Gene-gene functional relationships in Alzheimer's disease: CELF1 regulates KLC1 alternative splicing.

The causes of Alzheimer's disease (AD) are poorly understood, although many genes are known to be involved in this pathology. To gain insights into the underlying molecular mechanisms, it is essential to identify the relationships between individual AD genes. Previous work has shown that the splice variant E of KLC1 (KLC1_vE) promotes AD, and that the CELF1 gene, which encodes an RNA-binding protein involved in splicing regulation, is at a risk locus for AD. Here, we identified a functional link between CELF1 and KLC1 in AD pathogenesis. Transcriptomic data from human samples from different ethnic groups revealed that CELF1 mRNA levels are low in AD brains, and the splicing pattern of KLC1 is strongly correlated with CELF1 expression levels. Specifically, KLC1_vE is negatively correlated with CELF1. Depletion and overexpression experiments in cultured cells demonstrated that the CELF1 protein down-regulates KLC1_vE. In a cross-linking and immunoprecipitation sequencing (CLIP-seq) database, CELF1 directly binds to KLC1 RNA, following which it likely modulates terminal exon usage, hence KLC1_vE formation. These findings reveal a new pathogenic pathway where a risk allele of CELF1 is associated with reduced CELF1 expression, which up-regulates KLC1_vE to promote AD.

IgA deficiency destabilizes homeostasis toward intestinal microbes and increases systemic immune dysregulation.

The ability of most patients with selective immunoglobulin A (IgA) deficiency (SIgAD) to remain apparently healthy has been a persistent clinical conundrum. Compensatory mechanisms, including IgM, have been proposed, yet it remains unclear how secretory IgA and IgM work together in the mucosal system and, on a larger scale, whether the systemic and mucosal anti-commensal responses are redundant or have unique features. To address this gap in knowledge, we developed an integrated host-commensal approach combining microbial flow cytometry and metagenomic sequencing (mFLOW-Seq) to comprehensively define which microbes induce mucosal and systemic antibodies. We coupled this approach with high-dimensional immune profiling to study a cohort of pediatric patients with SIgAD and household control siblings. We found that mucosal and systemic antibody networks cooperate to maintain homeostasis by targeting a common subset of commensal microbes. In IgA-deficiency, we find increased translocation of specific bacterial taxa associated with elevated levels of systemic IgG targeting fecal microbiota. Associated features of immune system dysregulation in IgA-deficient mice and humans included elevated levels of inflammatory cytokines, enhanced follicular CD4 T helper cell frequency and activation, and an altered CD8 T cell activation state. Although SIgAD is clinically defined by the absence of serum IgA, the symptomatology and immune dysregulation were concentrated in the SIgAD participants who were also fecal IgA deficient. These findings reveal that mucosal IgA deficiency leads to aberrant systemic exposures and immune responses to commensal microbes, which increase the likelihood of humoral and cellular immune dysregulation and symptomatic disease in patients with IgA deficiency.

Arresting microbiome development limits immune system maturation and resistance to infection in mice.

Disruptions to the intestinal microbiome during weaning lead to negative effects on host immune function. However, the critical host-microbe interactions during weaning that are required for immune system development remain poorly understood. We find that restricting microbiome maturation during weaning stunts immune system development and increases susceptibility to enteric infection. We developed a gnotobiotic mouse model of the early-life microbiome Pediatric Community (PedsCom). These mice develop fewer peripheral regulatory T cells and less IgA, hallmarks of microbiota-driven immune system development. Furthermore, adult PedsCom mice retain high susceptibility to Salmonella infection, which is characteristic of young mice and children. Altogether, our work illustrates how the post-weaning transition in microbiome composition contributes to normal immune maturation and protection from infection. Accurate modeling of the pre-weaning microbiome provides a window into the microbial requirements for healthy development and suggests an opportunity to design microbial interventions at weaning to improve immune development in human infants.

Enterococci enhance Clostridioides difficile pathogenesis.

Enteric pathogens are exposed to a dynamic polymicrobial environment in the gastrointestinal tract1. This microbial community has been shown to be important during infection, but there are few examples illustrating how microbial interactions can influence the virulence of invading pathogens2. Here we show that expansion of a group of antibiotic-resistant, opportunistic pathogens in the gut-the enterococci-enhances the fitness and pathogenesis of Clostridioides difficile. Through a parallel process of nutrient restriction and cross-feeding, enterococci shape the metabolic environment in the gut and reprogramme C. difficile metabolism. Enterococci provide fermentable amino acids, including leucine and ornithine, which increase C. difficile fitness in the antibiotic-perturbed gut. Parallel depletion of arginine by enterococci through arginine catabolism provides a metabolic cue for C. difficile that facilitates increased virulence. We find evidence of microbial interaction between these two pathogenic organisms in multiple mouse models of infection and patients infected with C. difficile. These findings provide mechanistic insights into the role of pathogenic microbiota in the susceptibility to and the severity of C. difficile infection.

Insight Into Host-Microbe Interactions Using Microbial Flow Cytometry Coupled to Next-Generation Sequencing.

Antibody-based assays have been a cornerstone of infectious disease diagnostics for over 100 years [1]. These assays rely on the exquisite sensitivity and specificity of humoral response to almost all infections. While next-generation sequencing (NGS) has tremendous potential to improve diagnostics and uncover host-microbial relationships by directly identifying nucleic acids from infectious microbes, challenges and opportunities for new approaches remain. Here, we review a group of cutting-edge techniques that couple antibody responses with flow cytometry of antibody tagged microbes and NGS. These studies are bringing into focus the dynamic relationship between our immune systems and endogenous microbial communities, which are an important source of pathogens. For simplicity, we use the umbrella term mFLOW-Seq (microbial flow cytometry coupled to NGS) to describe these approaches.

Case Report: Immune Dysregulation Due to Toxoplasma gondii Reactivation After Allogeneic Hematopoietic Cell Transplant.

Disseminated toxoplasmosis is an uncommon but highly lethal cause of hyperferritinemic sepsis after hematopoietic cell transplantation (HCT). We report two cases of disseminated toxoplasmosis from two centers in critically ill adolescents after HCT: a 19-year-old who developed fever and altered mental status on day +19 after HCT and a 20-year-old who developed fever and diarrhea on day +52 after HCT. Both patients developed hyperferritinemia with multiple organ dysfunction syndrome and profound immune dysregulation, which progressed to death despite maximal medical therapies. Because disseminated toxoplasmosis is both treatable and challenging to diagnose, it is imperative that intensivists maintain a high index of suspicion for Toxoplasma gondii infection when managing immunocompromised children, particularly in those with known positive T. gondii serologies.

Intranasal Paclitaxel Alters Alzheimer's Disease Phenotypic Features in 3xTg-AD Mice.

BACKGROUND: Microtubule stabilizing drugs, commonly used as anti-cancer therapeutics, have been proposed for treatment of Alzheimer's disease (AD); however, many do not cross the blood-brain barrier. OBJECTIVE: This research investigated if paclitaxel (PTX) delivered via the intranasal (IN) route could alter the phenotypic progression of AD in 3xTg-AD mice. METHODS: We administered intranasal PTX in 3XTg-AD mice (3xTg-AD n = 15, 10 weeks and n = 10, 44 weeks, PTX: 0.6 mg/kg or 0.9%saline (SAL)) at 2-week intervals. After treatment, 3XTg-AD mice underwent manganese-enhanced magnetic resonance imaging to measure in vivo axonal transport. In a separate 3XTg-AD cohort, PTX-treated mice were tested in a radial water tread maze at 52 weeks of age after four treatments, and at 72 weeks of age, anxiety was assessed by an elevated-plus maze after 14 total treatments. RESULTS: PTX increased axonal transport rates in treated 3XTg-AD compared to controls (p≤0.003). Further investigation using an in vitro neuron model of Aß-induced axonal transport disruption confirmed PTX prevented axonal transport deficits. Confocal microscopy after treatment found fewer phospho-tau containing neurons (5.25±3.8 versus 8.33±2.5, p < 0.04) in the CA1, altered microglia, and reduced reactive astrocytes. PTX improved performance of 3xTg-AD on the water tread maze compared to controls and not significantly different from WT (Day 5, 143.8±43 versus 91.5±77s and Day 12, 138.3±52 versus 107.7±75s for SAL versus PTX). Elevated plus maze revealed that PTX-treated 3xTg-AD mice spent more time exploring open arms (Open arm 129.1±80 versus 20.9±31s for PTX versus SAL, p≤0.05). CONCLUSION: Taken collectively, these findings indicate that intranasal-administered microtubule-stabilizing drugs may offer a potential therapeutic option for treating AD.

Counter Regulation of Spic by NF-κB and STAT Signaling Controls Inflammation and Iron Metabolism in Macrophages.

Activated macrophages must carefully calibrate their inflammatory responses to balance efficient pathogen control with inflammation-mediated tissue damage, but the molecular underpinnings of this "balancing act" remain unclear. Using genetically engineered mouse models and primary macrophage cultures, we show that Toll-like receptor (TLR) signaling induces the expression of the transcription factor Spic selectively in patrolling monocytes and tissue macrophages by a nuclear factor κB (NF-κB)-dependent mechanism. Functionally, Spic downregulates pro-inflammatory cytokines and promotes iron efflux by regulating ferroportin expression in activated macrophages. Notably, interferon-gamma blocks Spic expression in a STAT1-dependent manner. High levels of interferon-gamma are indicative of ongoing infection, and in its absence, activated macrophages appear to engage a "default" Spic-dependent anti-inflammatory pathway. We also provide evidence for the engagement of this pathway in sterile inflammation. Taken together, our findings uncover a pathway wherein counter-regulation of Spic by NF-κB and STATs attune inflammatory responses and iron metabolism in macrophages.

Pharmacy-Led Quality Improvement Project on Pain Control Using Continuous Subcutaneous Infusion of Opioids in an Inpatient Hospice Unit.

OBJECTIVES: The purpose of this quality improvement (QI) project was to improve the overall process of implementing continuous subcutaneous infusion of opioids (CSCIOs) at the West Palm Beach Veterans Affairs Medical Center and characterize their use in the hospice unit. METHODS: A retrospective chart review from July 2014 to August 2017 was conducted to identify patients who had received CSCIO. Results were analyzed with descriptive statistics.The business philosphy, LEAN methodology "The 5 Whys" was utilized to identify the root causes for delayed infusion timeliness and corrections were implemented by August 2018. Follow-up retrospective time study completed from September 2018 to February 2019. RESULTS: Of the 107 patients identified, 7 were excluded and 100 were reviewed. The mean age was 73 years, 94% male, and 86% Caucasian. A total of 55 veterans received morphine with an average final infusion rate of 2.5 mg/h. A total of 45 Veterans received hydromorphone with a final infusion rate of 1.3 mg/h. The average infusion duration until death was 5 days. Pharmacy verified 94 (94%) orders and nursing verified 55 (55%) orders within 1 hour (gold standard). Sixteen (16%) patients received CSCIO within 1 hour. The 5 Whys identified nursing order verification and pharmacy lack of visual STAT order notification for priority as the potential sources for infusion timeliness improvement. The follow-up time study confirmed improvement in pharmacy delivery time from 29% to 75% on time. CONCLUSION: Pharmacist-led intervention directed to improve CSCIO processes in an inpatient hospice unit utilizing LEAN QI methodology increased timeliness of pharmacy CSCIO delivery.

Going Too Far Is the Same as Falling Short†: Kinesin-3 Family Members in Hereditary Spastic Paraplegia.

Proper intracellular trafficking is essential for neuronal development and function, and when any aspect of this process is dysregulated, the resulting "transportopathy" causes neurological disorders. Hereditary spastic paraplegias (HSPs) are a family of such diseases attributed to over 80 spastic gait genes (SPG), specifically characterized by lower extremity spasticity and weakness. Multiple genes in the trafficking pathway such as those relating to microtubule structure and function and organelle biogenesis are representative disease loci. Microtubule motor proteins, or kinesins, are also causal in HSP, specifically mutations in Kinesin-I/KIF5A (SPG10) and two kinesin-3 family members; KIF1A (SPG30) and KIF1C (SPG58). KIF1A is a motor enriched in neurons, and involved in the anterograde transport of a variety of vesicles that contribute to pre- and post-synaptic assembly, autophagic processes, and neuron survival. KIF1C is ubiquitously expressed and, in addition to anterograde cargo transport, also functions in retrograde transport between the Golgi and the endoplasmic reticulum. Only a handful of KIF1C cargos have been identified; however, many have crucial roles such as neuronal differentiation, outgrowth, plasticity and survival. HSP-related kinesin-3 mutants are characterized mainly as loss-of-function resulting in deficits in motility, regulation, and cargo binding. Gain-of-function mutants are also seen, and are characterized by increased microtubule-on rates and hypermotility. Both sets of mutations ultimately result in misdelivery of critical cargos within the neuron. This likely leads to deleterious cell biological cascades that likely underlie or contribute to HSP clinical pathology and ultimately, symptomology. Due to the paucity of histopathological or cell biological data assessing perturbations in cargo localization, it has been difficult to positively link these mutations to the outcomes seen in HSPs. Ultimately, the goal of this review is to encourage future academic and clinical efforts to focus on "transportopathies" through a cargo-centric lens.

Diet-induced microbial autofluorescence confounds flow cytometry of ex vivo isolated fecal microbes.

Microbial flow cytometry is a powerful emerging technology with a broad range of applications including the study of complex microbial communities. Immunologists are increasingly using this technology to study antibody responses against pathogenic and commensal microbes. We employed microbial flow cytometry to quantify the proportion of fecal microbes bound by six different Ig isotypes: IgA, IgM, IgG1, IgG2b, IgG2c, and IgG3. In healthy mammals, secretory IgA (sIgA) binds to a subset of commensal microbes in the gut whereas IgG is not typically found in the intestinal tract of healthy mammals. Unexpectedly, fecal microbes isolated from SPF C57BL/6 mice housed in the Hill facility and imported from the vendors The Jackson Laboratory and Taconic Biosciences showed a strong signal in the Brilliant Violet 711 (BV711) channel. Unstained fecal samples from these mice demonstrated that the BV711 signal was due to bacterial autofluorescence. We found that murine diets containing alfalfa induce ex vivo microbial autofluorescence in the far red spectrum, likely due to chlorophyll. Analysis of unstained intestinal microbes is an important step in microbial flow cytometry to identify diet-induced autofluorescence. We recommend fluorophores with emission spectra below 650 nm (e.g. BV421, PE).

A catalytic antioxidant for limiting amyloid-beta peptide aggregation and reactive oxygen species generation.

Alzheimer's disease (AD) is a multifaceted disease that is characterized by increased oxidative stress, metal-ion dysregulation, and the formation of intracellular neurofibrillary tangles and extracellular amyloid-ß (Aß) aggregates. In this work we report the large affinity binding of the iron(iii) 2,17-bis-sulfonato-5,10,15-tris(pentafluorophenyl)corrole complex FeL1 to the Aß peptide (K d ∼ 10-7) and the ability of the bound FeL1 to act as a catalytic antioxidant in both the presence and absence of Cu(ii) ions. Specific findings are that: (a) an Aß histidine residue binds axially to FeL1; (b) that the resulting adduct is an efficient catalase; (c) this interaction restricts the formation of high molecular weight peptide aggregates. UV-Vis and electron paramagnetic resonance (EPR) studies show that although the binding of FeL1 does not influence the Aß-Cu(ii) interaction (K d ∼ 10-10), bound FeL1 still acts as an antioxidant thereby significantly limiting reactive oxygen species (ROS) generation from Aß-Cu. Overall, FeL1 is shown to bind to the Aß peptide, and modulate peptide aggregation. In addition, FeL1 forms a ternary species with Aß-Cu(ii) and impedes ROS generation, thus showing the promise of discrete metal complexes to limit the toxicity pathways of the Aß peptide.

In vivo induction of membrane damage by ß-amyloid peptide oligomers.

Exposure to the ß-amyloid peptide (Aß) is toxic to neurons and other cell types, but the mechanism(s) involved are still unresolved. Synthetic Aß oligomers can induce ion-permeable pores in synthetic membranes, but whether this ability to damage membranes plays a role in the ability of Aß oligomers to induce tau hyperphosphorylation, or other disease-relevant pathological changes, is unclear. To examine the cellular responses to Aß exposure independent of possible receptor interactions, we have developed an in vivo C. elegans model that allows us to visualize these cellular responses in living animals. We find that feeding C. elegans E. coli expressing human Aß induces a membrane repair response similar to that induced by exposure to the CRY5B, a known pore-forming toxin produced by B. thuringensis. This repair response does not occur when C. elegans is exposed to an Aß Gly37Leu variant, which we have previously shown to be incapable of inducing tau phosphorylation in hippocampal neurons. The repair response is also blocked by loss of calpain function, and is altered by loss-of-function mutations in the C. elegans orthologs of BIN1 and PICALM, well-established risk genes for late onset Alzheimer's disease. To investigate the role of membrane repair on tau phosphorylation directly, we exposed hippocampal neurons to streptolysin O (SLO), a pore-forming toxin that induces a well-characterized membrane repair response. We find that SLO induces tau hyperphosphorylation, which is blocked by calpain inhibition. Finally, we use a novel biarsenical dye-tagging approach to show that the Gly37Leu substitution interferes with Aß multimerization and thus the formation of potentially pore-forming oligomers. We propose that Aß-induced tau hyperphosphorylation may be a downstream consequence of induction of a membrane repair process.

Use of Topical Morphine to Relieve Painful Pressure Ulcers.

INTRODUCTION: Topical morphine is a potential treatment option for painful pressure ulcers in hospice and palliative care patients who favor avoidance of systemic opioid therapy. CASE: A 65-year-old male African-American veteran with a painful stage 3 sacral pressure injury was hesitant to take systemic opioids to control his pain, as he wished to stay alert for family and friends. Topical morphine was initiated, and within 24 hours the patient reported a significant reduction in pain on the numeric rating scale. DISCUSSION: Palliative pharmacotherapy is focused on reducing the symptoms of disease while avoiding side effects that impair quality of life. Evidence suggests topical morphine can be an effective treatment option for painful pressure ulcers and can reduce the need for systemic opioids in select patients. CONCLUSION: In the palliative care setting, topical morphine may be considered for compassionate use when treatment with systemic analgesics is undesired, inadequate, or poorly tolerated.

Pharmacological Inhibition of O-GlcNAcase Enhances Autophagy in Brain through an mTOR-Independent Pathway.

The glycosylation of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is conserved among metazoans and is particularly abundant within brain. O-GlcNAc is involved in diverse cellular processes ranging from the regulation of gene expression to stress response. Moreover, O-GlcNAc is implicated in various diseases including cancers, diabetes, cardiac dysfunction, and neurodegenerative diseases. Pharmacological inhibition of O-GlcNAcase (OGA), the sole enzyme that removes O-GlcNAc, reproducibly slows neurodegeneration in various Alzheimer's disease (AD) mouse models manifesting either tau or amyloid pathology. These data have stimulated interest in the possibility of using OGA-selective inhibitors as pharmaceuticals to alter the progression of AD. The mechanisms mediating the neuroprotective effects of OGA inhibitors, however, remain poorly understood. Here we show, using a range of methods in neuroblastoma N2a cells, in primary rat neurons, and in mouse brain, that selective OGA inhibitors stimulate autophagy through an mTOR-independent pathway without obvious toxicity. Additionally, OGA inhibition significantly decreased the levels of toxic protein species associated with AD pathogenesis in the JNPL3 tauopathy mouse model as well as the 3×Tg-AD mouse model. These results strongly suggest that OGA inhibitors act within brain through a mechanism involving enhancement of autophagy, which aids the brain in combatting the accumulation of toxic protein species. Our study supports OGA inhibition being a feasible therapeutic strategy for hindering the progression of AD and other neurodegenerative diseases. Moreover, these data suggest more targeted strategies to stimulate autophagy in an mTOR-independent manner may be found within the O-GlcNAc pathway. These findings should aid the advancement of OGA inhibitors within the clinic.

Analysis of Inpatient Hospice Pharmacist Interventions Within a Veterans Affairs Medical Center.

Clinical pharmacy interventions have been shown to improve medication therapy, prevent undesirable side effects, and improve patients' clinical outcomes in a number of settings; however, limited data exist to characterize clinical pharmacy specialist (CPS) providers' interventions in an inpatient hospice Veteran Affairs (VA) setting. The primary objective of this quality improvement (QI) project was to quantify the number and types of pharmacy interventions implemented from the Pharmacists Achieve Results with Medications Documentation (PhARMD) tool for inpatient hospice patient encounters in a VA medical center. A total of 453 interventions during 185 patient care encounters were documented by CPS providers between September 1, 2016, and December 31, 2016. These interventions were documented across 32 unique patients, with an average of 14.2 interventions made per patient during this period. CPS providers frequently intervened to optimize pharmacotherapy for the treatment of pain (42.38%), terminal agitation (5.08%), and nausea (3.97%). Additionally, CPS providers played a significant role in the deprescribing of medication by discontinuing drugs no longer indicated (18.3%). These results substantiate the valuable contribution to patient care that the CPS providers make in optimizing symptom management and deprescribing at end-of-life. Future studies are needed to characterize the potential cost savings of CPS provider services in the inpatient hospice setting.

Impact of Antibiotics on Necrotizing Enterocolitis and Antibiotic-Associated Diarrhea.

Antibiotic treatment alters the composition and metabolic function of the intestinal microbiota. These alterations may contribute to the pathogenesis of necrotizing enterocolitis (NEC) and antibiotic-associated diarrhea (AAD). Recent studies are beginning to unravel the contribution of specific groups of microbes and their metabolic pathways to these diseases. Probiotics or other microbiota-targeted therapies may provide effect strategies to prevent and treat NEC and AAD.

Stochastic Subcellular Organization of Dense-Core Vesicles Revealed by Point Pattern Analysis.

Dense-core vesicles (DCVs) are regulated secretory organelles found in many types of neurons. In neurons of the hippocampus, their cargo includes proteins that mediate several pivotal processes, including differentiation and synaptic plasticity. Motivated by interest in DCV distribution and its impact on cargo action, we have used fluorescence microscopy and statistical analysis to develop a quantitative model of the subcellular organization of DCVs in hippocampal neurons that are spontaneously active (their most prevalent state). We also have tested the functionally motivated hypothesis that these organelles are synaptically enriched. Variance-to-mean ratio, frequency distribution, and Moran's autocorrelation analyses reveal that DCV distribution along shafts, and within synapses, follows Poisson statistics, establishing that stochastically dictated organization sustains cargo function. Occupancy in boutons exceeds that at nearby extrasynaptic axonal sites by approximately threefold, revealing significant local presynaptic enrichment. Widespread stochastic organization is consistent with the emerging functional importance of synaptically and extrasynaptically localized DCVs. Presynaptic enrichment is consistent with the established importance of protecting presynaptic sites from depletion of DCV cargo. These results enhance understanding of the link between DCV organization and mechanisms of cargo action, and they reinforce the emerging theme that randomness is a prevalent aspect of synaptic organization and composition.

Valproic Acid Suppositories for Management of Seizures for Geriatric Patients.

This case describes the use of valproic acid suppositories for secondary seizure prophylaxis in a geriatric veteran with a feeding and swallowing disorder. The effectiveness of valproic acid suppositories is outlined to reinforce the need for compounding pharmacies to have this formulation available to meet the needs of geriatric patients.