Acute Graft-Versus-Host Disease, Infections, Vascular Events and Drug Toxicities Affecting the Central Nervous System.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for patients with hematological malignancies. Acute Graft versus host diseases (GVHD) is a major immune mediated side effect of allo-HCT that can affect the central nervous system (CNS) in addition to post-allo-HCT vascular events, drug toxicity or infections. Here we summarize and discuss recent preclinical data on the CNS as a target of acute GVHD and the known mechanisms contributing to neurotoxicity with a focus on microglia and T cells. We also discuss open questions in the field and place the findings made in mouse models in a clinical context. While in mice the neurological deficits can be assessed in a controlled fashion, in patients the etiology of the CNS damage is difficult to attribute to acute GVHD versus infections, vascular events, and drug-induced toxicity. Ultimately, we discuss novel therapies for GVHD of the CNS. Our understanding of the biological mechanisms that lead to neurotoxicity after allo-HCT increased over the last decade. This review provides insights into CNS manifestations of GVHD versus other etiologies of CNS damage in mice and patients.

Cerebrovascular events in patients with isolated anti-phosphatidyl-serine/prothrombin antibodies.

The interest of extra-criteria antiphospholipid antibodies is growing, especially in patients negative for conventional antibodies. In this study we aimed to assess the clinical utility of anti-phosphatidyl-serine/prothrombin antibodies (aPS/PT) testing in patients negative for Beta2-Glycoprotein 1(ß2GPI)-dependent tests, for identifying antiphospholipid syndrome (APS) patients that developed cerebrovascular events (CVE). When screening APS patients attending our center, out of 119 aPS/PT IgG/IgM-positive patients, thus patients negative for aß2GPI and aCL, 42 patients (35%) tested negative for ß2GPI-dependent tests and were tested with thrombin generation assay (TGA). Ten patients (24%), with isolated aPS/PT IgG/IgM, had a history of CVE. Lupus anticoagulant (LA)-positive test was more frequently observed in patients with CVE (8/22 vs. 2/20; p = 0.045). Out of the 10 patients who experienced CVE, 3 patients were aPS/PT IgG positive (all LA positive), and 8 patients were aPS/PT IgM positive (6/8 LA positive). One patient was positive for both aPS/PT IgG and IgM. LA-positive patients had only high titers of aPS/PT IgG/IgM, all of them being ≥ 80 U/ml, while the 2 LA-negative patients were aPS/PT IgM positive with medium titers [40-60 U/ml]. LA-positive patients had significantly altered TGA profile when compared to those who were LA negative, considering all TGA parameters. LA-positive patients had significantly higher tLag (8.4 ± 3.3 min vs. 6.6 ± 1.8 min; p = 0.046), higher tPeak (14 ± 4.3 min vs. 11 ± 2.7 min; p = 0.015) and lower Peak (207 ± 152 nM vs. 356.3 ± 104.7 nM; p < 0.001) and lower AUC (2109.7 ± 1006.9 nM vs. 2772.5 ± 776.8 nM; p = 0.033). The use of aPS/PT might be of help in identifying patients with CVE and APS, as also confirmed by TGA testing.

Toll-Like Receptor Signaling Pathways: Novel Therapeutic Targets for Cerebrovascular Disorders.

Toll-like receptors (TLRs), a class of pattern recognition proteins, play an integral role in the modulation of systemic inflammatory responses. Cerebrovascular diseases (CVDs) are a group of pathological conditions that temporarily or permanently affect the brain tissue mostly via the decrease of oxygen and glucose supply. TLRs have a critical role in the activation of inflammatory cascades following hypoxic-ischemic events and subsequently contribute to neuroprotective or detrimental effects of CVD-induced neuroinflammation. The TLR signaling pathway and downstream cascades trigger immune responses via the production and release of various inflammatory mediators. The present review describes the modulatory role of the TLR signaling pathway in the inflammatory responses developed following various CVDs and discusses the potential benefits of the modulation of different TLRs in the improvement of functional outcomes after brain ischemia.

Location of recurrent cardiovascular events and anticardiolipin antibodies.

BACKGROUND: The relationship between anticardiolipin (aCL) antibodies and cardiovascular events is uncertain and may vary according to arterial location. MATERIALS AND METHODS: FRENA is an ongoing registry of stable outpatients with symptomatic coronary artery disease (CAD), cerebrovascular disease (CVD) or peripheral artery disease (PAD). The rate of subsequent ischaemic events was cross-referenced with the presence of aCL antibodies (any isotype, IgG or IgM). RESULTS: As of June 2017, 1387 stable outpatients were recruited. Of these, 120 (8.7%) showed positive levels of aCL antibodies. Over an average follow-up of 18 months, 250 patients developed subsequent events: 101 myocardial infarction, 57 ischaemic stroke and 92 critical leg events. Patients with positive aCL antibodies had a higher risk of distal artery events (a composite of ischaemic stroke or critical leg events) than patients with undetectable or low levels (rate ratio: 1.66; 95% CI: 1.07-2.60). However, an association with central coronary events was not found. The multivariate Cox analysis after adjustment for relevant clinical covariates showed that positivity of aCL antibodies is an independent risk factor for distal events (hazard ratio: 1.60; 95% CI: 1.01-2.55; P < .05). CONCLUSIONS: Positivity of aCL antibodies is associated with an increased risk of subsequent distal artery ischaemic events (cerebral or leg arteries) but not coronary artery events. Anticardiolipin antibodies appear to have a different relationship on the localisation of ischaemic events in patients with symptomatic artery disease.

Cytokines as therapeutic targets for cardio- and cerebrovascular diseases.

Despite major advances in prevention and treatment, cardiac and cerebral atherothrombotic complications still account for substantial morbidity and mortality worldwide. In this context, inflammation is involved in the chronic process leading atherosclerotic plaque formation and its complications, as well as in the maladaptive response to acute ischemic events. For this reason, modulation of inflammation is nowadays seen as a promising therapeutic strategy to counteract the burden of cardio- and cerebrovascular disease. Being produced and recognized by both inflammatory and vascular cells, the complex network of cytokines holds key functions in the crosstalk of these two systems and orchestrates the progression of atherothrombosis. By binding to membrane receptors, these soluble mediators trigger specific intracellular signaling pathways eventually leading to the activation of transcription factors and a deep modulation of cell function. Both stimulatory and inhibitory cytokines have been described and progressively reported as markers of disease or interesting therapeutic targets in the cardiovascular field. Nevertheless, cytokine inhibition is burdened by harmful side effects that will most likely prevent its chronic use in favor of acute administrations in well-selected subjects at high risk. Here, we summarize the current state of knowledge regarding the modulatory role of cytokines on atherosclerosis, myocardial infarction, and stroke. Then, we discuss evidence from clinical trials specifically targeting cytokines and the potential implication of these advances into daily clinical practice.

Hyperglycemic Condition Causes Pro-Inflammatory and Permeability Alterations Associated with Monocyte Recruitment and Deregulated NFκB/PPARγ Pathways on Cerebral Endothelial Cells: Evidence for Polyphenols Uptake and Protective Effect.

Hyperglycemia alters the function of cerebral endothelial cells from the blood-brain barrier, increasing the risk of cerebrovascular complications during diabetes. This study evaluated the protective effect of polyphenols on inflammatory and permeability markers on bEnd3 cerebral endothelial cells exposed to high glucose concentration. Results show that hyperglycemic condition increased nuclear factor kappa B (NFκB) activity, deregulated the expression of interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-10 (IL-10) and endothelial-leukocyte adhesion molecule (E-selectin) genes, raised MCP-1 secretion and elevated monocyte adhesion and transendothelial migration. High glucose decreased occludin, claudin-5, zona occludens-1 (ZO-1) and zona occludens-2 (ZO-2) tight junctions production and altered the endothelial permeability. Characterized polyphenolic extracts from the French medicinal plants Antirhea borbonica, Ayapana triplinervis, Dodonaea viscosa and Terminalia bentzoe, and their major polyphenols quercetin, caffeic, chlorogenic and gallic acids limited the pro-inflammatory and permeability alterations caused by high glucose. Peroxisome proliferator-activated receptor gamma (PPARγ) agonist also attenuated these damages while PPARγ antagonist aggravated them, suggesting PPARγ protective action. Interestingly, polyphenols improved PPARγ gene expression lowered by high glucose. Moreover, polyphenols were detected at the intracellular level or membrane-bound to cells, with evidence for breast cancer resistance protein (BCRP) efflux transporter role. Altogether, these findings emphasize the ability of polyphenols to protect cerebral endothelial cells in hyperglycemic condition and their relevance for pharmacological strategies aiming to limit cerebrovascular disorders in diabetes.

Libman-Sacks endocarditis and associated cerebrovascular disease: The role of medical therapy.

BACKGROUND: Libman-Sacks endocarditis in patients with systemic lupus erythematosus (SLE) is commonly complicated with embolic cerebrovascular disease (CVD) or valve dysfunction for which high-risk valve surgery is frequently performed. However, the role of medical therapy alone for Libman-Sacks endocarditis and associated acute CVD remains undefined. OBJECTIVE: To determine in this cross-sectional and longitudinal study if conventional anti-inflammatory and anti-thrombotic therapy may be an effective therapy in SLE patients with Libman-Sacks endocarditis and associated acute CVD. METHODS AND MATERIALS: 17 SLE patients with Libman-Sacks endocarditis detected by two-and-three-dimensional transesophageal echocardiography (TEE) and complicated with acute CVD [stroke/TIA, focal brain injury on MRI, or cognitive dysfunction] were treated with conventional anti-inflammatory and anti-thrombotic therapy for a median of 6 months and then underwent repeat TEE, transcranial Doppler, brain MRI, and neurocognitive testing for re-assessment of Libman-Sacks endocarditis and CVD. RESULTS: Valve vegetations decreased in number, diameter, and area (all p ≤0.01); associated valve regurgitation significantly improved (p = 0.04), and valve thickening did not progress (p = 0.56). In 13 (76%) patients, valve vegetations or valve regurgitation resolved or improved in number and size or by ≥1 degree, respectively, as compared to 4 (24%) patients in whom vegetations or valve regurgitation persisted unchanged or increased in size or by ≥1 degree (p = 0.03). Also, cerebromicroembolism, lobar and global gray and white matter cerebral perfusion, ischemic brain lesion load, and neurocognitive dysfunction resolved or significantly improved (all p ≤0.04). CONCLUSION: These preliminary data suggest that combined conventional anti-inflammatory and antithrombotic therapy may be an effective treatment for Libman-Sacks endocarditis and its associated CVD and may obviate the need for high-risk valve surgery.

The potential for immune checkpoint modulators in cerebrovascular injury and inflammation.

Introduction: Neuroinflammation has been linked to poor neurologic and functional outcomes in many cerebrovascular disorders. Immune checkpoints are upregulated in the setting of traumatic brain injury, intracerebral hemorrhage, ischemic stroke, central nervous systems vasculitis, and post-hemorrhagic vasospasm, and are potential mediators of pathologic inflammation. Burgeoning evidence suggests that immune checkpoint modulation is a promising treatment strategy to decrease immune cell recruitment, cytokine secretion, brain edema, and neurodegeneration.Areas covered: This review discusses the role of immune checkpoints in neuroinflammation, and the potential for therapeutic immune checkpoint modulation in inflammatory cerebrovascular disorders. A search of Pubmed and clinicaltrials.gov was performed to find relevant literature published within the last 50 years.Expert opinion: The clinical success of immune-activating checkpoint modulators in human cancers has shown the immense clinical potential of checkpoint-based immunotherapy. Given that checkpoint blockade can also precipitate a pathologic pro-inflammatory or autoimmune response, it is plausible that these pathways may also be targeted to quell aberrant inflammation. A limited but growing number of studies suggest that immune checkpoints play a critical role in regulating the immune response in the central nervous system in a variety of contexts, and that immune-deactivating checkpoint modulators may be a promising treatment strategy for acute and chronic neuroinflammation in cerebrovascular disorders.

Hypoperfusion is a potential inducer of immunosuppressive network in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease which causes a non-reversible cognitive impairment and dementia. The primary cause of late-onset AD remains unknown although its pathology was discovered over a century ago. Recently, the vascular hypothesis of AD has received backing from evidence emerging from neuroimaging studies which have revealed the presence of a significant hypoperfusion in the brain regions vulnerable to AD pathology. In fact, hypoxia can explain many of the pathological changes evident in AD pathology, e.g. the deposition of ß-amyloid plaques and chronic low-grade inflammation. Hypoxia-inducible factor-1α (HIF-1α) stimulates inflammatory responses and modulates both innate and adaptive immunity. It is known that hypoxia-induced inflammation evokes compensatory anti-inflammatory response involving tissue-resident microglia/macrophages and infiltrated immune cells. Hypoxia/HIF-1α induce immunosuppression by (i) increasing the expression of immunosuppressive genes, (ii) stimulating adenosinergic signaling, (iii) enhancing aerobic glycolysis, i.e. lactate production, and (iv) augmenting the secretion of immunosuppressive exosomes. Interestingly, it seems that these common mechanisms are also involved in the pathogenesis of AD. In AD pathology, an enhanced immunosuppression appears, e.g. as a shift in microglia/macrophage phenotypes towards the anti-inflammatory M2 phenotype and an increase in the numbers of regulatory T cells (Treg). The augmented anti-inflammatory capacity promotes the resolution of acute inflammation but persistent inflammation has crucial effects not only on immune cells but also harmful responses to the homeostasis of AD brain. I will examine in detail the mechanisms of the hypoperfusion/hypoxia-induced immunosuppressive state in general and especially, in its association with AD pathogenesis. These immunological observations support the vascular hypothesis of AD pathology.

Modeling blood-brain barrier pathology in cerebrovascular disease in vitro: current and future paradigms.

The complexity of the blood-brain barrier (BBB) and neurovascular unit (NVU) was and still is a challenge to bridge. A highly selective, restrictive and dynamic barrier, formed at the interface of blood and brain, the BBB is a "gatekeeper" and guardian of brain homeostasis and it also acts as a "sensor" of pathological events in blood and brain. The majority of brain and cerebrovascular pathologies are associated with BBB dysfunction, where changes at the BBB can lead to or support disease development. Thus, an ultimate goal of BBB research is to develop competent and highly translational models to understand mechanisms of BBB/NVU pathology and enable discovery and development of therapeutic strategies to improve vascular health and for the efficient delivery of drugs. This review article focuses on the progress being made to model BBB injury in cerebrovascular diseases in vitro.

Implicating endothelial cell senescence to dysfunction in the ageing and diseased brain.

Cerebrovascular endothelial cells (CECs) are integral components of both the blood-brain barrier (BBB) and the neurovascular unit (NVU). As the primary cell type of the BBB, CECs are responsible for the tight regulation of molecular transport between the brain parenchyma and the periphery. Additionally, CECs are essential in neurovascular coupling where they help regulate cerebral blood flow in response to regional increases in cellular demand in the NVU. CEC dysfunction occurs during both normative ageing and in cerebrovascular disease, which leads to increased BBB permeability and neurovascular uncoupling. This MiniReview compiles what is known about the molecular changes underlying CEC dysfunction, many of which are reminiscent of cells that have become senescent. In general, cellular senescence is defined as an irreversible growth arrest characterized by the acquisition of a pro-inflammatory secretory phenotype in response to DNA damage or other cellular stresses. We discuss evidence for endothelial cell senescence in ageing and cardiovascular disease, and how CEC senescence may contribute to age-related cerebrovascular dysfunction.

[Cerebrovascular events, headache and livedoracemosa - diagnosis at a glance?] / Zerebrovaskuläre Ereignisse, Kopfschmerzen und Livedo racemosa ­ Blickdiagnose?

Sneddon's syndrome is a rare disease characterized by cerebrovascular events and livedo racemosa. There are often autoimmunological comorbidities, especially antiphospholipid antibody syndrome. The underlying pathophysiology is still not fully clarified. A causal therapy does not exist. The reported case shows a patient with a thrombophilic form of Sneddon's syndrome with the main symptoms of headache and thromboembolic events. Symptoms, laboratory parameters, histology and differential diagnoses are explained.

Adjuvanted versus nonadjuvanted influenza vaccines and risk of hospitalizations for pneumonia and cerebro/cardiovascular events in the elderly.

Background: The higher effectiveness of MF59®-adjuvanted trivalent influenza vaccine (MF59-TIV) vs. nonadjuvanted TIV in preventing influenza-related hospitalizations was found considering few influenza seasons, local and heterogeneous settings. This study evaluated the relative vaccine effectiveness (rVE) of MF59-TIV vs. nonadjuvanted TIV on the risk of hospitalization for pneumonia and cerebro/cardiovascular events across 15 consecutive influenza seasons. Research design and methods: Using Health Search Database, a case-control study was nested in a cohort of elderly vaccinated with MF59-TIV or TIV. Conditional logistic regression was used to estimate the odds ratio with 95% confidence intervals (CI) of hospitalizations potentially related to influenza in patients vaccinated with MF59-TIV or TIV. Results: Of 43,000 patients vaccinated with MF59-TIV (66.2%) and TIV (33.8%) for the first time, 103 cases of hospitalization for pneumonia or cerebro/cardiovascular events (0.11 per 1,000 person-weeks) during 15 influenza seasons were identified. The MF59-TIV was associated with a reduced risk of hospitalizations for pneumonia and cerebro/cardiovascular events vs. TIV [rVE: 39% (95% CI: 4-61%)]. Conclusions: In a 15-season cohort of elderly, MF59-TIV seems to reduce the risk of hospitalizations for pneumonia and cerebro/cardiovascular events when compared with nonadjuvanted TIV. Our findings support the recommendation for MF59-TIV in the elderly population.

[Oxidative stress and inflammation as links in a chain in patients with chronic cerebrovascular diseases]. / Okislitel'nyi stress i vospalenie kak zven'ia odnoi tsepi u bol'nykh s khronicheskimi tserebrovaskuliarnymi zabolevaniiami.

Cerebrovascular diseases (CVD) are the main cause of death and permanent disability. The urgency of the problem of chronic CVD is associated with an increase of the absolute number of elderly and senile age in the population, a trend towards slowly increasing, sluggish pathological processes. It is obvious that any somatic disease in such patients is comorbid to cerebrovascular diseases that suggests a unified mechanism of the pathogenesis for both the main and concomitant diseases. The article notes that microangiopathy is the most common cause of CVD. The main etiopathogenetic factor affecting cerebral vessels of small caliber is endothelial dysfunction, systemic inflammation and oxidative stress. Understanding the molecular components that underlie functional abnormalities and damage of small blood vessels gives the key to the modern strategies in therapy, forming the foundation for an adequate pathogenetically justified therapy. This impact should be gradual, complex and aimed at correcting pathochemical disorders in general and neurotransmitter imbalance in particular. The drug dipyridamole, which has pleiotropic effects, can be considered as one of the pathogenetically justified means in complex drug therapy in patients with CVD.

Autologous CD34+ Cell Therapy for Ischemic Tissue Repair.

In 1997, the seminal manuscript by Asahara, Murohara, Isner et al outlined the evidence for the existence of circulating, bone marrow-derived cells capable of stimulating and contributing to the formation of new blood vessels. Consistent with the paradigm shift that this work represented, it triggered much scientific debate and controversy, some of which persists 2 decades later. In contrast, the clinical application of autologous CD34 cell therapy has been marked by a track record of consistent safety and clinical benefit in multiple ischemic conditions. In this review, we summarize the preclinical and clinical evidence from over 700 patients in clinical trials of CD34 cell therapy.

Cerebrovascular events after herpes zoster infection: a risk that should be not underestimated.

The occurrence of a cerebrovascular event after a herpes zoster (HZ) infection represents a nightmare in clinical practice, especially in those patients with concomitant cardiovascular comorbidities/risk factors and disease related per se to a higher risk of zoster infection. Moreover, the absence of a consensus opinion regarding a specific and adequate prevention of cerebrovascular events in these patients further complicates the treatment. Accumulating evidences demonstrated that HZ and HZ ophtalmicus (HZO) increase the risk of cerebrovascular events in the short-and long-term periods. Moreover, patient's ages < 40 years old, despite having fewer traditional cardiovascular comorbidities, demonstrated a higher risk of cerebrovascular events after both HZ and HZO infection. Further prospective studies are needed to analyse the role of antiviral treatments and vaccination in these subjects to clarify if they could be able to reduce the risk of stroke after a zoster infection. In the meanwhile, physicians must be aware of a higher risk of cerebrovascular events, especially in younger patients, with few cardiovascular risk factors, after an HZ infection.

Toll-like receptors in the pathogenesis of neuroinflammation.

Toll-like receptors (TLRs) are discovered as crucial pattern recognition receptors (PRRs) involved in the recognition of pathogen-associated molecular patterns (PAMPs). Later studies showed their involvement in the recognition of various damage/danger-associated molecular patterns (DAMPs) generated by host itself. Thus, TLRs are capable of recognizing wide-array of patterns/molecules derived from pathogens and host as well and initiating a proinflammatory immune response through the activation of NF-κB and other transcription factors causing synthesis of proinflammatory molecules. The process of neuroinflammation is seen under both sterile and infectious inflammatory diseases of the central nervous system (CNS) and may lead to the development of neurodegeneration. The present article is designed to highlight the importance of TLRs in the pathogenesis of neuroinflammation under diverse conditions. TLRs are expressed by various immune cells present in CNS along with neurons. However out of thirteen TLRs described in mammals, some are present and active in these cells, while some are absent and are described in detail in main text. The role of various immune cells present in the brain and their role in the pathogenesis of neuroinflammation depending on the type of TLR expressed is described. Thereafter the role of TLRs in bacterial meningitis, viral encephalitis, stroke, Alzheimer's disease (AD), Parkinson's disease (PD), and autoimmune disease including multiple sclerosis (MS) is described. The article is designed for both neuroscientists needing information regarding TLRs in neuroinflammation and TLR biologists or immunologists interested in neuroinflammation.

Cerebrovascular inflammation: A critical trigger for neurovascular injury?

The cerebrovascular system is not only inert bystandard that support the metabolic demands of the brain but also elicit the barrier functions against risk factors mediated neurovascular injury. The onsets of cerebrovascular inflammation are considered as stimuli that can provoke the host defense system and trigger the development of neurological disorders. Homeostasis of the brain function is regulated by the movement of endothelial, glial, and neuronal cells within the neurovascular unit (NVU), which acts as a "platform" for the coordinated action of anti- and pro-inflammatory mechanisms. The cerebrovascular system plays an integral role in the inflammatory response by either producing or expressing a variety of cytokines, adhesion molecules, metalloproteinases, and serine proteases. Excessive inflammatory cytokine production can further be affecting the blood-brain barrier (BBB) integrity and lead to brain tissue damage. In this review, we summarize the more recent evidence highlighting the importance of cerebrovascular injury in terms of risk prediction, and the mechanisms mediating the upregulation of inflammatory mediators in cerebrovascular dysfunction and neurodegeneration.

Effect of Interventional Therapy on IL-1ß, IL-6, and Neutrophil-Lymphocyte Ratio (NLR) Levels and Outcomes in Patients with Ischemic Cerebrovascular Disease.

BACKGROUND This study investigated the clinical effect of interventional therapy in ischemic cerebrovascular disease (ICD). MATERIAL AND METHODS A retrospective analysis was performed on 260 ICD patients who were divided into a control group (122 patients, conventional drug treatment) and an observation group (138 patients, interventional therapy plus conventional drug treatment). Enzyme-linked immunosorbent assay was used to examine the expression of IL-1ß, IL-6, and NLR. Furthermore, neurological deficit scores and Barthel index scores as well as the correlation of IL-1ß, IL-6 and NLR were examined in these 2 groups. RESULTS The expression of IL-1ß, IL-6, and NLR significantly decreased in both groups after 1 week or 4 weeks of treatment compared with before treatment (P<0.05). Significant differences in neurological impairment scores were detected between these 2 groups after 4 weeks of treatment (P<0.05), and the control group showed higher neurological deficit scores than did the observation group (P<0.05). Barthel index scores were significantly higher after treatment than before treatment in the control and observation group (P<0.05), and the control group had lower Barthel index scores than did the observation group (P<0.05). Pearson correlation analysis showed that IL-1ß, IL-6, and NLR expression were positively correlated in ICD patients (P<0.05). CONCLUSIONS Interventional surgery combined with conventional drug therapy can reduce serum IL-1ß and IL-6 levels, decrease neurological impairment, and improve the quality of life of patients. The combined treatment group showed better outcomes than did the group that received the drug alone; therefore, combined therapy is suitable for promoting better clinical outcomes.