Evaluation of a behavioural intervention to reduce perioperative midazolam administration to older adults.

Background: Older patients commonly receive benzodiazepines during anaesthesia despite guidelines recommending avoidance. Interventions to reduce perioperative benzodiazepine use are not well studied. We hypothesized an automated electronic medical record alert targeting anaesthesia providers would reduce administration of benzodiazepines to older adults undergoing general anaesthesia. Methods: We conducted a retrospective study of adults who underwent surgery at 5 hospitals within one US academic health system. One of the hospitals received an intervention consisting of provider education and an automated electronic medical record alert discouraging benzodiazepine administration to patients aged 70 years or older. We used difference-in-differences analysis to compare patterns of midazolam use 12-months before and after intervention at the intervention hospital, using the 4 non-intervention hospitals as contemporaneous comparators. Results: The primary analysis sample included 20,347 cases among patients aged 70 and older. At the intervention hospital, midazolam was administered in 454/4,240 (10.7%) cases pre-alert versus 250/3,750 (6.7%) post-alert (p<0.001). At comparator hospitals, respective rates were 3,186/6,366 (50.0%) versus 2,935/5,991 (49.0%) (p=0.24). After adjustment, the intervention was associated with a 3.2 percentage point (p.p.) reduction in the percentage of cases with midazolam administration (95% CI: (-5.2, -1.1); p=0.002). Midazolam dose was unaffected (adjusted mean difference -0.01 mg, 95% CI: (-0.20, 0.18); p=0.90). In 76,735 cases among patients aged 18-69, the percentage of cases with midazolam administration decreased by 6.9 p. p. (95% CI: (-8.0, -5.7); p<0.001). Conclusion: Provider-facing alerts in the intraoperative electronic medical record, coupled with education, can reduce midazolam administration to older patients presenting for surgery but may affect care of younger patients.

Modulation of PKM activity affects the differentiation of TH17 cells.

Small molecules that promote the metabolic activity of the pyruvate kinase isoform PKM2, such as TEPP-46 and DASA-58, limit tumorigenesis and inflammation. To understand how these compounds alter T cell function, we assessed their therapeutic activity in a mouse model of T cell-mediated autoimmunity that mimics multiple sclerosis (MS). TH17 cells are believed to orchestrate MS pathology, in part, through the production of two proinflammatory cytokines: interleukin-17 (IL-17) and GM-CSF. We found that both TEPP-46 and DASA-58 suppressed the development of IL-17-producing TH17 cells but increased the generation of those producing GM-CSF. This switch redirected disease pathology from the spinal cord to the brain. In addition, we found that activation of PKM2 interfered with TGF-ß1 signaling, which is necessary for the development of TH17 and regulatory T cells. Collectively, our data clarify the therapeutic potential of PKM2 activators in MS-like disease and how these agents alter T cell function.

The active contribution of OPCs to neuroinflammation is mediated by LRP1.

Oligodendrocyte progenitor cells (OPCs) account for about 5% of total brain and spinal cord cells, giving rise to myelinating oligodendrocytes that provide electrical insulation to neurons of the CNS. OPCs have also recently been shown to regulate inflammatory responses and glial scar formation, suggesting functions that extend beyond myelination. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifaceted phagocytic receptor that is highly expressed in several CNS cell types, including OPCs. Here, we have generated an oligodendroglia-specific knockout of LRP1, which presents with normal myelin development, but is associated with better outcomes in two animal models of demyelination (EAE and cuprizone). At a mechanistic level, LRP1 did not directly affect OPC differentiation into mature oligodendrocytes. Instead, animals lacking LRP1 in OPCs in the demyelinating CNS were characterized by a robust dampening of inflammation. In particular, LRP1-deficient OPCs presented with impaired antigen cross-presentation machinery, suggesting a failure to propagate the inflammatory response and thus promoting faster myelin repair and neuroprotection. Our study places OPCs as major regulators of neuroinflammation in an LRP1-dependent fashion.

Modulation of the sigma-1 receptor-IRE1 pathway is beneficial in preclinical models of inflammation and sepsis.

Sepsis is an often deadly complication of infection in which systemic inflammation damages the vasculature, leading to tissue hypoperfusion and multiple organ failure. Currently, the standard of care for sepsis is predominantly supportive, with few therapeutic options available. Because of increased sepsis incidence worldwide, there is an urgent need for discovery of novel therapeutic targets and development of new treatments. The recently discovered function of the endoplasmic reticulum (ER) in regulation of inflammation offers a potential avenue for sepsis control. Here, we identify the ER-resident protein sigma-1 receptor (S1R) as an essential inhibitor of cytokine production in a preclinical model of septic shock. Mice lacking S1R succumb quickly to hypercytokinemia induced by a sublethal challenge in two models of acute inflammation. Mechanistically, we find that S1R restricts the endonuclease activity of the ER stress sensor IRE1 and cytokine expression but does not inhibit the classical inflammatory signaling pathways. These findings could have substantial clinical implications, as we further find that fluvoxamine, an antidepressant therapeutic with high affinity for S1R, protects mice from lethal septic shock and dampens the inflammatory response in human blood leukocytes. Our data reveal the contribution of S1R to the restraint of the inflammatory response and place S1R as a possible therapeutic target to treat bacterial-derived inflammatory pathology.

Exploring Non-Metabolic Functions of Glycolytic Enzymes in Immunity.

At the beginning of the twentieth century, discoveries in cancer research began to elucidate the idiosyncratic metabolic proclivities of tumor cells (1). Investigators postulated that revealing the distinct nutritional requirements of cells with unchecked growth potential would reveal targetable metabolic vulnerabilities by which their survival could be selectively curtailed. Soon thereafter, researchers in the field of immunology began drawing parallels between the metabolic characteristics of highly proliferative cancer cells and those of immune cells that respond to perceived threats to host physiology by invading tissues, clonally expanding, and generating vast amounts of pro-inflammatory effector molecules to provide the host with protection. Throughout the past decade, increasing effort has gone into elucidating the biosynthetic and bioenergetic requirements of immune cells during inflammatory responses. It is now well established that, like tumor cells, immune cells must undergo metabolic adaptations to fulfill their effector functions (2, 3). Unraveling the metabolic adaptations that license inflammatory immune responses may lead to the development of novel classes of therapeutics for pathologies with prominent inflammatory components (e.g., autoimmunity). However, the translational potential of discoveries made toward this end is currently limited by the ubiquitous nature of the "pathologic" process being targeted: metabolism. Recent works have started to unravel unexpected non-metabolic functions for metabolic enzymes in the context of inflammation, including signaling and gene regulation. One way information gained through the study of immunometabolism may be leveraged for therapeutic benefit is by exploiting these non-canonical features of metabolic machinery, modulating their contribution to the immune response without impacting their basal metabolic functions. The focus of this review is to discuss the metabolically independent functions of glycolytic enzymes and how these could impact T cells, agents of the immune system that are commonly considered as orchestrators of auto-inflammatory processes.

Lineage-Specific Metabolic Properties and Vulnerabilities of T Cells in the Demyelinating Central Nervous System.

Multiple sclerosis (MS) is a disease that is characterized by immune-mediated destruction of CNS myelin. Current MS therapies aim to block peripheral immune cells from entering the CNS. Although these treatments limit new inflammatory activity in the CNS, no treatment effectively prevents long-term disease progression and disability accumulation in MS patients. One explanation for this paradox is that current therapies are ineffective at targeting immune responses already present in the CNS. To this end, we sought to understand the metabolic properties of T cells that mediate ongoing inflammation in the demyelinating CNS. Using experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice, a well-studied model of MS, we showed that the CD4+ and CD8+ T cells that invade the EAE CNS are highly glycolytic. Elevated glycolytic rates in T cells isolated from the EAE CNS correlate with upregulated expression of glycolytic machinery and is essential for inflammatory responses to myelin. Surprisingly, we found that an inhibitor of GAPDH, 3-bromopyruvic acid (3-BrPa), blocks IFN-γ, but not IL-17A, production in immune cells isolated from the EAE CNS. Indeed, in vitro studies confirmed that the production of IFN-γ by differentiated Th1 cells is more sensitive to 3-BrPa than is the production of IL-17A by Th17 cells. Finally, in transfer models of EAE, 3-BrPa robustly attenuates the encephalitogenic potential of EAE-driving immune cells. To our knowledge, these data are among the first to demonstrate the metabolic properties of T cells in the demyelinating CNS in vivo.

LRP1 expression in microglia is protective during CNS autoimmunity.

Multiple sclerosis is a devastating neurological disorder characterized by the autoimmune destruction of the central nervous system myelin. While T cells are known orchestrators of the immune response leading to MS pathology, the precise contribution of CNS resident and peripheral infiltrating myeloid cells is less well described. Here, we explore the myeloid cell function of Low-density lipoprotein receptor-related protein-1 (LRP1), a scavenger receptor involved in myelin clearance and the inflammatory response, in the context of Multiple sclerosis. Supporting its central role in Multiple sclerosis pathology, we find that LRP1 expression is increased in Multiple sclerosis lesions in comparison to the surrounding healthy tissue. Using two genetic mouse models, we show that deletion of LRP1 in microglia, but not in peripheral macrophages, negatively impacts the progression of experimental autoimmune encephalomyelitis, an animal model of Multiple sclerosis. We further show that the increased disease severity in experimental autoimmune encephalomyelitis is not due to haplodeficiency of the Cx3cr1 locus. At the cellular level, microglia lacking LRP1 adopt a pro-inflammatory phenotype characterized by amoeboid morphology and increased production of the inflammatory mediator TNF-α. We also show that LRP1 functions as a robust inhibitor of NF-kB activation in myeloid cells via a MyD88 dependent pathway, potentially explaining the increase in disease severity observed in mice lacking LRP1 expression in microglia. Taken together, our data suggest that the function of LRP1 in microglia is to keep these cells in an anti-inflammatory and neuroprotective status during inflammatory insult, including experimental autoimmune encephalomyelitis and potentially in Multiple sclerosis.

Erratum to: Safety and feasibility of oral immunotherapy to multiple allergens for food allergy.

[This corrects the article DOI: 10.1186/1710-1492-10-1.].

Phase 1 results of safety and tolerability in a rush oral immunotherapy protocol to multiple foods using Omalizumab.

BACKGROUND: Up to 30% of patients with food allergies have clinical reactivity to more than one food allergen. Although there is currently no cure, oral immunotherapy (OIT) is under investigation. Pilot data have shown that omalizumab may hasten the ability to tolerate over 4 g of food allergen protein. OBJECTIVE: To evaluate the safety and dose tolerability of a Phase 1 Single Site OIT protocol using omalizumab to allow for a faster and safe desensitization to multiple foods simultaneously. METHODS: Participants with multiple food allergies received OIT for up to 5 allergens simultaneously with omalizumab (rush mOIT). Omalizumab was administered for 8 weeks prior to and 8 weeks following the initiation of a rush mOIT schedule. Home reactions were recorded with diaries. RESULTS: Twenty-five (25) participants were enrolled in the protocol (median age 7 years). For each included food, participants had failed an initial double-blind placebo-controlled food challenge at a protein dose of 100 mg or less. After pre-treatment with omalizumab, 19 participants tolerated all 6 steps of the initial escalation day (up to 1250 mg of combined food proteins), requiring minimal or no rescue therapy. The remaining 6 were started on their highest tolerated dose as their initial daily home doses. Participants reported 401 reactions per 7,530 home doses (5.3%) with a median of 3.2 reactions per 100 doses. Ninety-four percent (94%) of reactions were mild. There was one severe reaction. Participants reached their maintenance dose of 4,000 mg protein per allergen at a median of 18 weeks. CONCLUSION: These phase 1 data demonstrate that rush OIT to multiple foods with 16 weeks of treatment with omalizumab could allow for a fast desensitization in subjects with multiple food allergies. Phase 2 randomized controlled trials are needed to better define safety and efficacy parameters of multi OIT experimental treatments with and without omalizumab.

Identification of STAT5A and STAT5B target genes in human T cells.

Signal transducer and activator of transcription (STAT) comprises a family of universal transcription factors that help cells sense and respond to environmental signals. STAT5 refers to two highly related proteins, STAT5A and STAT5B, with critical function: their complete deficiency is lethal in mice; in humans, STAT5B deficiency alone leads to endocrine and immunological problems, while STAT5A deficiency has not been reported. STAT5A and STAT5B show peptide sequence similarities greater than 90%, but subtle structural differences suggest possible non-redundant roles in gene regulation. However, these roles remain unclear in humans. We applied chromatin immunoprecipitation followed by DNA sequencing using human CD4(+) T cells to detect candidate genes regulated by STAT5A and/or STAT5B, and quantitative-PCR in STAT5A or STAT5B knock-down (KD) human CD4(+) T cells to validate the findings. Our data show STAT5A and STAT5B play redundant roles in cell proliferation and apoptosis via SGK1 interaction. Interestingly, we found a novel, unique role for STAT5A in binding to genes involved in neural development and function (NDRG1, DNAJC6, and SSH2), while STAT5B appears to play a distinct role in T cell development and function via DOCK8, SNX9, FOXP3 and IL2RA binding. Our results also suggest that one or more co-activators for STAT5A and/or STAT5B may play important roles in establishing different binding abilities and gene regulation behaviors. The new identification of these genes regulated by STAT5A and/or STAT5B has major implications for understanding the pathophysiology of cancer progression, neural disorders, and immune abnormalities.

Safety and feasibility of oral immunotherapy to multiple allergens for food allergy.

BACKGROUND: Thirty percent of children with food allergy are allergic to more than one food. Previous studies on oral immunotherapy (OIT) for food allergy have focused on the administration of a single allergen at the time. This study aimed at evaluating the safety of a modified OIT protocol using multiple foods at one time. METHODS: Participants underwent double-blind placebo-controlled food challenges (DBPCFC) up to a cumulative dose of 182 mg of food protein to peanut followed by other nuts, sesame, dairy or egg. Those meeting inclusion criteria for peanut only were started on single-allergen OIT while those with additional allergies had up to 5 foods included in their OIT mix. Reactions during dose escalations and home dosing were recorded in a symptom diary. RESULTS: Forty participants met inclusion criteria on peanut DBPCFC. Of these, 15 were mono-allergic to peanut and 25 had additional food allergies. Rates of reaction per dose did not differ significantly between the two groups (median of 3.3% and 3.7% in multi and single OIT group, respectively; p = .31). In both groups, most reactions were mild but two severe reactions requiring epinephrine occurred in each group. Dose escalations progressed similarly in both groups although, per protocol design, those on multiple food took longer to reach equivalent doses per food (median +4 mo.; p < .0001). CONCLUSIONS: Preliminary data show oral immunotherapy using multiple food allergens simultaneously to be feasible and relatively safe when performed in a hospital setting with trained personnel. Additional, larger, randomized studies are required to continue to test safety and efficacy of multi-OIT. TRIAL REGISTRATION: Clinicaltrial.gov NCT01490177.

Differentiating the roles of STAT5B and STAT5A in human CD4+ T cells.

STAT5A and STAT5B are highly homologous proteins whose distinctive roles in human immunity remain unclear. However, STAT5A sufficiency cannot compensate for STAT5B defects, and human STAT5B deficiency, a rare autosomal recessive primary immunodeficiency, is characterized by chronic lung disease, growth failure and autoimmunity associated with regulatory T cell (Treg) reduction. We therefore hypothesized that STAT5A and STAT5B play unique roles in CD4(+) T cells. Upon knocking down STAT5A or STAT5B in human primary T cells, we found differentially regulated expression of FOXP3 and IL-2R in STAT5B knockdown T cells and down-regulated Bcl-X only in STAT5A knockdown T cells. Functional ex vivo studies in homozygous STAT5B-deficient patients showed reduced FOXP3 expression with impaired regulatory function of STAT5B-null Treg cells, also of increased memory phenotype. These results indicate that STAT5B and STAT5A act partly as non-redundant transcription factors and that STAT5B is more critical for Treg maintenance and function in humans.

Markers of antigen presentation and activation on eosinophils and T cells in the esophageal tissue of patients with eosinophilic esophagitis.

OBJECTIVES: Evidence suggests eosinophils may be acting as antigen-presenting cells (APCs) by presenting antigen to T cells. We investigated the surface proteins of eosinophils and T cells in the esophageal biopsies of patients with eosinophilic esophagitis (EoE), patients with gastroesophageal reflux disease (GERD), and healthy controls (HCs). METHODS: : Subjects were categorized as EoE, GERD, or HC. In esophageal tissue, EG2+ eosinophils were stained for the APC markers, CD40 or CD80, via immunohistochemistry. CD3+ T cells were stained for costimulatory markers, CD40L or CD28, and for activation markers, CD69 or CD134, via immunofluorescence or immunohistochemistry. RESULTS: Eosinophils stained with CD40 and CD80. The number of EG2+CD40+ cells was increased in EoE (mean 19.1±14.8 cells/high-power field [HPF], n=11), compared with GERD (mean 0.13±0.19 cells/HPF, n=5, P<0.01) and HC (mean 0.3±0.7 cells/HPF, n=5, P<0.01). There was an elevation in EG2+CD80+ cells in EoE (mean 18.1±16.2 cells/HPF, n=10), GERD (mean 1.7±2.8 cells/HPF, n=6, P<0.01), or HC (mean 0.8±1.3 cells/HPF, n=6, P<0.01). CD3+ T cells stained with CD40L (not quantified). CD3+ T cells stained with CD28 at elevated levels in EoE (mean 14±8.7 cells/HPF, n=9) versus GERD (mean 3.3±1.2 cells/HPF, n=6, P<0.05) or HC (mean 3.0±3.2 cells/HPF, n=7, P<0.01). The number of CD3+CD69+ cells was highest in EoE (mean 14.8±7.5 cells/HPF, n=6) versus GERD (mean 0.8±0.9 cells/HPF, n=6, P<0.001) or HC (mean 2.7±2.5 cells/HPF, n=6, P<0.001). CONCLUSIONS: We show that esophageal eosinophils express CD40 and CD80, and T cells with CD40L, CD28, and CD69. The number of double-stained cells was higher in EoE in comparison to control groups. These data support the hypothesis that eosinophils in EoE may act as APCs, activating T cells.

Immunomodulatory effect of vancomycin on Treg in pediatric inflammatory bowel disease and primary sclerosing cholangitis.

Vancomycin has been shown to affect tumor necrosis factor-alpha (TNF-α) pathways as an immunomodulator; this is thought to be separate from its function as an antibiotic [1]. Previous studies have shown that oral vancomycin (OV) is an effective treatment for concomitant primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) in children [2, 3]. Since both diseases are associated with immune dysfunction, we hypothesized that vancomycin's therapeutic effect in IBD and PSC occurs through immunomodulation. Therefore, we examined the in vivo immunological changes that occur during OV treatment of 14 children with PSC and IBD. Within 3 months of OV administration, peripheral gamma-glutamyl transpeptidase (GGT) and alanine aminotransferase (ALT) concentrations, white blood cell (WBC) counts, and neutrophil counts normalized from elevated levels before treatment. Patients also demonstrated improved biliary imaging studies, liver biopsies and IBD symptoms and biopsies. Additionally, plasma transforming growth factor beta (TGF-ß) levels were increased without concurrent shifts in Th1-or Th2-associated cytokine production. Peripheral levels of CD4 + CD25hiCD127lo and CD4 + FoxP3+ regulatory T (Treg) cells also increased in OV-treated PSC + IBD patients compared to pretreatment levels. A unique case study shows that the therapeutic effects of OV in the treatment of PSC + IBD do not always endure after OV discontinuation, with relapse of PSC associated with a decrease in blood Treg levels; subsequent OV retreatment was then associated with a rise in blood Treg levels and normalization of liver function tests (LFTs). Taken together, these studies support immune-related pathophysiology of PSC with IBD, which is responsive to OV.

Personal omics profiling reveals dynamic molecular and medical phenotypes.

Personalized medicine is expected to benefit from combining genomic information with regular monitoring of physiological states by multiple high-throughput methods. Here, we present an integrative personal omics profile (iPOP), an analysis that combines genomic, transcriptomic, proteomic, metabolomic, and autoantibody profiles from a single individual over a 14 month period. Our iPOP analysis revealed various medical risks, including type 2 diabetes. It also uncovered extensive, dynamic changes in diverse molecular components and biological pathways across healthy and diseased conditions. Extremely high-coverage genomic and transcriptomic data, which provide the basis of our iPOP, revealed extensive heteroallelic changes during healthy and diseased states and an unexpected RNA editing mechanism. This study demonstrates that longitudinal iPOP can be used to interpret healthy and diseased states by connecting genomic information with additional dynamic omics activity.

Eotaxin and FGF enhance signaling through an extracellular signal-related kinase (ERK)-dependent pathway in the pathogenesis of Eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is characterized by the inflammation of the esophagus and the infiltration of eosinophils into the esophagus, leading to symptoms such as dysphagia and stricture formation. Systemic immune indicators like eotaxin and fibroblast growth factor were evaluated for possible synergistic pathological effects. Moreover, blood cells, local tissue, and plasma from EoE and control subjects were studied to determine if the localized disease was associated with a systemic effect that correlated with presence of EoE disease. METHOD: Real-time polymerase chain reaction from peripheral blood mononuclear cells (PBMC), immunohistochemistry from local esophageal biopsies, fluid assays on plasma, and fluorescence-activated cell sorting on peripheral blood cells from subjects were used to study the systemic immune indicators in newly diagnosed EoE (n = 35), treated EoE (n = 9), Gastroesophageal reflux disease (GERD) (n = 8), ulcerative colitis (n = 5), Crohn's disease (n = 5), and healthy controls (n = 8). RESULT: Of the transcripts tested for possible immune indicators, we found extracellular signal-regulated kinase (ERK), Bcl-2, bFGF (basic fibroblast growth factor), and eotaxin levels were highly upregulated in PBMC and associated with disease presence of EoE. Increased FGF detected by immunohistochemistry in esophageal tissues and in PBMC was correlated with low levels of pro-apoptotic factors (Fas, Caspase 8) in PBMC from EoE subjects. Plasma-derived bFGF was shown to be the most elevated and most specific in EoE subjects in comparison to healthy controls and disease control subjects. CONCLUSION: We describe for the first time a possible mechanism by which increased FGF is associated with inhibiting apoptosis in local esophageal tissues of EoE subjects as compared to controls. Eotaxin and FGF signaling pathways share activation through the ERK pathway; together, they could act to increase eosinophil activation and prolong the half-life of eosinophils in local tissues of the esophagus in EoE subjects.