Differential responsivity of neurons in perirhinal cortex, lateral entorhinal cortex, and dentate gyrus during time-bridging learning.

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.

Identification of single nucleotide polymorphisms in hematopoietic cell transplant patients affecting early recognition of, and response to, endotoxin.

Hematopoietic cell transplant (HCT) is a life-saving therapy for many malignant and non-malignant bone marrow diseases. Associated morbidities are often due to transplant-related toxicities and infections, exacerbated by regimen-induced immune suppression and systemic incursion of bacterial products. Patients undergoing myeloablative conditioning for HCT become endotoxemic and display blood/plasma changes consistent with lipopolysaccharide (LPS)-induced systemic innate immune activation. Herein, we addressed whether patients scheduled for HCT display differences in recognition/response to LPS ex vivo traceable to specific single nucleotide polymorphisms (SNPs). Two SNPs of LPS binding protein (LBP) were associated with changes in plasma LBP levels, with one LBP SNP also associating with differences in efficiency of extraction and transfer of endotoxin to myeloid differentiation factor-2 (MD-2), a step needed for activation of TLR4. None of the examined SNPs of CD14, bactericidal/permeability-increasing protein (BPI), TLR4 or MD-2 were associated with corresponding protein plasma levels or endotoxin delivery to MD-2, but CD14 and BPI SNPs significantly associated with differences in LPS-induced TNF-α release ex vivo and infection frequency, respectively. These findings suggest that specific LBP, CD14 and BPI SNPs might be contributory assessments in studies where clinical outcome may be affected by host response to endotoxin and bacterial infection.

Perirhinal and postrhinal, but not lateral entorhinal, cortices are essential for acquisition of trace eyeblink conditioning.

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.

Imidazoquinoline Toll-like receptor 8 agonists activate human newborn monocytes and dendritic cells through adenosine-refractory and caspase-1-dependent pathways.

BACKGROUND: Newborns have frequent infections and manifest impaired vaccine responses, motivating a search for neonatal vaccine adjuvants. Alum is a neonatal adjuvant but might confer a T(H)2 bias. Toll-like receptor (TLR) agonists are candidate adjuvants, but human neonatal cord blood monocytes demonstrate impaired T(H)1-polarizing responses to many TLR agonists caused by plasma adenosine acting through cyclic AMP. TLR8 agonists, including imidazoquinolines (IMQs), such as the small synthetic 3M-002, induce adult-level TNF from neonatal monocytes, but the scope and mechanisms of IMQ-induced activation of neonatal monocytes and monocyte-derived dendritic cells (MoDCs) have not been reported. OBJECTIVE: We sought to characterize IMQ-induced activation of neonatal monocytes and MoDCs. METHODS: Neonatal cord and adult peripheral blood monocytes and MoDCs were cultured in autologous plasma; levels of alum- and TLR agonist-induced cytokines and costimulatory molecules were measured. TLR8 and inflammasome function were assayed by using small interfering RNA 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-1ß from monocytes. 3M-002 induced robust TLR pathway transcriptome activation and T(H)1-polarizing cytokine production in neonatal and adult monocytes and MoDCs, signaling through TLR8 in an adenosine/cyclic AMP-refractory manner. Newborn MoDCs displayed impaired LPS/ATP-induced caspase-1-mediated IL-1ß production but robust 3M-002-induced caspase-1-mediated inflammasome activation independent of exogenous ATP. TLR8 IMQs induced robust TNF and IL-1ß in whole blood of rhesus macaques at birth and infancy. CONCLUSIONS: IMQ TLR8 agonists engage adenosine-refractory TLR8 and inflammasome pathways to induce robust monocyte and MoDC activation and represent promising neonatal adjuvants.

Bactericidal/permeability-increasing protein (rBPI21) and fluoroquinolone mitigate radiation-induced bone marrow aplasia and death.

Identification of safe, effective treatments to mitigate toxicity after extensive radiation exposure has proven challenging. Only a limited number of candidate approaches have emerged, and the U.S. Food and Drug Administration has yet to approve any agent for a mass-casualty radiation disaster. Because patients undergoing hematopoietic stem cell transplantation undergo radiation treatment that produces toxicities similar to radiation-disaster exposure, we studied patients early after such treatment 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 and tested this idea in mice. A single 7-Gy radiation dose, which killed 95% of the mice 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 antibacterial activity, including that against endotoxin-bearing Gram-negative bacteria. Compared to either fluoroquinolone alone or vehicle plus fluoroquinolone, the combined rBPI21 plus fluoroquinolone treatment improved survival, accelerated hematopoietic recovery, and promoted expansion of stem and progenitor cells. The observed efficacy of rBPI21 plus fluoroquinolone initiated 24 hours after lethal irradiation, combined with their established favorable bioactivity and safety profiles in critically ill humans, suggests the potential clinical use of this radiation mitigation strategy and supports its further evaluation.

Endotoxin-directed innate immunity in tracheal aspirates of mechanically ventilated human neonates.

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 toward 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. TA supernatants (n = 42; GA = 23-40 wk, postnatal age = 1-71 d) were assayed for endotoxin (Limulus amoebocyte lysate assay) and endotoxin-modulating proteins: bactericidal/ permeability-increasing protein (BPI), LPS-binding protein (LBP), and soluble cell differentiation antigen 14 (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 real-time PCR (qRT-PCR). 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 (TLR) 4, CD14, and myeloid differentiation protein 2 (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. TAs from intubated neonates demonstrate endotoxin accumulation and mobilization of endotoxin-directed innate immunity, potentially contributing to pulmonary inflammation.

Innate immunity of the human newborn is polarized toward a high ratio of IL-6/TNF-alpha production in vitro and in vivo.

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 pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha 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-alpha 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-alpha 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-alpha production is likely to profoundly modulate both innate and adaptive immune responses in the human newborn.

Unique efficacy of Toll-like receptor 8 agonists in activating human neonatal antigen-presenting cells.

Newborns are prone to microbial infection and have poor memory responses to multiple antigens. We have previously shown that human neonatal blood monocytes exhibit impaired TNF-alpha responses to most known TLR agonists, including the pure TLR7 agonist imiquimod. Surprisingly, however, neonatal TNF-alpha responses to the imiquimod congener R-848 (TLR 7/8) were fully intact. We now show that TLR8 agonists, including R-848 (TLR7/8), the imidazoquinoline congeners 3M-003 (TLR7/8) and 3M-002 (TLR8), as well as single-stranded viral RNAs (TLR8) induced robust production of the Th1-polarizing cytokines TNF-alpha and IL-12 from neonatal antigen-presenting cells (APCs) that substantially exceeds responses induced by TLR-2, -4, or -7 (alone) agonists. TLR8 agonists also effectively induced up-regulation of the costimulatory molecule CD40 on neonatal and adult myeloid dendritic cells (DCs). The strong activity of TLR8 agonists correlates with their induction of p38 MAP kinase phosphorylation and with degradation of IkappaB-alpha in both neonatal and adult monocytes. We conclude that TLR8 agonists are uniquely efficacious in activating costimulatory responses in neonatal APCs and suggest that these agents are promising candidate adjuvants for enhancing immune responses in human newborns.