Jonathan R. Weinstein

Prior Antiplatelet Therapy, Platelet Infusion Therapy, and Outcome after Intracerebral Hemorrhage

BACKGROUND Recent studies examining the effect of prior antiplatelet therapy (APT) on outcome in patients with spontaneous intracerebral hemorrhage (ICH) have shown conflicting results. The effect of platelet infusion therapy (PIT) on outcome in patients with ICH taking APT is unknown. METHODS We reviewed records of patients with ICH admitted to a single hospital, excluding those with international normalized ratio greater than or equal to 1.5. Baseline characteristics were compared by APT status in all patients and by PIT status in the subgroup of patients on APT. We used multivariate analyses to generate propensity and prognostic scores for exposures (APT and PIT) and outcomes (favorable outcome and hospital death), respectively. We examined the associations between exposures and outcomes and adjusted these for propensity and/or prognostic scores. We then validated our findings with a sensitivity analysis. RESULTS Of 368 patients identified, 121 (31.3%) were taking APT, mostly aspirin. Patients on APT were older and more likely to have vascular comorbidities than those not. The APT group also had a higher initial Glasgow Coma Scale score at presentation. In analyses adjusted for both propensity and prognostic scores, APT was associated with a higher likelihood of hospital death (odds ratio [OR] 2.4; 95% confidence interval [CI] 1.1-5.6); PIT did not prevent hospital death (OR 1.2; 95% CI 0.3-5.5) or increase favorable outcome (OR 1.4; 95% CI 0.4-5.4). CONCLUSIONS In patients with ICH, APT is associated with an increased risk of hospital death. In the subgroup of patients on APT, PIT did not prevent death or improve outcome.

Do-Not-Attempt-Resuscitation Orders and Prognostic Models for Intraparenchymal Hemorrhage

Objectives:Statistical models predicting outcome after intraparenchymal hemorrhage include patients irrespective of do-not-attempt-resuscitation orders. We built a model to explore how the inclusion of patients with do-not-attempt-resuscitation orders affects intraparenchymal hemorrhage prognostic models. Design:Retrospective, observational cohort study from May 2001 until September 2003. Setting:University-affiliated tertiary referral hospital in Seattle, WA. Patients:Four hundred twenty-four consecutive patients with spontaneous intraparenchymal hemorrhage. Measurements and Main Results:We retrospectively abstracted information from medical records of intraparenchymal hemorrhage patients admitted to a single hospital. Using multivariate logistic regression of presenting clinical characteristics, but not do-not-attempt-resuscitation status, we generated a prognostic score for favorable outcome (defined as moderate disability or better at discharge). We compared observed probability of favorable outcome with that predicted, stratified by do-not-attempt-resuscitation status. We then generated a modified prognostic score using only non-do-not-attempt-resuscitation patients. Records of 424 patients were reviewed: 44% had favorable outcome, 43% had a do-not-attempt-resuscitation order, and 38% died in hospital. The observed and predicted probability of favorable outcome agreed well with all patients taken together. The observed probability of favorable outcome was significantly higher than predicted in non-do-not-attempt-resuscitation patients and significantly lower in do-not-attempt-resuscitation patients. Results were similar when applying a previously published and validated prognostic score. Our modified prognostic score was no longer pessimistic in non-do-not-attempt-resuscitation patients but remained overly optimistic in do-not-attempt-resuscitation patients. Conclusions:Although our prognostic model was well-calibrated when assessing all intraparenchymal hemorrhage patients, predictions were significantly pessimistic in patients without and optimistic in those with do-not-attempt-resuscitation orders. Such pessimism may drive decisions not to attempt resuscitation in patients in whom a favorable outcome may have been possible, thereby creating a self-fulfilling prophecy. To be most useful in clinical decision making, intraparenchymal hemorrhage prognostic models should be calibrated to large intraparenchymal hemorrhage cohorts in whom do-not-attempt-resuscitation orders were not used.

Lipopolysaccharide is a frequent and significant contaminant in microglia-activating factors

Lipopolysaccharide (LPS/endotoxin) is a potent immunologic stimulant. Many commercial‐grade reagents used in research are not screened for LPS contamination. LPS induces a wide spectrum of proinflammatory responses in microglia, the immune cells of the brain. Recent studies have demonstrated that a broad range of endogenous factors including plasma‐derived proteins and bioactive phospholipids can also activate microglia. However, few of these studies have reported either the LPS levels found in the preparations used or the effect of LPS inhibitors such as polymyxin B (PMX) on factor‐induced responses. Here, we used the Limulus amoebocyte lysate assay to screen a broad range of commercial‐ and pharmaceutical‐grade proteins, peptides, lipids, and inhibitors commonly used in microglia research for contamination with LPS. We then characterized the ability of PMX to alter a representative set of factor‐induced microglial activation parameters including surface antigen expression, metabolic activity/proliferation, and NO/cytokine/chemokine release in both the N9 microglial cell line and primary microglia. Significant levels of LPS contamination were detected in a number of commercial‐grade plasma/serum‐ and nonplasma/serum‐derived proteins, phospholipids, and synthetic peptide preparations, but not in pharmaceutical‐grade recombinant proteins or pharmacological inhibitors. PMX had a significant inhibitory effect on the microglia‐activating potential of a number of commercial‐, but not pharmaceutical‐grade, protein preparations. Novel PMX‐resistant responses to α2‐macroglobulin and albumin were incidentally observed. Our results indicate that LPS is a frequent and significant contaminant in commercial‐grade preparations of previously reported microglia‐activating factors. Careful attention to LPS levels and appropriate controls are necessary for future studies in the neuroinflammation field.

The p53 Transcription Factor Modulates Microglia Behavior through MicroRNA-Dependent Regulation of c-Maf

Neuroinflammation occurs in acute and chronic CNS injury, including stroke, traumatic brain injury, and neurodegenerative diseases. Microglia are specialized resident myeloid cells that mediate CNS innate immune responses. Disease-relevant stimuli, such as reactive oxygen species (ROS), can influence microglia activation. Previously, we observed that p53, a ROS-responsive transcription factor, modulates microglia behaviors in vitro and in vivo, promoting proinflammatory functions and suppressing downregulation of the inflammatory response and tissue repair. In this article we describe a novel mechanism by which p53 modulates the functional differentiation of microglia both in vitro and in vivo. Adult microglia from p53-deficient mice have increased expression of the anti-inflammatory transcription factor c-Maf. To determine how p53 negatively regulates c-Maf, we examined the impact of p53 on known c-Maf regulators. MiR-155 is a microRNA that targets c-Maf. We observed that cytokine-induced expression of miR-155 was suppressed in p53-deficient microglia. Furthermore, Twist2, a transcriptional activator of c-Maf, is increased in p53-deficient microglia. We identified recognition sites in the 3′ untranslated region of Twist2 mRNA that are predicted to interact with two p53-dependent microRNAs: miR-34a and miR-145. In this article, we demonstrate that miR-34a and -145 are regulated by p53 and negatively regulate Twist2 and c-Maf expression in microglia and the RAW macrophage cell line. Taken together, these findings support the hypothesis that p53 activation induced by local ROS or accumulated DNA damage influences microglia functions and that one specific molecular target of p53 in microglia is c-Maf.

Lentiviral transduction of microglial cells

Microglial cells are the resident immune cells of the central nervous system. Their function resembles that of tissue macrophages and, as such, they share many properties with both peripheral macrophages and monocytes. One striking similarity is the difficulty with which these cells can be genetically manipulated via transfection or transduction. We have sought to overcome this challenge and generate stably transduced microglial cell lines. Based on encouraging results from macrophages, we hypothesized that lentiviral vectors might provide a valuable tool in the transduction of microglial cells. Using a lentiviral‐based vector system expressing enhanced green fluorescent protein (eGFP) under the control of the murine stem cell virus promoter (MSCV), we found that multiplicities of infection (MOIs) of 1, 10, and 100 transduce >70%, >88%, and >95% of the cells, respectively. From the pool of transduced cells, we established lines of N9 and BV‐2 microglial cells with distinct fluorescence intensities. Using real time‐polymerase chain reaction (PCR), we correlated the integrated eGFP copy numbers to eGFP fluorescence measured by flow cytometry. When mixed, up to three lines with different eGFP intensities could be separated by flow cytometry and fluorescence microscopy. Neither infection nor transgene expression influenced microglial activation as assessed by nitric oxide (NO) production, cytokine release, and surface antigen expression. Our findings that microglial cells are easily transduced by lentiviral based vectors will facilitate research depending on genetic manipulation and help generate transgenic cell lines. In addition, the availability of microglial cell lines with defined fluorescence properties could replace elaborate staining procedures for microglial identification in co‐culture experiments.

Injury‐Related Factors and Conditions Down‐Regulate the Thrombin Receptor (PAR‐1) in a Human Neuronal Cell Line

Abstract: Previous studies have demonstrated that thrombin can induce potent effects on neural cell morphology, biochemistry, and viability. Nearly all of these effects are mediated by proteolytic activation of the thrombin receptor (PAR‐1). Mechanisms of PAR‐1 regulation in several nonneural cell types have been shown to be novel and cell type specific; however, little is known about PAR‐1 regulation in neural cells. In the present study, PAR‐1 cell surface expression and regulation were examined in a transformed retinoblast (Ad12 HER 10) cell line using radioiodinated anti‐PAR‐1 monoclonal antibodies ATAP2, which recognizes intact and cleaved receptors, and SPAN12, which is specific for the intact form of the receptor. Scatchard analysis revealed high‐affinity, specific binding to a single affinity class of receptors: KD = 3.13 and 5.25 nM, Bmax = 190.1 and 67.8 fmol/mg of protein for 125I‐ATAP2 and 125I‐SPAN12, respectively. Specificity for PAR‐1 was confirmed by demonstrating rapid and near complete decreases for both antibodies following treatment with thrombin or PAR‐1 activating peptide (SFLLRN). Differential antibody binding was used to demonstrate rapid and near complete thrombin‐induced PAR‐1 cleavage and internalization, with protein synthesis‐dependent replacement of intact receptors occurring over longer time intervals, but only minimal recycling of cleaved receptors. A variety of factors and conditions were screened for their effects on PAR‐1 expression. Significant decreases in PAR‐1 expression were induced by the protein kinase C activator phorbol 12‐myristate 13‐acetate (87% at 3 h), the phospholipid inflammatory mediator lysophosphatidic acid (32% at 3 h), and the injury‐related condition hypoglycemia (64 and 100% at 24 h in the absence and presence of dibutyryl cyclic AMP, respectively). The effect of hypoglycemia was shown by RNase protection to be at least partially pretranslational. Finally, thrombins ability to enhance hypoglycemia‐induced cell killing correlated temporally with PAR‐1 cell surface expression.

Unraveling thrombin's true microglia-activating potential: markedly disparate profiles of pharmaceutical-grade and commercial-grade thrombin preparations

Microglia are the resident immune cells of the CNS. Brain injury triggers microglial activation, leading to proliferation, changes in antigenic profile, NO production and cytokine release. It is widely believed that serum factors inundating the injured tissue can prompt this activation, leading to long‐term phenotypic changes. We and others have recently reported that commercial‐grade preparations of thrombin, a serine protease known for its central function in blood coagulation, activate microglial cells. Recent findings, however, have called into question the involvement of thrombin itself in the induction of microglial cytokine release and led us to systematically re‐investigate the ability of the protease to induce a broad spectrum of microglial activation parameters. We used a pharmaceutical‐grade recombinant human thrombin (rh‐thr) and compared it with a commercial‐grade plasma‐derived bovine thrombin (pb‐thr) preparation that has been used extensively in the literature, including in our own earlier report. We investigated the effect of these two thrombin preparations on proliferation, NO production, interleukin‐6 and tumour necrosis factor‐α release, intracellular calcium signaling and cell surface expression of CD95 (Fas) and CD40. Pb‐thr induced robust responses in all variables tested. In contrast, rh‐thr triggered calcium signals and induced small but significant changes in the expression of cell surface antigens, but had no effect on proliferation, NO production or cytokine release. Control studies assured equivalent thrombin potencies and excluded both species‐specific effects and endotoxin (lipopolysaccharide) contamination as possible causes of the disparity. Our results indicate a substantially more restricted role for thrombin itself in microglial activation than previously appreciated, but point to several potentially important co‐stimulatory effects. In addition, these results suggest that previous studies examining thrombins activation of microglia should be cautiously re‐interpreted.

Regulation of Fcγ Receptors and Immunoglobulin G-Mediated Phagocytosis in Mouse Microglia

As resident macrophages in the CNS, microglia can transform from a surveillance state to an activated phenotype in response to brain injury. During this transition microglia become highly capable phagocytic cells. Invading pathogens undergo opsonization with immunoglobulins and microglia recognize these opsonized pathogens through interaction with their cognate F(c) receptors. In mice, both FcgammaRI and FcgammaRIIb receptors are involved in IgG-mediated phagocytosis of opsonzied pathogens. At sites of inflammation, microglial activity is regulated by T-cell derived cytokines. Here we first investigated the effects of IFN-gamma, IL-4, IL-13 and GM-CSF on expression of FcgammaRI and FcgammaRIIb mRNA levels in both primary microglia and microglial cell line N9. Using quantitative real-time PCR we show that IFN-gamma induced a 4-fold increase in the mRNA level of FcgammaRI but did not induce changes in FcgammaRIIb expression. IL-4 and IL-13 induced approximately 2-fold increases in expression of FcgammaRIIb mRNA, but had no effect on FcgammaRI expression. GM-CSF increased both FcgammaRI and FcgammaRIIb mRNA expression. We then characterized the ability of these same cytokines to regulate phagocytosis of immune complexes composed of IgG and the bacteria Staphylococcus aureus. IFN-gamma and GM-CSF both induced approximately 2-fold increases in IgG-mediated phagocytosis whereas IL-4 and IL-13 both decreased IgG-mediated phagocytosis by about one-third. None of the cytokines influenced basal levels of phagocytosis. These findings demonstrate a highly selective cytokine-induced regulation of both phagocytosis-related Fcgamma receptor subtypes and IgG-mediated phagocytosis itself in microglia. This selective regulation has implications for our understanding of the pathophysiology of CNS infection and autoimmune disease.

Neuroimmune Response in Ischemic Preconditioning.

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon in which a brief period of cerebral ischemia confers transient tolerance to subsequent ischemic challenge. Research on IPC has implicated cellular, molecular, and systemic elements of the immune response in this phenomenon. Potent molecular mediators of IPC include innate immune signaling pathways such as Toll-like receptors and type 1 interferons. Brain ischemia results in release of pro- and anti-inflammatory cytokines and chemokines that orchestrate the neuroinflammtory response, resolution of inflammation, and transition to neurological recovery and regeneration. Cellular mediators of IPC include microglia, the resident central nervous system immune cells, astrocytes, and neurons. All of these cell types engage in cross-talk with each other using a multitude of signaling pathways that modulate activation/suppression of each of the other cell types in response to ischemia. As the postischemic neuroimmune response evolves over time there is a shift in function toward provision of trophic support and neuroprotection. Peripheral immune cells infiltrate the central nervous system en masse after stroke and are largely detrimental, with a few subtypes having beneficial, protective effects, though the role of these immune cells in IPC is largely unknown. The role of neural progenitor cells in IPC-mediated neuroprotection is another active area of investigation as is the role of microglial proliferation in this setting. A mechanistic understanding of these molecular and cellular mediators of IPC may not only facilitate more effective direct application of IPC to specific clinical scenarios, but also, more broadly, reveal novel targets for therapeutic intervention in stroke.

CD14 is a key organizer of microglial responses to CNS infection and injury.

Microglia, innate immune cells of the CNS, sense infection and damage through overlapping receptor sets. Toll‐like receptor (TLR) 4 recognizes bacterial lipopolysaccharide (LPS) and multiple injury‐associated factors. We show that its co‐receptor CD14 serves three non‐redundant functions in microglia. First, it confers an up to 100‐fold higher LPS sensitivity compared to peripheral macrophages to enable efficient proinflammatory cytokine induction. Second, CD14 prevents excessive responses to massive LPS challenges via an interferon β‐mediated feedback. Third, CD14 is mandatory for microglial reactions to tissue damage‐associated signals. In mice, these functions are essential for balanced CNS responses to bacterial infection, traumatic and ischemic injuries, since CD14 deficiency causes either hypo‐ or hyperinflammation, insufficient or exaggerated immune cell recruitment or worsened stroke outcomes. While CD14 orchestrates functions of TLR4 and related immune receptors, it is itself regulated by TLR and non‐TLR systems to thereby fine‐tune microglial damage‐sensing capacity upon infectious and non‐infectious CNS challenges. GLIA 2016;64:635–649.