor, provides dose-dependent efficacy and sustained 24-hour blood pressure control in patients with hypertension.

OBJECTIVES: This dose-ranging study evaluated the antihypertensive efficacy and tolerability of aliskiren in patients with mild-to-moderate hypertension. BACKGROUND: Low blood pressure (BP) control rates among patients with hypertension indicate a need for improved treatment options. This study investigates aliskiren, the first in a new antihypertensive class called renin inhibitors. METHODS: Patients with mean sitting diastolic BP 95 to 109 mm Hg were randomized to aliskiren 150, 300, or 600 mg or placebo once daily for 8 weeks. Patients completing this treatment phase entered a 2-week treatment-free withdrawal period. Office BP was recorded at baseline, weeks 2, 4, 6, and 8 of treatment, and 4 days and 2 weeks after cessation of treatment. A subgroup of patients underwent ambulatory BP monitoring. RESULTS: In total, 672 patients were randomized to treatment. After 8 weeks, aliskiren 150, 300, and 600 mg significantly reduced mean sitting BP (systolic/diastolic) by 13.0/10.3, 14.7/11.1, and 15.8/12.5 mm Hg, respectively, versus 3.8/4.9 mm Hg with placebo (all p < 0.0001 for systolic and diastolic BP). The BP-lowering effect of aliskiren persisted for up to 2 weeks after treatment withdrawal. Aliskiren significantly reduced mean 24-h ambulatory BP (p < 0.0001 vs. placebo with all doses) exhibiting smooth, sustained effects and high trough-to-peak ratios. Aliskiren was well tolerated; overall adverse event rates were 40.1%, 46.7%, and 52.4% with aliskiren 150, 300, and 600 mg, respectively, and 43.0% with placebo. Few patients discontinued treatment due to adverse events. CONCLUSIONS: Aliskiren provides significant antihypertensive efficacy in patients with hypertension, with no rebound effects on blood pressure after treatment withdrawal.

Bifluorophoric molecules as fluorescent beacons for antibody-antigen binding.

The quenching of fluorescence (up to 98%) by anti-fluorescein antibodies is well documented in the literature. Here we report a system where, instead of quenching, bifluorophoric molecules are designed to increase in fluorescence upon binding by an anti-fluorescein antibody. Bifluorophoric molecules are made of fluorescein (F) linked to tetramethylrhodamine (T) via varying numbers of methylene units, denoted as F-(CH(2))(n)-T. These F-(CH(2))(n)-T conjugates are almost nonfluorescent when free in solution due to intramolecular dimerization and stacking. Upon binding to an anti-fluorescein antibody, however, up to 110-fold increase in fluorescence was observed from the rhodamine moiety. This increase is believed to result from intramolecular dimer dissociation that dequenches the rhodamine fluorescence. Fluorescein fluorescence, on the other hand, remains quenched due to binding and intramolecular resonance energy transfer. Moreover, the excitation wavelength was at the absorption maxima of fluorescein, giving a Stoke's shift of about 90 nm. This system couples directly molecular recognition with a concurrent increase in fluorescence emission, obviating wash and incubation steps required by most assays. It is an important molecular reporter system for developing homogeneous assays.

How well can a T-cell epitope replace its parent carrier protein? A dose-response study.

PURPOSE: This work examines the effectiveness of synthetic peptide immunogens derived from immunodominant T-cell epitopes as replacements for their intact parent protein in vaccines. METHODS: Fluorescein was conjugated to hen egg lysozyme (FL-HEL, positive control) and three synthetic peptide immunogens: (a) murine B10.A (H-2a) immunodominant T-cell epitope of HEL [FL-(T-cell epitope)]; (b) multiple antigenic peptide (MAP) multimer of this epitope ([FL-(T epitope)]n-MAP, n = 2-4); and (c) negative control MAP with T-cell epitope residues replaced with glycine [(FL-Gly18)4-MAP]. The dose response of each immunogen was examined over a 300-fold range in B10.A mice. The immune response was monitored using antifluorescein ELISA assays. RESULTS: FL-(T epitope)'s immune response correlated positively with dose, with maximum response comparable to that of [FL-(T epitope)]n-MAP, or FL-HEL. This trend was consistent across 1 degrees, 2 degrees, and 3 degrees responses, although interanimal variability was higher in the latter two because of an all-or-none response in mice immunized with this peptide. [FL-(T epitope)]n-MAP's immune response was consistently high and nearly dose independent, a trend observed across 1 degrees, 2 degrees, and 3 degrees responses. FL-HEL's immune response correlated negatively to dose in the 1 degrees response but was nearly dose independent in the 2 degrees and 3 degrees responses. The magnitude of these latter responses was comparable to that observed for [FL-(T epitope)]n-MAP. (FL-Gly18)4-MAP did not elicit an immune response except at the highest dose. This trend was consistent across 1 degrees, 2 degrees, and 3 degrees responses. CONCLUSIONS: The monomeric epitope was 300-fold less potent than its parent carrier protein, but increasing immunogen valency using MAP technology compensated totally for reduced potency. (FL-Gly18)4-MAP's lack of response at all but the highest dose strongly suggests that a specific immunodominant T-cell epitope sequence for HEL is necessary for successful peptide mimicry of HEL. This work also demonstrates the importance of quality assessment of commercial MAP core resins.

How well can an idiotope peptide mimic replace its parent idiotype in a synthetic peptide vaccine?

PURPOSE: To determine whether a vaccine consisting of an idiotope peptide mimic of the third complementarity-determining region of the immunoglobulin heavy chain (CDR-H3) is an effective substitute for its parent idiotype. Such peptide vaccines could ultimately be used for targeting pathological B lymphocytes. METHODS: Hen egg lysozyme (HEL) conjugates of the Fab' fragment of monoclonal anti-fluorescein antibody 9-40 (Fab'-HEL) or a peptide mimic of the 9-40 CDR-H3 (referred to as the "B epitope" or "Bep," the conjugate is referred to as "Bep-HEL") were injected into separate cohorts of B10.A mice. Two additional control cohorts were injected with either the Bep peptide alone or a noncovalent mixture of Bep and HEL. Sera were assayed for both anti-idiotope and anti-idiotype activity by enzyme-linked immunosorbant assay (ELISA). Primary, secondary, and tertiary immune responses were examined. RESULTS: Both the Bep-HEL idiotope and the Fab-HEL idiotype immunogens elicited homologous, allogenic immune responses. No cross-reactivity was observed between anti-idiotope and anti-idiotype responses after primary immunization. With secondary immunization, 50% of mice immunized with the Bep-HEL conjugate exhibited a cross-reactive anti-idiotype response. Conversely, 100% of mice immunized with the Fab'-HEL conjugate exhibited a marginal, but statistically significant cross-reactive anti-idiotope response. Upon tertiary immunization, 100% of mice immunized with Bep-HEL exhibited a cross-reactive anti-idiotype response, and 55.6% of mice immunized with the Fab'-HEL conjugate exhibited a cross-reactive anti-idiotope response. CONCLUSIONS: Covalent coupling of a xenogenic carrier protein to an idiotype immunogen or its peptide mimic significantly enhances the intensity of homologous, allogenic anti-idiotype or anti-idiotope immune responses. Multiple immunizations are necessary to induce cross-reactivity between the peptide mimic and its parent idiotype.

Single-chain polymorphism analysis in long QT syndrome using planar waveguide fluorescent biosensors.

Rapid detection of single nucleotide polymorphisms (SNPs) has potential applications in both genetic screening and pharmacogenomics. Planar waveguide fluorescent biosensor technology was employed to detect SNPs using a simple hybridization assay with the complementary strand ("capture oligo") immobilized on the waveguide. This technology allows real-time measurements of DNA hybridization kinetics. Under normal conditions, both the wild-type sequence and the SNP-containing sequence will hybridize with the capture oligo, but with different reaction kinetics and equilibrium duplex concentrations. A "design of experiments" approach was used to maximize the differences in the kinetics profiles of the two. Nearly perfect discrimination can be achieved at short times (2 min) with temperatures that destabilize or melt the heteroduplex while maintaining the stability of the homoduplex. The counter ion content of the solvent was shown to have significant effect not only on the melting point of the heteroduplex and the homoduplex but also on the hybridization rate. Changes in both the stability and the difference between the hybridization rates of the hetero- and homoduplex were observed with varying concentrations of three different cations (Na(+), K(+), Mg(2+)). With the difference in hybridization rates maximized, discrimination between the hetero- and the homoduplex can be obtained at lower, less rigorous temperatures at hybridization times of 7.5 min or longer.