Microbial colonization programs are structured by breastfeeding and guide healthy respiratory development.

Breastfeeding and microbial colonization during infancy occur within a critical time window for development, and both are thought to influence the risk of respiratory illness. However, the mechanisms underlying the protective effects of breastfeeding and the regulation of microbial colonization are poorly understood. Here, we profiled the nasal and gut microbiomes, breastfeeding characteristics, and maternal milk composition of 2,227 children from the CHILD Cohort Study. We identified robust colonization patterns that, together with milk components, predict preschool asthma and mediate the protective effects of breastfeeding. We found that early cessation of breastfeeding (before 3 months) leads to the premature acquisition of microbial species and functions, including Ruminococcus gnavus and tryptophan biosynthesis, which were previously linked to immune modulation and asthma. Conversely, longer exclusive breastfeeding supports a paced microbial development, protecting against asthma. These findings underscore the importance of extended breastfeeding for respiratory health and highlight potential microbial targets for intervention.

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.

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.

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).

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.

Atlas stumbled: kinesin light chain-1 variant E triggers a vicious cycle of axonal transport disruption and amyloid-ß generation in Alzheimer's disease.

Substantial evidence implicates fast axonal transport (FAT) defects in neurodegeneration. In Alzheimer's disease (AD), it is controversial whether transport defects cause or arise from amyloid-ß (Aß)-induced toxicity. Using a novel, unbiased genetic screen, Morihara et al. identified kinesin light chain-1 splice variant E (KLC1vE) as a modifier of Aß accumulation. Here, we propose three mechanisms to explain this causal role. First, KLC1vE reduces APP transport, leading to Aß accumulation. Second, reduced transport of APP by KLC1vE triggers an ER stress response that activates the amyloidogenic pathway. Third, KLC1vE impairs transport of other KLC1 cargos that regulate amyloidogenesis, promoting Aß retention within the secretory pathway. Collectively, KLC1vE perpetuates a vicious cycle of Aß generation, kinase dysregulation, and global FAT impairment that inevitably leads to cellular toxicity. These concepts implicate alternative splicing of KLC1 in AD and suggest that the reciprocal influence of transport mechanisms on disease states contributes to neurodegeneration.

Transcriptome analysis of distinct mouse strains reveals kinesin light chain-1 splicing as an amyloid-ß accumulation modifier.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-ß (Aß). The genes that govern this process, however, have remained elusive. To this end, we combined distinct mouse strains with transcriptomics to directly identify disease-relevant genes. We show that AD model mice (APP-Tg) with DBA/2 genetic backgrounds have significantly lower levels of Aß accumulation compared with SJL and C57BL/6 mice. We then applied brain transcriptomics to reveal the genes in DBA/2 that suppress Aß accumulation. To avoid detecting secondarily affected genes by Aß, we used non-Tg mice in the absence of Aß pathology and selected candidate genes differently expressed in DBA/2 mice. Additional transcriptome analysis of APP-Tg mice with mixed genetic backgrounds revealed kinesin light chain-1 (Klc1) as an Aß modifier, indicating a role for intracellular trafficking in Aß accumulation. Aß levels correlated with the expression levels of Klc1 splice variant E and the genotype of Klc1 in these APP-Tg mice. In humans, the expression levels of KLC1 variant E in brain and lymphocyte were significantly higher in AD patients compared with unaffected individuals. Finally, functional analysis using neuroblastoma cells showed that overexpression or knockdown of KLC1 variant E increases or decreases the production of Aß, respectively. The identification of KLC1 variant E suggests that the dysfunction of intracellular trafficking is a causative factor of Aß pathology. This unique combination of distinct mouse strains and model mice with transcriptomics is expected to be useful for the study of genetic mechanisms of other complex diseases.

SLP76 and SLP65: complex regulation of signalling in lymphocytes and beyond.

SLP76 and SLP65 are adaptor proteins that lack intrinsic enzymatic activity but contain multiple protein-binding domains. These proteins are essential for signalling downstream of integrins and receptors that contain immunoreceptor tyrosine-based activation motifs. The absence of these adaptor proteins profoundly affects various lineages in the haematopoietic compartment and severely compromises vascular development, highlighting their importance as regulators of signalling cascades. In this Review, we discuss the role of SLP76 and SLP65 in several signalling pathways in haematopoietic cells, with an emphasis on recent studies that provide insight into their mechanisms of action.

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.

Activity-dependent secretion of progranulin from synapses.

The secreted growth factor progranulin (PGRN) has been shown to be important for regulating neuronal survival and outgrowth, as well as synapse formation and function. Mutations in the PGRN gene that result in PGRN haploinsufficiency have been identified as a major cause of frontotemporal dementia (FTD). Here we demonstrate that PGRN is colocalized with dense-core vesicle markers and is co-transported with brain-derived neurotrophic factor (BDNF) within axons and dendrites of cultured hippocampal neurons in both anterograde and retrograde directions. We also show that PGRN is secreted in an activity-dependent manner from synaptic and extrasynaptic sites, and that the temporal profiles of secretion are distinct in axons and dendrites. Neuronal activity is also shown to increase the recruitment of PGRN to synapses and to enhance the density of PGRN clusters along axons. Finally, treatment of neurons with recombinant PGRN is shown to increase synapse density, while decreasing the size of the presynaptic compartment and specifically the number of synaptic vesicles per synapse. Together, this indicates that activity-dependent secretion of PGRN can regulate synapse number and structure.

Comparison of gnotobiotic communities reveals milk-adapted metabolic functions and unexpected amino acid metabolism by the pre-weaning microbiome.

The intestinal microbiome during infancy and childhood has distinct metabolic functions and microbial composition compared to adults. We recently published a gnotobiotic mouse model of the pre-weaning microbiome (PedsCom), which retains a pre-weaning configuration during the transition from a milk-based diet to solid foods, leads to a stunted immune system, and increases susceptibility to enteric infection. Here, we compared the phylogenetic and metabolic relationships of the PedsCom consortium to two adult-derived gnotobiotic communities, Altered Schaedler Flora and Oligo-Mouse Microbiota 12 (Oligo-MM12). We find that PedsCom contains several unique functions relative to these adult-derived mouse consortia, including differences in carbohydrate and lipid metabolism genes. Notably, amino acid degradation metabolic modules are more prevalent among PedsCom isolates, which is in line with the ready availability of these nutrients in milk. Indeed, metabolomic analysis revealed significantly lower levels of total free amino acids and lower levels of specific amino acids abundant in milk (e.g. glutamine and glutamic acid) in the intestinal contents of adult PedsCom colonized mice compared to Oligo-MM12 controls. Metabolomic analysis of pre-weaning intestinal contents also showed lower levels of amino acids that are replete in milk compared to germ-free controls. Thus, enhanced amino acid metabolism is a prominent feature of the pre-weaning microbiome that may facilitate design of early-life microbiome interventions.

Maternal and neonatal IgG against Klebsiella pneumoniae are associated with broad protection from neonatal sepsis: a case-control study of hospitalized neonates in Botswana.

Sepsis is the leading postnatal cause of neonatal mortality worldwide. Globally Klebsiella pneumoniae is the leading cause of sepsis in hospitalized neonates. This study reports development and evaluation of ELISA for anti-Klebsiella IgG using dried blood spot samples and evaluates the association of anti-Klebsiella IgG (anti-Kleb IgG) antibodies in maternal and neonatal samples and the risk of neonatal sepsis. Neonates and their mothers were enrolled at 0-96 hours of life in the neonatal unit of a tertiary referral hospital in Gaborone, Botswana and followed until death or discharge to assess for episodes of blood culture-confirmed neonatal sepsis. Neonates with sepsis had significantly lower levels of Kleb-IgG compared to neonates who did not develop sepsis (Mann-Whitney U, p=0.012). Similarly, samples from mothers of neonates who developed sepsis tended to have less Kleb-IgG compared to mothers of controls (p=0.06). The inverse correlation between Kleb-IgG levels and all-cause bacteremia suggests that maternal Kleb-IgG is broadly protective through cross-reactivity with common bacterial epitopes. These data support the continued use of immunoglobulin assays using DBS samples to explore the role of passive immunity on neonatal sepsis risk and reaffirm the critical need for research supporting the development of maternal vaccines for neonatal sepsis.

Eastern equine encephalitis in children, Massachusetts and New Hampshire,USA, 1970-2010.

We describe the clinical, laboratory, and radiographic characteristics of 15 cases of eastern equine encephalitis in children during 1970-2010. The most common clinical and laboratory features were fever, headache, seizures, peripheral leukocytosis, and cerebrospinal fluid neutrophilic pleocytosis. Radiographic lesions were found in the basal ganglia, thalami, and cerebral cortex. Clinical outcomes included severe neurologic deficits in 5 (33%) patients, death of 4 (27%), full recovery of 4 (27%), and mild neurologic deficits in 2 (13%). We identify an association between a short prodrome and an increased risk for death or for severe disease.

Cooperation of adapter molecules in proximal signaling cascades during allergic inflammation.

Activation of mast cells through their high-affinity immunoglobulin E receptor (FcepsilonRI) plays an important role in allergic disorders. Other mast cell-activating stimuli, such as Toll-like receptor (TLR) ligands, synergize with FcepsilonRI to enhance allergic inflammation. Thus, there is much interest in understanding how signaling occurs downstream of these receptors. One key event for FcepsilonRI-mediated mast cell activation is the inducible formation of multimolecular proximal signaling complexes. These complexes are nucleated by adapter proteins, scaffolds that localize various signaling molecules through their multiple molecule-binding domains. Here we review recent findings in proximal signaling cascades with an emphasis on how adapter molecules cooperate with each other to generate an optimal signal in mast cells, and we discuss how signals crosstalk between FcepsilonRI and TLRs in enhancing mast cell activation. Deciphering the molecular mechanisms leading to mast cell activation will hopefully bring new ideas for the development of novel therapeutics to control allergic diseases.

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.

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.

Impaired degranulation but enhanced cytokine production after Fc epsilonRI stimulation of diacylglycerol kinase zeta-deficient mast cells.

Calcium and diacylglycerol are critical second messengers that together effect mast cell degranulation after allergen cross-linking of immunoglobulin (Ig)E-bound FcepsilonRI. Diacylglycerol kinase (DGK)zeta is a negative regulator of diacylglycerol-dependent signaling that acts by converting diacylglycerol to phosphatidic acid. We reported previously that DGKzeta-/- mice have enhanced in vivo T cell function. Here, we demonstrate that these mice have diminished in vivo mast cell function, as revealed by impaired local anaphylactic responses. Concordantly, DGKzeta-/- bone marrow-derived mast cells (BMMCs) demonstrate impaired degranulation after Fc epsilonRI cross-linking, associated with diminished phospholipase Cgamma activity, calcium flux, and protein kinase C-betaII membrane recruitment. In contrast, Ras-Erk signals and interleukin-6 production are enhanced, both during IgE sensitization and after antigen cross-linking of Fc epsilonRI. Our data demonstrate dissociation between cytokine production and degranulation in mast cells and reveal the importance of DGK activity during IgE sensitization for proper attenuation of Fc epsilonRI signals.

Providing support through life's final chapter for those who made it home.

Military personnel are exposed to unique environmental hazards and psychological stressors during their service to our nation. As a result, military service personnel are at high risk not only for physical injury but for psychological trauma as well that may result in post-traumatic stress disorder, depression, substance abuse, and homelessness. These medical and psychosocial issues may hasten the development of life-limiting illnesses and may complicate the delivery of end-of-life care. Community-based hospice agencies often lack the resources and expertise to address the special needs of veterans. This article highlights the efforts of the Department of Veterans Affairs to provide comprehensive and co-ordinated end-of-life support for "those who served."

Amyloid-beta peptide oligomers disrupt axonal transport through an NMDA receptor-dependent mechanism that is mediated by glycogen synthase kinase 3beta in primary cultured hippocampal neurons.

Disruption of axonal transport is a hallmark of several neurodegenerative diseases, including Alzheimer's disease (AD). Even though defective transport is considered an early pathologic event, the mechanisms by which neurodegenerative insults impact transport are poorly understood. We show that soluble oligomers of the amyloid-beta peptide (AbetaOs), increasingly recognized as the proximal neurotoxins in AD pathology, induce disruption of organelle transport in primary hippocampal neurons in culture. Live imaging of fluorescent protein-tagged organelles revealed a marked decrease in axonal trafficking of dense-core vesicles and mitochondria in the presence of 0.5 microm AbetaOs. NMDA receptor (NMDAR) antagonists, including d-AP5, MK-801, and memantine, prevented the disruption of trafficking, thereby identifying signals for AbetaO action at the cell membrane. Significantly, both pharmacological inhibition of glycogen synthase kinase-3beta (GSK-3beta) and transfection of neurons with a kinase-dead form of GSK-3beta prevented the transport defect. Finally, we demonstrate by biochemical and immunocytochemical means that AbetaOs do not affect microtubule stability, indicating that disruption of transport involves a more subtle mechanism than microtubule destabilization, likely the dysregulation of intracellular signaling cascades. Results demonstrate that AbetaOs negatively impact axonal transport by a mechanism that is initiated by NMDARs and mediated by GSK-3beta and establish a new connection between toxic Abeta oligomers and AD pathology.