Safety and efficacy of factor XIa inhibition with milvexian for secondary stroke prevention (AXIOMATIC-SSP): a phase 2, international, randomised, double-blind, placebo-controlled, dose-finding trial.

BACKGROUND: People with factor XI deficiency have lower rates of ischaemic stroke than the general population and infrequent spontaneous bleeding, suggesting that factor XI has a more important role in thrombosis than in haemostasis. Milvexian, an oral small-molecule inhibitor of activated factor XI, added to standard antiplatelet therapy, might reduce the risk of non-cardioembolic ischaemic stroke without increasing the risk of bleeding. We aimed to estimate the dose-response of milvexian for recurrent ischaemic cerebral events and major bleeding in patients with recent ischaemic stroke or transient ischaemic attack (TIA). METHODS: AXIOMATIC-SSP was a phase 2, randomised, double-blind, placebo-controlled, dose-finding trial done at 367 hospitals in 27 countries. Eligible participants aged 40 years or older, with acute (<48 h) ischaemic stroke or high-risk TIA, were randomly assigned by a web-based interactive response system in a 1:1:1:1:1:2 ratio to receive one of five doses of milvexian (25 mg once daily, 25 mg twice daily, 50 mg twice daily, 100 mg twice daily, or 200 mg twice daily) or matching placebo twice daily for 90 days. All participants received clopidogrel 75 mg daily for the first 21 days and aspirin 100 mg daily for the first 90 days. Investigators, site staff, and participants were masked to treatment assignment. The primary efficacy endpoint was the composite of ischaemic stroke or incident covert brain infarct on MRI at 90 days, assessed in all participants allocated to treatment who completed a follow-up MRI brain scan, and the primary analysis assessed the dose-response relationship with Multiple Comparison Procedure-Modelling (MCP-MOD). The main safety outcome was major bleeding at 90 days, assessed in all participants who received at least one dose of the study drug. This trial is registered with ClinicalTrials.gov (NCT03766581) and the EU Clinical Trials Register (2017-005029-19). FINDINGS: Between Jan 27, 2019, and Dec 24, 2021, 2366 participants were randomly allocated to placebo (n=691); milvexian 25 mg once daily (n=328); or twice-daily doses of milvexian 25 mg (n=318), 50 mg (n=328), 100 mg (n=310), or 200 mg (n=351). The median age of participants was 71 (IQR 62-77) years and 859 (36%) were female. At 90 days, the estimates of the percentage of participants with either symptomatic ischaemic stroke or covert brain infarcts were 16·8 (90·2% CI 14·5-19·1) for placebo, 16·7 (14·8-18·6) for 25 mg milvexian once daily, 16·6 (14·8-18·3) for 25 mg twice daily, 15·6 (13·9-17·5) for 50 mg twice daily, 15·4 (13·4-17·6) for 100 mg twice daily, and 15·3 (12·8-19·7) for 200 mg twice daily. No significant dose-response was observed among the five milvexian doses for the primary composite efficacy outcome. Model-based estimates of the relative risk with milvexian compared with placebo were 0·99 (90·2% CI 0·91-1·05) for 25 mg once daily, 0·99 (0·87-1·11) for 25 mg twice daily, 0·93 (0·78-1·11) for 50 mg twice daily, 0·92 (0·75-1·13) for 100 mg twice daily, and 0·91 (0·72-1·26) for 200 mg twice daily. No apparent dose-response was observed for major bleeding (four [1%] of 682 participants with placebo, two [1%] of 325 with milvexian 25 mg once daily, two [1%] of 313 with 25 mg twice daily, five [2%] of 325 with 50 mg twice daily, five [2%] of 306 with 100 mg twice daily, and five [1%] of 344 with 200 mg twice daily). Five treatment-emergent deaths occurred, four of which were considered unrelated to the study drug by the investigator. INTERPRETATION: Factor XIa inhibition with milvexian, added to dual antiplatelet therapy, did not substantially reduce the composite outcome of symptomatic ischaemic stroke or covert brain infarction and did not meaningfully increase the risk of major bleeding. Findings from our study have informed the design of a phase 3 trial of milvexian for the prevention of ischaemic stroke in patients with acute ischaemic stroke or TIA. FUNDING: Bristol Myers Squibb and Janssen Research & Development.

Bacterial pore-forming cytolysins induce neuronal damage in a rat model of neonatal meningitis.

BACKGROUND: Group B Streptococcus (GBS) and Streptococcus pneumoniae (SP) are leading causes of bacterial meningitis in neonates and children. Each pathogen produces a pore-forming cytolytic toxin, ß-hemolysin/cytolysin (ß-h/c) by GBS and pneumolysin by SP. The aim of this study was to understand the role of these pore-forming cytotoxins, in particular of the GBS ß-h/c, as potential neurotoxins in experimental neonatal meningitis. METHODS: Meningitis was induced in 7- and 11-day-old rats by intracisternal injection of wild type (WT) GBS or SP and compared with isogenic ß-h/c- or pneumolysin-deficient mutants, or a double mutant of SP deficient in pneumolysin and hydrogen peroxide production. RESULTS: GBS ß-h/c and SP pneumolysin contributed to neuronal damage, worsened clinical outcome and weight loss, but had no influence on the early kinetics of leukocyte influx and bacterial growth in the cerebrospinal fluid. In vitro, ß-h/c-induced neuronal apoptosis occurred independently of caspase-activation and was not preventable by the broad spectrum caspase-inhibitor z-VAD-fmk. CONCLUSIONS: These data suggest that both cytolytic toxins, the GBS ß-h/c and SP pneumolysin, contribute to neuronal damage in meningitis and extend the concept of a key role for bacterial pore-forming cytolysins in the pathogenesis and sequelae of neonatal meningitis.

Bacterial inhibition of phosphatidylcholine synthesis triggers apoptosis in the brain.

Streptococcus pneumoniae is the most common cause of bacterial meningitis of high mortality and morbidity. Neurological sequelae include paralysis, mental retardation, and learning disorders. In humans, neurons of the hippocampus undergo apoptosis as a result of meningitis. Phosphatidylcholine (PtdCho) is an essential component of mammalian cell membranes and PtdCho deficiency, either due to chemicals or altered nutrition, leads to apoptosis, especially in hippocampal neurons. We show that apoptosis of a variety of brain cells after pneumococcal infection arises from inhibition of PtdCho biosynthesis, the first such activity described for a bacterium. Apoptosis inhibitors did not prevent the bacterial-dependent inhibition of PtdCho biosynthesis. Supplementation with exogenous lyso-phosphatidylcholine prevents cell death and treatment of mice with cytidine diphosphocholine attenuates hippocampal damage during meningitis, even after the onset of infection. We conclude that bacterial inhibition of PtdCho biosynthesis activates an apoptotic cascade that is a causative event in pathogenesis and amenable to therapeutic intervention.

TRAIL limits excessive host immune responses in bacterial meningitis.

Apart from potential roles in anti-tumor surveillance, the TNF-related apoptosis-inducing ligand (TRAIL) has important regulatory functions in the host immune response. We studied antiinflammatory effects of endogenous and recombinant TRAIL (rTRAIL) in experimental meningitis. Following intrathecal application of pneumococcal cell wall, a TLR2 ligand, we found prolonged inflammation, augmented clinical impairment, and increased apoptosis in the hippocampus of TRAIL(-/-) mice. Administration of rTRAIL into the subarachnoid space of TRAIL(-/-) mice or reconstitution of hematopoiesis with wild-type bone marrow cells reversed these effects, suggesting an autoregulatory role of TRAIL within the infiltrating leukocyte population. Importantly, intrathecal application of rTRAIL in wild-type mice with meningitis also decreased inflammation and apoptosis. Moreover, patients suffering from bacterial meningitis showed increased intrathecal synthesis of TRAIL. Our findings provide what we believe is the first evidence that TRAIL may act as a negative regulator of acute CNS inflammation. The ability of TRAIL to modify inflammatory responses and to reduce neuronal cell death in meningitis suggests that it may be used as a novel antiinflammatory agent in invasive infections.

A vicious cycle involving release of heat shock protein 60 from injured cells and activation of toll-like receptor 4 mediates neurodegeneration in the CNS.

Infection, ischemia, trauma, and neoplasia elicit a similar inflammatory response in the CNS characterized by activation of microglia, the resident CNS monocyte. The molecular events leading from CNS injury to the activation of innate immunity is not well understood. We show here that the intracellular chaperone heat shock protein 60 (HSP60) serves as a signal of CNS injury by activating microglia through a toll-like receptor 4 (TLR4)-dependent and myeloid differentiation factor 88 (MyD88)-dependent pathway. HSP60 is released from CNS cells undergoing necrotic or apoptotic cell death and specifically binds to microglia. HSP60-induced synthesis of neurotoxic nitric oxide by microglia is dependent on TLR4. HSP60 induces extensive axonal loss and neuronal death in CNS cultures from wild-type but not TLR4 or MyD88 loss-of-function mutant mice. This is the first evidence of an endogenous molecular pathway common to many forms of neuronal injury that bidirectionally links CNS inflammation with neurodegeneration.

Bacterial programmed cell death of cerebral endothelial cells involves dual death pathways.

Major barriers separating the blood from tissue compartments in the body are composed of endothelial cells. Interaction of bacteria with such barriers defines the course of invasive infections, and meningitis has served as a model system to study endothelial cell injury. Here we report the impressive ability of Streptococcus pneumoniae, clinically one of the most important pathogens, to induce 2 morphologically distinct forms of programmed cell death (PCD) in brain-derived endothelial cells. Pneumococci and the major cytotoxins H2O2 and pneumolysin induce apoptosis-like PCD independent of TLR2 and TLR4. On the other hand, pneumococcal cell wall, a major proinflammatory component, causes caspase-driven classical apoptosis that is mediated through TLR2. These findings broaden the scope of bacterial-induced PCD, link these effects to innate immune TLRs, and provide insight into the acute and persistent phases of damage during meningitis.

Heterogeneous effects of distinct tocopherol analogues on NO release, cell volume, and cell death in microglial cells.

Tocopherols (vitamin E) are potent antioxidants as well as modulators of enzymes involved in signal transduction, like nitric oxide synthase (NOS). In primary murine microglial cells and in the microglial cell line BV-2, alpha-, gamma-, and delta-tocopherol and alpha-tocopherol acid succinate, respectively, promote nitric oxide (NO) release. The NOS inhibitors aminoguanidine and N(G)-methyl-L-arginine (L-NMMA) suppressed alpha- and gamma-tocopherol-induced NO release, but had no significant effect on delta-tocopherol- and alpha-tocopherol acid succinate-induced NO release. In BV-2 cells, but not in primary microglial cells, gamma- and delta-tocopherol and alpha-tocopherol acid succinate, respectively, led to cell death, characterized by exposition of phosphatidylserine on the cell surface, chromatin condensation, changes in cell volume, and formation of blebs on the cell surface. Aminoguanidine, L-NMMA, and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) enhanced apoptosis in gamma-tocopherol-exposed cells and suppressed apoptosis in delta-tocopherol-treated cells, but had no effect on cells supplemented with alpha-tocopherol acid succinate. The NO donors sodium nitroprusside and 2-(N,N-diethylamino)-diazenolate 2-oxide enhanced apoptosis in gamma- or delta-tocopherol-treated cells, but rescued cells from alpha-tocopherol acid succinate-induced cell death.

Bacterial hydrogen peroxide contributes to cerebral hyperemia during early stages of experimental pneumococcal meningitis.

Alterations of blood flow contribute to major clinical complications in invasive infections such as sepsis and bacterial meningitis. As a unique feature streptococci -- in particular, Streptococcus pneumoniae, the most frequent pathogen in bacterial meningitis -- release hydrogen peroxide (H(2)O(2)) because of the absence of functional catalase. In a 6 h rat model of experimental meningitis, we studied the impact of bacterial H(2)O(2) production on regional cerebral blood flow (rCBF) and intracranial pressure (ICP). Compared to wild-type D39 pneumococci, the increase of rCBF was diminished in meningitis induced by the H(2)O(2) defective SpxB(-) mutant (maximum increase, 135% +/- 17% versus 217% +/- 23% of the individual baseline; P<0.01) or after treatment of D39-induced meningitis with H(2)O(2)-degrading catalase or with tetraethylammonium (TEA), a blocker of calcium-sensitive potassium channels, which mediate H(2)O(2)-induced vasodilation. Catalase did not significantly reduce the remaining rCBF increase caused by SpxB(-), supporting the predominant role of bacterial H(2)O(2). We conclude that in addition to host-sided mediators, bacterial-derived H(2)O(2) acts as a potent vasodilator, which accounts for a certain proportion of the early cerebral hyperperfusion in pneumococcal meningitis.

Pneumococcal pneumolysin and H(2)O(2) mediate brain cell apoptosis during meningitis.

Pneumococcus is the most common and aggressive cause of bacterial meningitis and induces a novel apoptosis-inducing factor-dependent (AIF-dependent) form of brain cell apoptosis. Loss of production of two pneumococcal toxins, pneumolysin and H(2)O(2), eliminated mitochondrial damage and apoptosis. Purified pneumolysin or H(2)O(2) induced microglial and neuronal apoptosis in vitro. Both toxins induced increases of intracellular Ca(2+) and triggered the release of AIF from mitochondria. Chelating Ca(2+) effectively blocked AIF release and cell death. In experimental pneumococcal meningitis, pneumolysin colocalized with apoptotic neurons of the hippocampus, and infection with pneumococci unable to produce pneumolysin and H(2)O(2) significantly reduced damage. Two bacterial toxins, pneumolysin and, to a lesser extent, H(2)O(2), induce apoptosis by translocation of AIF, suggesting new neuroprotective strategies for pneumococcal meningitis.

Innate sensors for Gram-positive bacteria.

More than half of invasive bacterial infections are Gram-positive in origin. This class of bacteria has neither endotoxins nor an outer membrane, yet it generates some of the most powerful inflammatory responses known in medicine. Some recent seminal studies go a long way toward settling the controversies that surround the process by which Gram-positive bacterial surfaces trigger the human immune system. Although the components of the cell wall are now chemically defined in exquisite detail and the interaction with the toll-like receptor 2 pathway has been discovered, it is only very recently that definitive studies combining these advanced biochemical and cell biological tools have been carried out. It is these breakthrough studies that have finally confirmed the paradigm of innate sensors for Gram-positive bacteria.

Cellular damage in bacterial meningitis: an interplay of bacterial and host driven toxicity.

Bacterial meningitis is still an important infectious disease causing death and disability. Invasive bacterial infections of the CNS generate some of the most powerful inflammatory responses known in medicine. Although the components of bacterial cell surfaces are now chemically defined in exquisite detail and the interaction with several receptor pathways has been discovered, it is only very recently that studies combining these advanced biochemical and cell biological tools have been done. Additional to the immunological response direct bacterial toxicity has been identified as an important contributor to neuronal damage. A detailed understanding of the complex interaction of bacterial toxicity and host response may generate opportunities for innovative and specific neuroprotective therapies.

Toll-like receptor 2 mediates CNS injury in focal cerebral ischemia.

Toll-like receptors (TLR) recognize molecular structures associated with pathogens as well as host-derived components and initiate an inflammatory innate immune response. Microglia represent the resident immune host defense and are the major inflammatory cell type in the central nervous system (CNS). We show here that TLR2-deficient mice develop a decreased CNS injury compared to wild type mice in a model of focal cerebral ischemia. TLR2 mRNA is up-regulated in wild type mice during cerebral ischemia. In ischemic brains, TLR2 protein is expressed in lesion-associated microglia. Absence of TLR2 does not affect the recruitment of granulocytes to the infarct region. We conclude that TLR2 in microglia propagates stroke-induced CNS injury.

The role of lipopolysaccharide-binding protein in modulating the innate immune response.

Lipopolysaccharide-binding protein (LBP) has a well-established role in Gram-negative infection. New data suggest a more expanded role for LBP as a general recognition molecule. Several bacterial surface components from Gram-positive pathogens are also recognized by this molecule. LBP may also serve as a clinical marker in severe infections and may carry therapeutic potential.

Triptans reduce the inflammatory response in bacterial meningitis.

Severe headache and meningism provide clear evidence for the activation of trigeminal neurotransmission in meningitis. The authors assessed the antiinflammatory potential of 5HT1B/D/F receptor agonists (triptans), which inhibit the release of proinflammatory neuropeptides from perivascular nerve fibers. In a 6-hour rat model of pneumococcal meningitis, zolmitriptan and naratriptan reduced the influx of leukocytes into the cerebrospinal fluid, and attenuated the increase of regional cerebral blood flow. Elevated intracranial pressure as well as the brain water content at 6 hours was reduced by triptans. These effects were partially reversed by a specific 5HT1D as well as by a specific 5HT1B receptor antagonist. Meningitis caused a depletion of calcitonin gene-related peptide (CGRP) and substance P from meningeal nerve fibers, which was prevented by zolmitriptan and naratriptan. In line with these findings, patients with bacterial meningitis had significantly elevated CGRP levels in the cerebrospinal fluid. In a mouse model of pneumococcal meningitis, survival and clinical score at 24 hours were significantly improved by triptan treatment. The findings suggest that, besides mediating meningeal nociception, meningeal nerve fibers contribute to the inflammatory cascade in the early phase of bacterial meningitis. Adjunctive treatment with triptans may open a new therapeutic approach in the acute phase of bacterial meningitis.

Timing the valve replacement in infective endocarditis involving the brain.

Neurological complications are very frequent in patients with infective endocarditis (20-40 %). In these patients it is unclear at what time a valve replacement should be performed. In order to develop a data based recommendation we studied 12 patients of our own and analyzed 228 patients from the literature. We included patients with valve replacement after a neurological complication of endocarditis and documented the time between manifestation and operation and the outcome. Based on these 240 patients we calculated the risk of neurological deterioration after the valve replacement. After brain infarction this risk is 20% within three days, 20-50% between day 4 and 14, but declines to < 10% after 14 days and < 1% after 4 weeks. Valve replacement within the first four weeks after intracranial hemorrhage has been reported to be successful only in individual cases. The risk of deteriorating declines later to 15%. Based on these limited data we suggest that valve replacement in patients with brain infarction should be considered within the first 72 hours if they have severe heart failure, otherwise after four weeks. Only a few selected patients with intracranial hemorrhage and progressive heart failure might benefit from valve replacement within the first four weeks. For all other neurological complications there are no reliable data. We propose a structured approach depending on cardiac and neurological complications and the time course of the disease.

Brain parenchymal TNF-α and IL-1ß induction in experimental pneumococcal meningitis.

Triggers of brain inflammation in pneumococcal meningitis are unknown. TNF-α and IL-1ß were upregulated (real time PCR and in situ hybridization) in neurons and astrocytes time-dependently and maximally in the hippocampus during murine pneumococcal meningitis. Upregulation of TNF-α and IL-1ß mRNA in the brain parenchyma was independent of cerebrospinal fluid leukocytosis, pneumococcal pneumolysin and H2O2, but it was potently induced by pneumococcal cell wall (PCW) fragments. Brain TNF-α mRNA was downregulated by a matrix metalloproteinases inhibitor. PCW fragments were located in the brain parenchyma. In conclusion, PCW fragments and matrix metalloproteinases trigger cytokine induction in the brain parenchyma during pneumococcal meningitis.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in central nervous system inflammation.

In a wide variety of acute and chronic central nervous system (CNS) disorders, inflammatory processes contribute to the damage of brain cells and progression of the disease. Along with other regulatory cytokines, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is involved in the pathology of multiple sclerosis (MS) and murine experimental autoimmune encephalomyelitis (EAE), bacterial meningitis (BM), HIV encephalitis (HIVE), stroke and Alzheimer's disease (AD). In these conditions, TRAIL is released within the brain mainly by activated microglia and leukocytes infiltrating from the blood stream. TRAIL promotes apoptosis of parenchymal cells in MS/EAE, HIVE, AD and stroke through interaction with TRAIL death receptors expressed on these cells. Frequently, cells in the diseased brain display increased susceptibility to apoptosis induction by TRAIL due to upregulation of death receptors and downregulation of decoy receptors. On the other hand, TRAIL inhibits the proliferation of encephalitogenic T cells in EAE, and it is involved in the clearance of infected brain macrophages in HIVE and of activated neutrophils in BM by interaction with their death receptors. Especially in BM, the ability of TRAIL to limit an acute granulocyte-driven inflammation carries significant neuroprotective potential. Given the diversity of beneficial and harmful effects in the immune and nervous system, TRAIL is a double-edged sword in diseases involving CNS inflammation.

Pneumococcal cell wall-induced meningitis impairs adult hippocampal neurogenesis.

Bacterial meningitis is a major infectious cause of neuronal degeneration in the hippocampus. Neurogenesis, a continuous process in the adult hippocampus, could ameliorate such loss. Yet the high rate of sequelae from meningitis suggests that this repair mechanism is inefficient. Here we used a mouse model of nonreplicative bacterial meningitis to determine the impact of transient intracranial inflammation on adult neurogenesis. Experimental meningitis resulted in a net loss of neurons, diminished volume, and impaired neurogenesis in the dentate gyrus for weeks following recovery from the insult. Inducible nitric oxide synthase (iNOS) immunoreactivity was prominent in microglia in nonproliferating areas of the dentate gyrus and hilus region after meningitis induction. Treatment with the specific iNOS inhibitor N6-(1-iminoethyl)-L-lysine restored neurogenesis in experimental meningitis. These data suggest that local central nervous system inflammation in and of itself suppresses adult neurogenesis by affecting both proliferation and neuronal differentiation. Repair of cognitive dysfunction following meningitis could be improved by intervention to interrupt these actively suppressive effects.

Pneumolysin causes neuronal cell death through mitochondrial damage.

Bacterial toxins such as pneumolysin are key mediators of cytotoxicity in infections. Pneumolysin is a pore-forming toxin released by Streptococcus pneumoniae, the major cause of bacterial meningitis. We found that pneumolysin is the pneumococcal factor that accounts for the cell death pathways induced by live bacteria in primary neurons. The pore-forming activity of pneumolysin is essential for the induction of mitochondrial damage and apoptosis. Pneumolysin colocalized with mitochondrial membranes, altered the mitochondrial membrane potential, and caused the release of apoptosis-inducing factor and cell death. Pneumolysin induced neuronal apoptosis without activating caspase-1, -3, or -8. Wild-type pneumococci also induced apoptosis without activation of caspase-3, whereas pneumolysin-negative pneumococci activated caspase-3 through the release of bacterial hydrogen peroxide. Pneumolysin caused upregulation of X-chromosome-linked inhibitor of apoptosis protein and inhibited staurosporine-induced caspase activation, suggesting the presence of actively suppressive mechanisms on caspases. In conclusion, our results indicate additional functions of pneumolysin as a mitochondrial toxin and as a determinant of caspase-independent apoptosis. Considering this, blocking of pneumolysin may be a promising cytoprotective strategy in pneumococcal meningitis and other infections.

TLR2 mediates neuroinflammation and neuronal damage.

Innate immunity relies on pattern recognition receptors to detect the presence of infectious pathogens. In the case of Gram-positive bacteria, binding of bacterial lipopeptides to TLR2 is currently regarded as an important mechanism. In the present study, we used the synthetic bacterial lipopeptide Pam3CysSK4, a selective TLR2 agonist, to induce meningeal inflammation in rodents. In a 6-h rat model, intrathecal application of Pam3CysSK4 caused influx of leukocytes into the cerebrospinal fluid (CSF) and induced a marked increase of regional cerebral blood flow and intracranial pressure. In wild-type mice, we observed CSF pleocytosis and an increased number of apoptotic neurons in the dentate gyrus 24 h after intrathecal challenge. Inflammation and associated neuronal loss were absent in TLR2 knockout mice. In purified neurons, cytotoxicity of Pam3CysSK4 itself was not observed. Exposure of microglia to Pam3CysSK4 induced neurotoxic properties in the supernatant of wild-type, but not TLR2-deficient microglia. We conclude that TLR2-mediated signaling is sufficient to induce the host-dependent key features of acute bacterial meningitis. Therefore, synthetic lipopeptides are a highly specific tool to study mechanisms of TLR2-driven neurodegeneration in vivo.