No abstract

Human newborns are susceptible to microbial infection related to incompletely defined aspects of the neonatal immune system. To characterize neonatal innate immunity, we studied production of two early response cytokines in response to Toll-like receptor (TLR)-activating microbial stimuli in vitro: the proinflammatory cytokine tumor necrosis factor (TNF)-␣ and IL-6, a multifunctional cytokine with antiinflammatory and Th2-polarizing properties. Neonatal cord blood responses to multiple TLR agonists, including poly dI:dC (TLR3), lipopolysaccharide (LPS) (TLR4), flagellin (TLR5), and CpG DNA (TLR9), are characterized by a higher IL-6/TNF-␣ ratio than in adult peripheral blood. Robust LPS-induced IL-6 production is due to both neonatal cellular (monocyte-) and humoral (serum-) factors. Remarkably, serum collected from newborns during the first week of life demonstrates higher IL-6/TNF-␣ ratios than does cord blood, associated with elevations of the IL-6-inducible acute phase reactants CRP and LPS-binding protein in the first days of life. A high ratio of stimulus-induced IL-6/TNF-␣ production is likely to profoundly modulate both innate and adaptive immune responses in the human newborn.

Background Newborns suffer frequent infection and manifest impaired vaccine responses, motivating a search for neonatal vaccine adjuvants. Alum is a neonatal adjuvant, but may confer a Th2 bias. Toll-like receptor (TLR) agonists are candidate adjuvants, but human neonatal cord blood monocytes (Mos) demonstrate impaired Th1-polarizing responses to many TLR agonists due to plasma adenosine acting via cAMP. TLR8 agonists, including imidazoquinolines (IMQs) such as the small synthetic 3M-002, induce adult-level TNF from neonatal Mos, but the scope and mechanisms of IMQ-induced activation of neonatal Mos and Mo-derived dendritic cells (MoDCs) have not been reported. Objectives To characterize IMQ-induced activation of neonatal Mos and MoDCs. Methods Neonatal cord and adult peripheral blood Mos and MoDCs were cultured in autologous plasma; Alum- and TLR agonist-induced cytokines and co-stimulatory molecules were measured. TLR8 and inflammasome function were assayed using siRNA and western blotting/caspase-1 inhibitory peptide, respectively. The ontogeny of TLR8 agonist–induced cytokine responses was defined in Rhesus macaque whole blood ex vivo. Results IMQs were more potent and effective than Alum at inducing TNF and IL-1β from Mos. 3M-002 induced robust TLR pathway transcriptome activation and Th1-polarizing cytokine production in neonatal and adult Mos and MoDCs, signaling via TLR8 in an adenosine/cAMP- refractory manner. Newborn MoDCs displayed impaired LPS/ATP-induced caspase-1-mediated IL-1β production, but robust 3M-002-induced caspase-1-mediated inflammasome activation independent of exogenous ATP. TLR8-IMQs induced robust TNF and IL-1β in whole blood of Rhesus macaques at birth and infancy. Conclusions IMQ TLR8 agonists engage adenosine-refractory TLR8 and inflammasome pathways to induce robust Mo and MoDC activation and represent promising neonatal adjuvants.

Identification of safe, effective treatment strategies to mitigate toxicity after extensive radiation exposure has proven challenging. Only a limited number of candidate approaches have emerged, and the Federal Drug Administration has yet to approve any agent for a mass-casualty radiation disaster indication. As preparative treatments for hematopoietic stem cell transplantation (HSCT) produce toxicities similar to such radiation exposures, we studied patients early after myeloablative HSCT to identify new approaches to this problem. Patients rapidly developed endotoxemia and reduced plasma bactericidal/permeability-increasing protein (BPI), a potent endotoxin-neutralizing protein, in association with neutropenia. We hypothesized that a treatment supplying similar endotoxin-neutralizing activity might replace the BPI deficit and mitigate radiation toxicity. We tested this idea in mice. A single 7 Gy radiation dose, which was 95% lethal by 30 days, was followed 24 hours later by twice daily subcutaneous injections of the recombinant BPI fragment rBPI21 or vehicle alone for 14 or 30 days, with or without an oral fluoroquinolone antibiotic with broad-spectrum anti-bacterial activity including that against endotoxin-bearing Gram-negative bacteria. Compared to either fluoroquinolone alone or vehicle/fluoroquinolone, combined rBPI21/fluoroquinolone treatment improved survival, accelerated hematopoietic recovery and promoted expansion of stem and progenitor cells. The observed efficacy of rBPI21 and fluoroquinolones initiated 24 hours after lethal irradiation, combined with their favorable bioactivity and safety profiles in critically-ill humans, suggest the potential clinical utility of this radiation mitigation strategy and support its further evaluation.

Many studies have focused on the function of hippocampal region CA1 as a critical site for associative memory, but much less is known about changes in the afferents to CA1. Here we report the activity of multiple single neurons from perirhinal and entorhinal cortex and from dentate gyrus during trace eyeblink conditioning as well as consolidated recall, and in pseudo‐conditioned control rabbits. We also report an analysis of theta activity filtered from the local field potential (LFP). Our results show early associative changes in single‐neuron firing rate as well as theta oscillations in lateral entorhinal cortex (EC) and dentate gyrus (DG), and increases in the number of responsive neurons in perirhinal cortex. In both EC and DG, a subset of neurons from conditioned animals exhibited an elevated baseline firing rate and large responses to the conditioned stimulus and trace period. A similar population of cells has been seen in DG and in medial, but not lateral, EC during spatial tasks, suggesting that lateral EC contains cells responsive to a temporal associative task. In contrast to recent studies in our laboratory that found significant CA1 contributions to long‐term memory, the activity profiles of neurons within EC and DG were similar for conditioned and pseudoconditioned rabbits during post‐consolidation sessions. Collectively these results demonstrate that individual subregions of medial temporal lobe differentially support new and remotely acquired memories. Neuron firing profiles were similar on training trials when conditioned responses were and were not exhibited, demonstrating that these temporal lobe regions represent the CS–US association and do not control the behavioral response. The analysis of theta activity revealed that theta power was modulated by the conditioning stimuli in both the conditioned and pseudoconditioned groups and that although both groups exhibited a resetting of phase to the corneal airpuff, only the conditioned group exhibited a resetting of phase to the whisker conditioned stimulus.

The acquisition of temporal associative tasks such as trace eyeblink conditioning is hippocampus-dependent, while consolidated performance is not. The parahippocampal region mediates much of the input and output of the hippocampus, and perirhinal (PER) and entorhinal (EC) cortices support persistent spiking, a possible mediator of temporal bridging between stimuli. Here we show that lesions of the perirhinal or postrhinal cortex severely impair the acquisition of trace eyeblink conditioning, while lateral EC lesions do not. Our findings suggest that direct projections from the PER to the hippocampus are functionally important in trace acquisition, and support a role for PER persistent spiking in time-bridging associations.

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Background Mechanical ventilation of preterm infants is associated with pulmonary inflammation. Intubated infants often develop bacterial tracheal colonization but little is known about endotoxin in tracheal aspirates (TAs) or the mobilization of innate immunity towards endotoxin, a potent stimulus that contributes to inflammatory disease. We characterized mobilization of endotoxin-directed innate immunity in TAs from an observational cohort of mechanically ventilated neonates. Methods TA supernatants (n=42; GA=23-40 weeks, postnatal age=1-71 days) were assayed for endotoxin (Limulus amoebocyte lysate assay) and endotoxin-modulating proteins: bactericidal/permeability-increasing protein (BPI), LPS-binding protein (LBP) and soluble CD14 (sCD14). TA cellular BPI was measured by ELISA, western blot, flow cytometry, and bactericidal assay. TA mRNAs encoding endotoxin-modulating proteins were measured by quantitative RT-PCR. Results Endotoxin in TA supernatants was proportional to both postnatal age and polymorphonuclear leukocytes (PMN). Neonatal TAs were rich in PMN containing BPI and expressed mRNAs encoding Toll-like receptor 4 (TLR4), CD14, and MD-2. Extracellular BPI was consistently detectable and correlated with TA PMN and GA. Both extracellular- and cellular-BPI increased during the first postnatal week. TA extracellular BPI, LBP, and sCD14 were positively correlated. Conclusions TAs from intubated neonates demonstrate endotoxin accumulation and mobilization of endotoxin-directed innate immunity, potentially contributing to pulmonary inflammation.