CD4+ T cell expression of the IL-10 receptor is necessary for facial motoneuron survival after axotomy.

BACKGROUND: After peripheral nerve transection, facial motoneuron (FMN) survival depends on an intact CD4+ T cell population and a central source of interleukin-10 (IL-10). However, it has not been determined previously whether CD4+ T cells participate in the central neuroprotective IL-10 cascade after facial nerve axotomy (FNA). METHODS: Immunohistochemical labeling of CD4+ T cells, pontine vasculature, and central microglia was used to determine whether CD4+ T cells cross the blood-brain barrier and enter the facial motor nucleus (FMNuc) after FNA. The importance of IL-10 signaling in CD4+ T cells was assessed by performing adoptive transfer of IL-10 receptor beta (IL-10RB)-deficient CD4+ T cells into immunodeficient mice prior to injury. Histology and qPCR were utilized to determine the impact of IL-10RB-deficient T cells on FMN survival and central gene expression after FNA. Flow cytometry was used to determine whether IL-10 signaling in T cells was necessary for their differentiation into neuroprotective subsets. RESULTS: CD4+ T cells were capable of crossing the blood-brain barrier and associating with reactive microglial nodules in the axotomized FMNuc. Full induction of central IL-10R gene expression after FNA was dependent on CD4+ T cells, regardless of their own IL-10R signaling capability. Surprisingly, CD4+ T cells lacking IL-10RB were incapable of mediating neuroprotection after axotomy and promoted increased central expression of genes associated with microglial activation, antigen presentation, T cell co-stimulation, and complement deposition. There was reduced differentiation of IL-10RB-deficient CD4+ T cells into regulatory CD4+ T cells in vitro. CONCLUSIONS: These findings support the interdependence of IL-10- and CD4+ T cell-mediated mechanisms of neuroprotection after axotomy. CD4+ T cells may potentiate central responsiveness to IL-10, while IL-10 signaling within CD4+ T cells is necessary for their ability to rescue axotomized motoneuron survival. We propose that loss of IL-10 signaling in CD4+ T cells promotes non-neuroprotective autoimmunity after FNA.

Neuroendocrine regulation of inflammation.

The interaction between the sympathetic nervous system and the immune system has been documented over the last several decades. In this review, the neuroanatomical, cellular, and molecular evidence for neuroimmune regulation in the maintenance of immune homeostasis will be discussed, as well as the potential impact of neuroimmune dysregulation in health and disease.

Temporospatial Analysis and New Players in the Immunology of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive loss of lower and upper motor neurons (MN) leading to muscle weakness, paralysis and eventually death. Although a highly varied etiology results in ALS, it broadly manifests itself as sporadic and familial forms that have evident similarities in clinical symptoms and disease progression. There is a tremendous amount of knowledge on molecular mechanisms leading to loss of MNs and neuromuscular junctions (NMJ) as major determinants of disease onset, severity and progression in ALS. Specifically, two main opposing hypotheses, the dying forward and dying back phenomena, exist to account for NMJ denervation. The former hypothesis proposes that the earliest degeneration occurs at the central MNs and proceeds to the NMJ, whereas in the latter, the peripheral NMJ is the site of precipitating degeneration progressing backwards to the MN cell body. A large body of literature strongly indicates a role for the immune system in disease onset and progression via regulatory involvement at the level of both the central and peripheral nervous systems (CNS and PNS). In this review, we discuss the earliest reported immune responses with an emphasis on newly identified immune players in mutant superoxide dismutase 1 (mSOD1) transgenic mice, the gold standard mouse model for ALS.

Th17 Cell Response in SOD1G93A Mice following Motor Nerve Injury.

An increased risk of ALS has been reported for veterans, varsity athletes, and professional football players. The mechanism underlying the increased risk in these populations has not been identified; however, it has been proposed that motor nerve injury may trigger immune responses which, in turn, can accelerate the progression of ALS. Accumulating evidence indicates that abnormal immune reactions and inflammation are involved in the pathogenesis of ALS, but the specific immune cells involved have not been clearly defined. To understand how nerve injury and immune responses may contribute to ALS development, we investigated responses of CD4(+) T cell after facial motor nerve axotomy (FNA) at a presymptomatic stage in a transgenic mouse model of ALS (B6SJL SOD1(G93A)). SOD1(G93A) mice, compared with WT mice, displayed an increase in the basal activation state of CD4(+) T cells and higher frequency of Th17 cells, which were further enhanced by FNA. In conclusion, SOD1(G93A) mice exhibit abnormal CD4(+) T cell activation with increased levels of Th17 cells prior to the onset of neurological symptoms. Motor nerve injury exacerbates Th17 cell responses and may contribute to the development of ALS, especially in those who carry genetic susceptibility to this disease.

A novel role of microglial NADPH oxidase in mediating extra-synaptic function of norepinephrine in regulating brain immune homeostasis.

Although the peripheral anti-inflammatory effect of norepinephrine (NE) is well documented, the mechanism by which this neurotransmitter functions as an anti-inflammatory/neuroprotective agent in the central nervous system (CNS) is unclear. This article aimed to determine the anti-inflammatory/neuroprotective effects and underlying mechanisms of NE in inflammation-based dopaminergic neurotoxicity models. In mice, NE-depleting toxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) was injected at 6 months of lipopolysaccharide (LPS)-induced neuroinflammation. It was found that NE depletion enhanced LPS-induced dopaminergic neuron loss in the substantia nigra. This piece of in vivo data prompted us to conduct a series of studies in an effort to elucidate the mechanism as to how NE affects dopamine neuron survival by using primary midbrain neuron/glia cultures. Results showed that submicromolar concentrations of NE dose-dependently protected dopaminergic neurons from LPS-induced neurotoxicity by inhibiting microglia activation and subsequent release of pro-inflammatory factors. However, NE-elicited neuroprotection was not totally abolished in cultures from ß2-adrenergic receptor (ß2-AR)-deficient mice, suggesting that novel pathways other than ß2-AR are involved. To this end, It was found that submicromolar NE dose-dependently inhibited NADPH oxidase (NOX2)-generated superoxide, which contributes to the anti-inflammatory and neuroprotective effects of NE. This novel mechanism was indeed adrenergic receptors independent since both (+) and (-) optic isomers of NE displayed the same potency. We further demonstrated that NE inhibited LPS-induced NOX2 activation by blocking the translocation of its cytosolic subunit to plasma membranes. In summary, we revealed a potential physiological role of NE in maintaining brain immune homeostasis and protecting neurons via a novel mechanism.

Adrenergic regulation of IgE involves modulation of CD23 and ADAM10 expression on exosomes.

Soluble CD23 plays a role in the positive regulation of an IgE response. Engagement of the ß2 adrenergic receptor (ß2AR) on a B cell is known to enhance the level of both soluble CD23 and IgE, although the mechanism by which this occurs is not completely understood. In this study, we report that, in comparison with a CD40 ligand/IL-4-primed murine B cell alone, ß2AR engagement on a primed B cell increased gene expression of a disintegrin and metalloproteinase (ADAM)10, which is the primary sheddase of CD23, as well as protein expression of both CD23 and ADAM10, in a protein kinase A- and p38 MAPK-dependent manner, and promoted the localization of these proteins to exosomes as early as 2 d after priming, as determined by both Western blot and flow cytometry and confirmed by electron microscopy. In comparison with isolated exosomes released from primed B cells alone, the transfer of exosomes released from ß2AR agonist-exposed primed B cells to cultures of recipient primed B cells resulted in an increase in the level of IgE produced per cell, without affecting the number of cells producing IgE, as determined by ELISPOT. These effects still occurred when a ß2AR antagonist was added along with the transfer to block residual agonist, and they failed to occur when exosomes were isolated from ß2AR-deficient B cells. These findings suggest that the mechanism responsible for mediating the ß2AR-induced increase in IgE involves a shuttling of the ß2AR-induced increase in CD23 and ADAM10 proteins to exosomes that subsequently mediate an increase in IgE.

Prohibitins and the cytoplasmic domain of CD86 cooperate to mediate CD86 signaling in B lymphocytes.

CD86 engagement on a CD40L/IL-4-primed murine B cell activates signaling intermediates that promote NF-κB activation to increase Oct-2 and mature IgG1 mRNA and protein expression, as well as the rate of IgG1 transcription, without affecting class switch recombination. One of the most proximal signaling intermediates identified is phospholipase Cγ2, a protein reported to bind tyrosine residues, which are absent in the cytoplasmic domain of CD86. Using a proteomics-based identification approach, we show that the tyrosine-containing transmembrane adaptor proteins prohibitin (Phb)1 and Phb2 bind to CD86. The basal expression of Phb1/2 and association with CD86 was low in resting B cells, whereas the level of expression and association increased primarily after priming with CD40. The CD86-induced increase in Oct-2 and IgG1 was less when either Phb1/2 expression was reduced by short hairpin RNA or the cytoplasmic domain of CD86 was truncated or mutated at serine/threonine protein kinase C phosphorylation sites, which did not affect Phb1/2 binding to CD86. Using this approach, we also show that Phb1/2 and the CD86 cytoplasmic domain are required for the CD86-induced phosphorylation of IκBα, which we previously reported leads to NF-κB p50/p65 activation, whereas only Phb1/2 was required for the CD86-induced phosphorylation of phospholipase Cγ2 and protein kinase Cα/ß(II), which we have previously reported leads to NF-κB (p65) phosphorylation and subsequent nuclear translocation. Taken together, these findings suggest that Phb1/2 and the CD86 cytoplasmic domain cooperate to mediate CD86 signaling in a B cell through differential phosphorylation of distal signaling intermediates required to increase IgG1.

SOD1(G93A) transgenic mouse CD4(+) T cells mediate neuroprotection after facial nerve axotomy when removed from a suppressive peripheral microenvironment.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving motoneuron (MN) axonal withdrawal and cell death. Previously, we established that facial MN (FMN) survival levels in the SOD1(G93A) transgenic mouse model of ALS are reduced and nerve regeneration is delayed, similar to immunodeficient RAG2(-/-) mice, after facial nerve axotomy. The objective of this study was to examine the functionality of SOD1(G93A) splenic microenvironment, focusing on CD4(+) T cells, with regard to defects in immune-mediated neuroprotection of injured MN. We utilized the RAG2(-/-) and SOD1(G93A) mouse models, along with the facial nerve axotomy paradigm and a variety of cellular adoptive transfers, to assess immune-mediated neuroprotection of FMN survival levels. We determined that adoptively transferred SOD1(G93A) unfractionated splenocytes into RAG2(-/-) mice were unable to support FMN survival after axotomy, but that adoptive transfer of isolated SOD1(G93A) CD4(+) T cells could. Although WT unfractionated splenocytes adoptively transferred into SOD1(G93A) mice were able to maintain FMN survival levels, WT CD4(+) T cells alone could not. Importantly, these results suggest that SOD1(G93A) CD4(+) T cells retain neuroprotective functionality when removed from a dysfunctional SOD1(G93A) peripheral splenic microenvironment. These results also indicate that the SOD1(G93A) central nervous system microenvironment is able to re-activate CD4(+) T cells for immune-mediated neuroprotection when a permissive peripheral microenvironment exists. We hypothesize that a suppressive SOD1(G93A) peripheral splenic microenvironment may compromise neuroprotective CD4(+) T cell activation and/or differentiation, which, in turn, results in impaired immune-mediated neuroprotection for MN survival after peripheral axotomy in SOD1(G93A) mice.

The beta2-adrenergic receptor on T and B lymphocytes: do we understand it yet?

The role played by the beta2-adrenergic receptor (ß(2)AR) in regulating the level of T and B lymphocyte function has been studied for over half a century. During this time, we have learned that T and B lymphocytes express almost exclusively the ß(2)AR, and that the level of expression on a specific lymphocyte subset differs due to epigenetic regulation by histone and DNA methylation. We have also learned that engagement of the ß(2)AR on lymphocytes, by either norepinephrine or a selective pharmacologic ligand, regulates the level of lymphocyte activity differentially, depending on the time of receptor engagement in relation to the activation and differentiation state of the cell, the molecular signaling pathway activated, and the cytokine microenvironment. The challenge now is to determine if we understand enough about how this receptor functions on lymphocytes to predict the relevance of such regulation to overall immune homeostasis and the development/progression of human disease.

CD4+ T cell-mediated neuroprotection is independent of T cell-derived BDNF in a mouse facial nerve axotomy model.

BACKGROUND: The production of neurotrophic factors, such as BDNF, has generally been considered an important mechanism of immune-mediated neuroprotection. However, the ability of T cells to produce BDNF remains controversial. METHODS: In the present study, we examined mRNA and protein of BDNF using RT-PCR and western blot, respectively, in purified and reactivated CD4(+) T cells. In addition, to determine the role of BDNF derived from CD4(+) T cells, the BDNF gene was specifically deleted in T cells using the Cre-lox mouse model system. RESULTS: Our results indicate that while both mRNA expression and protein secretion of BDNF in reactivated T cells were detected at 24 h, only protein could be detected at 72 h after reactivation. The results suggest a transient up-regulation of BDNF mRNA in reactivated T cells. Furthermore, in contrast to our hypothesis that the BDNF expression is necessary for CD4(+) T cells to mediate neuroprotection, mice with CD4(+) T cells lacking BDNF expression demonstrated a similar level of facial motoneuron survival compared to their littermates that expressed BDNF, and both levels were comparable to wild-type. The results suggest that the deletion of BDNF did not impair CD4(+) T cell-mediated neuroprotection. CONCLUSION: Collectively, while CD4(+) T cells are a potential source of BDNF after nerve injury, production of BDNF is not necessary for CD4(+) T cells to mediate their neuroprotective effects.

CD8+ T cells negatively regulate IL-4-dependent, IgG1-dominant posttransplant alloantibody production.

We have previously reported that CD8(+) T cells significantly influence Ab production based on the observation that posttransplant alloantibody levels in CD8-deficient murine hepatocyte transplant recipients are markedly enhanced. However, the precise mechanisms contributing to enhanced alloantibody production in the absence of CD8(+) T cells is not understood. We hypothesized that alloactivated CD8(+) T cells inhibit Ab production by skewing toward a proinflammatory cytokine profile, whereas when these cells are absent, an anti-inflammatory cytokine profile shifts the alloimmune response toward alloantibody production. To investigate this possibility, alloantibody isotype profiles were examined in CD8-deficient and wild-type hepatocyte recipients. We found that IgG1 (IL-4-dependent isotype) was the dominant alloantibody isotype in wild-type recipients as well as in CD8-deficient recipients, although the amount of alloantibody in the latter group was substantially higher. Utilizing real-time PCR we found that CD4(+) T cells from wild-type recipients significantly upregulated IFN-γ but not IL-4 mRNA. In contrast, in the absence of CD8(+) T cells, CD4(+) T cells switched to significantly upregulate IL-4 mRNA, while IFN-γ was downregulated. IL-4 knockout mice do not produce any posttransplant alloantibody. However, adoptive transfer of wild-type CD4(+) T cells into CD8-depleted IL-4 knockout mice restores high alloantibody levels observed in CD8-depleted wild-type recipients. This suggests that IL-4-producing CD4(+) T cells are critical for posttransplant alloantibody production. Additionally, this CD8-mediated regulation of posttransplant alloantibody production is IFN-γ-dependent. Further elucidation of the mechanisms by which CD8(+) T cells influence Ab production will significantly contribute to development of therapies to manipulate humoral responses to Ag.

Immune cell-mediated neuroprotection is independent of estrogen action through estrogen receptor-alpha.

It has been well documented that both estrogen and immune cells (CD4+ T cells) mediate neuroprotection in the mouse facial nerve axotomy model. Estrogen has been shown to play an important role in regulating the immune response. However, it is unclear whether immune cell-mediated neuroprotection is dependent on estrogen signaling. In this study, using FACS staining, we confirmed that the majority of CD4+ T cells express high levels of estrogen receptor-alpha (ERα), suggesting that CD4+ T cell-mediated neuroprotection may be modulated by estrogen signaling. We previously found that immunodeficient Rag-2KO mice showed a significant increase in axotomy-induced facial motoneuron death compared to immunocompetent wild-type mice. Therefore, we investigated axotomy-induced facial motoneuron loss in immunodeficient Rag-2KO mice that received 17ß-estradiol treatment or adoptive transfer of immune cells from mice lacking functional ERα. Our results indicate that while estradiol treatment failed to rescue facial motoneurons from axotomy-induced cell death in Rag-2KO mice, immune cells lacking ERα successfully restored facial motoneuron survival in Rag-2 KO mice to a wild-type level. Collectively, we concluded that CD4+ T cell-mediated neuroprotection is independent of estrogen action through ERα.

Cellular Sources and Neuroprotective Roles of Interleukin-10 in the Facial Motor Nucleus after Axotomy.

Facial motoneuron (FMN) survival is mediated by CD4+ T cells in an interleukin-10 (IL-10)-dependent manner after facial nerve axotomy (FNA), but CD4+ T cells themselves are not the source of this neuroprotective IL-10. The aims of this study were to (1) identify the temporal and cell-specific induction of IL-10 expression in the facial motor nucleus and (2) elucidate the neuroprotective capacity of this expression after axotomy. Immunohistochemistry revealed that FMN constitutively produced IL-10, whereas astrocytes were induced to make IL-10 after FNA. Il10 mRNA co-localized with microglia before and after axotomy, but microglial production of IL-10 protein was not detected. To determine whether any single source of IL-10 was critical for FMN survival, Cre/Lox mouse strains were utilized to selectively knock out IL-10 in neurons, astrocytes, and microglia. In agreement with the localization data reflecting concerted IL-10 production by multiple cell types, no single cellular source of IL-10 alone could provide neuroprotection after FNA. These findings suggest that coordinated neuronal and astrocytic IL-10 production is necessary for FMN survival and has roles in neuronal homeostasis, as well as neuroprotective trophism after axotomy.

Epigenetic regulation of beta2-adrenergic receptor expression in T(H)1 and T(H)2 cells.

We showed previously that murine naive CD4(+) T cells and T(H)1 cell clones express the beta2-adrenergic receptor (ß(2)AR), while T(H)2 cell clones do not. We report here that naive CD4(+) T cells that differentiated for 1-5 days under T(H)1 driving conditions increased ß(2)AR gene expression, while cells cultured under T(H)2 driving conditions decrease ß(2)AR gene expression. Chromatin immunoprecipitation revealed that the increase in ß(2)AR gene expression in T(H)1 cells is mediated by an increase in histone 3 (H3) and H4 acetylation, as well as an increase in histone 3 lysine 4 (H3K4) methylation. Conversely, the decrease in ß(2)AR gene expression in T(H)2 cells is mediated by a decrease in H3 and H4 acetylation and a decrease in H3K4 methylation, as well as an increase H3K9 and H3K27 methylation. The histone changes could be detected as early as 3 days of differentiating conditions. Genomic bisulfite sequencing showed that the level of methylated CpG dinucleotides within the promoter of the ß(2)AR gene was increased in T(H)2 cells as compared to naive and T(H)1 cells. Collectively, these results suggest that epigenetic mechanisms mediate maintenance and repression, respectively, of the ß(2)AR gene expression in T(H)1- and T(H)2-driven cells, providing a potential mechanism by which the level of ß(2)AR expression might be modulated pharmacologically within immune cells and other cell types in which the expression profile may change during a disease process.

IL-10 within the CNS is necessary for CD4+ T cells to mediate neuroprotection.

We have previously shown that immunodeficient mice exhibit significant facial motoneuron (FMN) loss compared to wild-type (WT) mice after a facial nerve axotomy. Interleukin-10 (IL-10) is known as a regulatory cytokine that plays an important role in maintaining the anti-inflammatory environment within the central nervous system (CNS). IL-10 is produced by a number of different cells, including Th2 cells, and may exert an anti-apoptotic action on neurons directly. In the present study, the role of IL-10 in mediating neuroprotection following facial nerve axotomy in Rag-2- and IL-10-deficient mice was investigated. Results indicate that IL-10 is neuroprotective, but CD4+ T cells are not the requisite source of IL-10. In addition, using real-time PCR analysis of laser microdissected brainstem sections, results show that IL-10 mRNA is constitutively expressed in the facial nucleus and that a transient, significant reduction of IL-10 mRNA occurs following axotomy under immunodeficient conditions. Dual labeling immunofluorescence data show, unexpectedly, that the IL-10 receptor (IL-10R) is constitutively expressed by facial motoneurons, but is selectively induced in astrocytes within the facial nucleus after axotomy. Thus, a non-CD4+ T cell source of IL-10 is necessary for modulating both glial and neuronal events that mediate neuroprotection of injured motoneurons, but only with the cooperation of CD4+ T cells, providing an avenue of novel investigation into therapeutic approaches to prevent or reverse motoneuron diseases, such as amyotrophic lateral sclerosis (ALS).

Design and synthesis of boron-containing PDE4 inhibitors using soft-drug strategy for potential dermatologic anti-inflammatory application.

PDE4 inhibitors are a validated approach as anti-inflammatory agents but are limited by systemic side effects including emesis. We report a soft-drug strategy incorporating a carboxylic ester group into boron-containing PDE4 inhibitors leading to the discovery of a series of benzoxaborole compounds with good potency (for example IC(50)=47 nM of compound 2) and low emetic activity. These compounds are intended for dermatological use further limiting possible systemic side effects.

Stress hormones collaborate to induce lymphocyte apoptosis after high level spinal cord injury.

Post-traumatic immune suppression renders individuals with spinal cord injury (SCI) susceptible to infection. Normally, proper immune function is regulated by collaboration between the sympathetic nervous system (SNS) and hypothalamic-pituitary-adrenal (HPA) axis and involves the controlled release of glucocorticoids (GCs) and norepinephrine (NE). Recently, we showed that after high thoracic (T3) SCI, aberrant levels of GCs and NE accumulate in the blood and spleen, respectively. These changes are associated with splenic atrophy, splenic leucopenia, increased intrasplenic caspase 3 levels, and suppressed B lymphocyte function. As GCs boost SNS function, in part by increasing the expression and affinity of beta2 adrenergic receptors (beta2ARs) while simultaneously preventing beta2AR down-regulation, we predicted that surges in stress hormones (i.e., GCs and NE) in the blood and spleen of mice with high-level SCI would act concurrently to adversely affect lymphocyte function and survival. Here, we show that post-SCI concentrations of GCs enhance the sensitivity of lymphocytes to beta2AR stimulation causing an increase in intracellular Bcl-2 interacting mediator of cell death (Bim) and subsequent apoptosis. In vivo, the combined antagonism of GC receptors and beta2ARs significantly diminished lymphocyte Bim levels and SCI-induced splenic lymphopenia. Together, these data suggest that pharmacological antagonists of the HPA/SNS axes should be considered as adjunct therapies for ameliorating post-traumatic immune suppression in quadriplegics and high paraplegics.

Discovery and structure-activity study of a novel benzoxaborole anti-inflammatory agent (AN2728) for the potential topical treatment of psoriasis and atopic dermatitis.

A series of phenoxy benzoxaboroles were synthesized and screened for their inhibitory activity against PDE4 and cytokine release. 5-(4-Cyanophenoxy)-2,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2728) showed potent activity both in vitro and in vivo. This compound is now in clinical development for the topical treatment of psoriasis and being pursued for the topical treatment of atopic dermatitis.

Improving Care for Infants With Neonatal Abstinence Syndrome: A Multicenter, Community Hospital-Based Study.

BACKGROUND: For infants with neonatal abstinence syndrome (NAS) in children's hospitals, treatment protocols emphasizing nonpharmacologic care have revealed improved hospital outcomes. We sought to improve NAS care within the community hospital setting through the implementation of an Eat, Sleep, Console (ESC) protocol. METHODS: Using a multidisciplinary quality improvement approach, we implemented an ESC protocol at 2 community hospitals. Primary outcomes were to decrease length of stay (LOS) by 20% and decrease scheduled morphine use to <20%. Balancing measures included transfer to a higher level of care and unplanned 30-day readmissions. Data were extracted over 2 years, from 2017 through 2018. Interventions included an emphasis on nonpharmacologic care, the initiation of 1-time morphine dosing, flexible weaning schedules for infants on morphine, and the use of ESC scoring. Data were analyzed by using statistical process control. RESULTS: A total of 304 NAS patients were admitted from January 2017 to December 2018, with 155 during the postintervention period. After implementation, mean LOS decreased from 9.0 to 6.2 days, and morphine use decreased from 57% to 23%, both with special cause variation. There were 2 unplanned readmissions in the postintervention period compared with 1 preintervention and no transfers to higher level of care in either period. CONCLUSIONS: Implementation of a nonpharmacologic care protocol within 2 community hospitals led to significant and sustained improvement in LOS and morphine exposure without compromising safety. In this study, we illustrate that evidence-based practice can be successfully implemented and sustained within community hospitals treating infants with NAS.

Phenotype of CD4+ T cell subsets that develop following mouse facial nerve axotomy.

We have previously shown that CD4(+) T helper (Th) 2 cells, but not Th1 cells, participate in the rescue of mouse facial motoneurons (FMN) from axotomy-induced cell death. Recently, a number of other CD4(+) T cell subsets have been identified in addition to the Th1 and Th2 effector subsets, including Th17, inducible T regulatory type 1 (Tr1), and naturally thymus-born Foxp3(+) regulatory (Foxp3(+) Treg) cells. These subsets regulate the nature of a T cell-mediated immune response. Th1 and Th17 cells are pro-inflammatory subsets, while Th2, Tr1, and Foxp3(+) Treg cells are anti-inflammatory subsets. Pro-inflammatory responses in the central nervous system are thought to be neurodestructive, while anti-inflammatory responses are considered neuroprotective. However, it remains to be determined if another CD4(+) T cell subset, other than the Th2 cell, develops after peripheral nerve injury and participates in FMN survival. In the present study, we used FACS analysis to determine the temporal frequency of Th1, Th17, Th2, Tr1 and Foxp3(+) Treg CD4(+) T cell subset development in C57BL/6 wild type mice after facial nerve transection at the stylomastoid foramen in the mouse. The results indicate that all of the known CD4(+) T cell subsets develop and expand in number within the draining lymph node, with a peak in number primarily at 7 days postoperative (dpo), followed by a decline at 9 dpo. In addition to the increase in subset frequency over time, FACS analysis of individual cells showed that the level of cytokine expressed per cell also increased for interferon-gamma (IFN-gamma), interleukin (IL)-10 and IL-17, but not IL-4. Additional control double-cytokine labeling experiments were done which indicate that, at 7dpo, the majority of cells indeed have committed to a specific phenotype and express only 1 cytokine. Collectively, our findings indicate for the first time that there is no preferential activation and expansion of any single CD4(+) T cell subset after peripheral nerve injury but, rather, that both pro-inflammatory and anti-inflammatory CD4(+) T cells develop.