Method for detecting inflammasome proteins as biomarkers of neurological disorders

Abstract

To provide methods for detecting inflammasome proteins as biomarkers of neurological disorders.SOLUTION: The present invention provides compositions and methods for detecting components of the inflammasome in a sample from a subject as markers for brain injuries such as multiple sclerosis, stroke or traumatic brain injury. Methods of using such inflammasome markers to determine prognosis, direct treatment and monitor response to treatment for the subject with a brain injury such as multiple sclerosis, stroke, mild cognitive impairment or traumatic brain injury are also described. Presented herein are inflammasome components useful as biomarkers with high sensitivity and specificity for various neurological or psychiatric conditions and methods of their use.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 696,549, filed July 11, 2018, and U.S. Provisional Application No. 62 / 560,963, filed September 20, 2017, each of which is incorporated by reference in its entirety for all purposes.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with U.S. government support under Grant Nos. 5R42NS086274-03 and NS086274 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention.

[0003] Field The present invention relates generally to the fields of immunology and medicine. More particularly, the present invention relates to compositions and methods for detecting ASC (apoptosis-associated speck-like protein containing a caspase-activation recruitment domain (CARD)) activity, caspase-1, IL-18, IL-1β, NOD-like receptors (NLRs) and Absent in Melanoma 2 (AIM2)-like receptors (ALRs) and other inflammasome proteins in samples obtained from mammals as biomarkers for neurological disorders such as multiple sclerosis (MS), stroke, mild cognitive impairment (MCI) or traumatic brain injury (TBI).

[0004] Sequence Listing Statement The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference. The text file containing the sequence listing is named UNMI_014_00WO_SeqList_ST25.txt. The text file is approximately 1.1 KB, was created on September 20, 2018, and has been submitted electronically via EFS-Web. [Background technology]

[0005] background Multiple sclerosis (MS) is a progressive autoimmune disorder affecting the central nervous system (CNS). Pathologically, it is characterized by demyelination and the presence of inflammatory lesions in the spinal cord and brain (Compston A. The pathogenesis and basis for treatment in multiple sclerosis. Clin Neurol Neurosurg. 2004;106:246-8). Clinically, patients with MS experience blurred vision, muscle weakness, fatigue, dizziness, and balance and gating problems (Compston A. The pathogenesis and basis for treatment in multiple sclerosis. Clin Neurol Neurosurg. 2004;106:246-8). There are 400,000 patients with MS in the United States alone, and approximately 2 million patients worldwide (Compston A. The pathogenesis and basis for treatment in multiple sclerosis. Clin Neurol Neurosurg. 2004;106:246-8).

[0006] Since the 1960s, immunoglobulin (Ig) G oligoclonal bands (OCBs) have been used as a classic biomarker in the diagnosis of MS (Stangel M, Fredrikson S, Meinl E, Petzold A, Stuve O and Tumani H. The utility of cerebrospinal fluid analysis in patients with multiple sclerosis. Nat Rev Neurol. 2013;9:267-76). However, the specificity of IgG-OCB is only 61%, and as a result, other diagnostic criteria are required to clinically determine the diagnosis of MS (Teunissen CE, Malekzadeh A, Leurs C, Bridel C and Killestein J. Body fluid biomarkers for multiple sclerosis--the long road to clinical application. Nat Rev Neurol. 2015;11:585-96). Currently, CSF-restricted IgG-OCB is a good predictor of conversion from CIS to CDMS, independent of MRI (Tintore M, Rovira A, Rio J, Tur C, Pelayo R, Nos C, Tellez N, Perkal H, Comabella M, Sastre-Garriga J and Montalban X. Do oligoclonal bands add information to MRI in first attacks of multiple sclerosis? Neurology. 2008;70:1079-83).Similar results have been obtained when analyzing IgM-OCB (Villar LM, Masjuan J, Gonzalez-Porque P, Plaza J, Sadaba MC, Roldan E, Bootello A and Alvarez-Cermeno JC. Intrathecal IgM synthesis predicts the onset of new relapses and a worse disease course in MS. Neurology. 2002;59:555-9). An important area of ​​research in the field of MS is the identification of suitable biomarkers for predicting who is at risk of developing MS, biomarkers of disease progression or worsening, and biomarkers of treatment response and prognosis.

[0007] Each year, 17.5 million deaths are related to cardiovascular disease, of which 6.7 million occur as a result of stroke (Mendis S, Davis S and Norrving B. Organizational update: the world health organization global status report on noncommunicable diseases 2014; one more landmark step in the combat against stroke and vascular disease. Stroke. 2015;46:e121-2). Despite several large-scale studies of stroke biomarkers, there is currently no gold standard biomarker used in the care of stroke patients. There remains a need for biomarkers that provide high sensitivity and specificity for stroke. The Centers for Disease Control (CDC) defines traumatic brain injury (TBI) as "a disruption of the brain's normal function that can be caused by a bump, blow, or impact to the head, or a penetrating head injury." As of 2010, the CDC recorded 823.7 TBI-related emergency department visits, hospitalizations, and deaths per 100,000 individuals in the United States (Centers for Disease Control, "Traumatic Brain Injury and Concussion" website, https: / / www.cdc.gov / traumaticbraininjury / index.html, as of June 21, 2018). Key areas of research in the field of TBI include identifying appropriate biomarkers for risk of developing a TBI, disease diagnosis, progression, or worsening, and treatment response and prognosis. Previous research on inflammasomes has demonstrated that inflammasome proteins can be used as biomarkers following traumatic brain injury. Inflammasomes are multiprotein complexes of the innate immune response that are involved in the activation of caspase-1 and the processing of the proinflammatory cytokines IL-1beta and IL-18. Inflammasomes contribute to the inflammatory response, particularly after injury to the brain and spinal cord. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Compston A. The pathogenesis and basis for treatment in multiple sclerosis. Clin Neurol Neurosurg. 2004;106:246-8 [Non-patent document 2] Stangel M, Fredrikson S, Meinl E, Petzold A, Stuve O and Tumani H. The utility of cerebrospinal fluid analysis in patients with multiple sclerosis. Nat Rev Neurol. 2013;9:267-76

Table 3

Fashion 4

Wood 5

Wood 6

[0009] Much of the interest has arisen regarding the topic of the boundary or transition state between normal aging and dementia or Alzheimer's disease (AD).This state has received several descriptors, including mild cognitive impairment (MCI), early dementia and dissociated memory dysfunction.Subjects with mild cognitive impairment (MCI) have memory dysfunction that exceeds what is expected for age and education, but are not yet dementia.These subjects have been the focus of many predictive studies and early intervention trials.However, the diagnostic criteria for MCI have generally not been elucidated, and there is no biomarker.

[0010] Thus, presented herein are inflammasome components useful as biomarkers with high sensitivity and specificity for various neurological or psychiatric conditions and methods of their use to address the above-identified needs. [Means for solving the problem]

[0011] overview In one aspect, the present disclosure provides a method for evaluating a patient suspected of having multiple sclerosis (MS), comprising: measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with MS, wherein the protein signature comprises an elevated level of the at least one inflammasome protein; and selecting the patient as having MS if the patient exhibits the presence of the protein signature. In some cases, the patient exhibits clinical symptoms consistent with MS. In some cases, the MS is relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS), or progressive relapsing MS (PRMS). In some cases, the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). In some cases, the level of the at least one inflammasome protein in the protein signature is measured by immunoassay using one or more antibodies against the at least one inflammasome protein in the protein signature.In some cases, the at least one inflammasome protein is interleukin-18 (IL-18), IL-1 beta, apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof.In some cases, the at least one inflammasome protein includes each of caspase-1, IL-18, IL-1 beta, and ASC.In some cases, the at least one inflammasome protein includes ASC.In some cases, the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD), or a portion of the PYD or CARD domain of the ASC protein.In some cases, the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control. In some cases, the biological sample obtained from the control is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). In some cases, the control is a healthy individual, and the healthy individual is an individual who does not exhibit clinical symptoms consistent with MS. In some cases, the at least one inflammasome protein includes ASC, and the level of ASC is at least 50% higher than the level of ASC in the biological sample obtained from the control. In some cases, the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values. In some cases, the biological sample obtained from the patient is serum, and the patient is selected as having MS with at least 80%, 85%, 90%, 95%, 99%, or 100% sensitivity and at least 90% specificity. In some cases, the biological sample is serum, and the patient is selected as having MS with at least 80%, 85%, 90%, 95%, 99%, or 100% specificity. In some cases, the biological sample is serum, and the patient is selected as having MS with at least 90% sensitivity and at least 80% specificity. In some cases, the at least one inflammasome protein comprises ASC. In some cases, a cutoff value for determining the sensitivity, specificity, or both is selected from Table 7. In some cases, the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval.

[0012] In another aspect, the present invention provides a method for evaluating a patient suspected of having a stroke, comprising: measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with stroke or stroke-related injury, wherein the protein signature comprises an elevated level of the at least one inflammasome protein; and selecting the patient as having a stroke if the patient exhibits the presence of the protein signature. In some cases, the patient exhibits clinical symptoms consistent with stroke, and the stroke is ischemic stroke, transient ischemic stroke, or hemorrhagic stroke. In some cases, the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). In some cases, the level of the at least one inflammasome protein in the protein signature is measured by immunoassay using one or more antibodies against the at least one inflammasome protein in the protein signature.In some cases, the at least one inflammasome protein is interleukin-18 (IL-18), IL-1 beta, apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof.In some cases, the at least one inflammasome protein includes each of caspase-1, IL-18, IL-1 beta, and ASC.In some cases, the at least one inflammasome protein includes ASC.In some cases, the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD), or a portion of the PYD or CARD domain of the ASC protein. In some cases, the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control.In some cases, the biological sample obtained from the subject is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). In some cases, the subject is a healthy individual, and the healthy individual is an individual who does not exhibit clinical symptoms consistent with MS. In some cases, the at least one inflammasome protein comprises ASC, and the level of ASC in a serum sample obtained from the subject is at least 70% higher than the level of ASC in a serum sample obtained from the subject. In some cases, the at least one inflammasome protein comprises ASC, and the level of ASC in a serum-derived EV sample obtained from the subject is at least 110% higher than the level of ASC in a serum-derived EV sample obtained from the control. In some cases, the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values. In some cases, the biological sample obtained from the patient is serum, and the patient is selected as having a stroke with at least 80%, 85%, 90%, 95%, 99%, or 100% sensitivity and at least 90% specificity. In some cases, the biological sample is serum, and the patient is selected as having a stroke with at least 80%, 85%, 90%, 95%, 99%, or 100% specificity. In some cases, the biological sample is serum, and the patient is selected as having a stroke with at least 100% sensitivity and at least 95% specificity. In some cases, the at least one inflammasome protein comprises ASC. In some cases, the cutoff value for determining the sensitivity, specificity, or both is selected from Table 8. In some cases, the biological sample obtained from the patient is serum-derived EVs, and the patient is selected as having a stroke with at least 80%, 85%, 90%, 95%, 99%, or 100% sensitivity and at least 90% specificity. In some cases, the biological sample is serum-derived EVs, and the patient is selected as having a stroke with at least 80%, 85%, 90%, 95%, 99%, or 100% specificity.In some cases, the biological sample is serum-derived EVs, and the patient is selected as having a stroke with at least 100% sensitivity and at least 100% specificity. In some cases, the at least one inflammasome protein includes ASC. In some cases, a cutoff value for determining the sensitivity, specificity, or both is selected from Table 9. In some cases, the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval.

[0013] In yet another aspect, provided herein is a method of treating a patient diagnosed with multiple sclerosis (MS), comprising administering to the patient a standard of care treatment for MS, wherein the diagnosis of MS is made by detecting elevated levels of at least one inflammasome protein in a biological sample obtained from the patient. In some cases, the MS is relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS), or progressive relapsing MS (PRMS). In some cases, the standard of care treatment is selected from a treatment aimed at modifying disease outcome, managing relapses, managing symptoms, or any combination thereof. In some cases, the treatment aimed at modifying disease outcome is selected from beta-interferon, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, mitoxantrone, ocrelizumab, alemtuzumab, daclizumab, and natalizumab.

[0014] In yet another aspect, provided herein is a method of treating a patient diagnosed with stroke or stroke-related injury, comprising administering to the patient a standard of care treatment for stroke or stroke-related injury, wherein the diagnosis of stroke or stroke-related injury is made by detecting an elevated level of at least one inflammasome protein in a biological sample obtained from the patient. In some cases, the stroke is an ischemic stroke, a transient ischemic stroke, or a hemorrhagic stroke. In some cases, the stroke is an ischemic stroke or a transient ischemic stroke, and the standard of care treatment is selected from tissue plasminogen activator (tPA), an antiplatelet agent, an anticoagulant, carotid angioplasty, carotid endarterectomy, intra-arterial thrombolysis, and mechanical clot removal in cerebral ischemia (MERCI), or a combination thereof. In some cases, the stroke is a hemorrhagic stroke, and the standard of care treatment is aneurysm clipping, coil embolization, or arteriovenous malformation (AVM) repair. In some cases, the increase in the level of the at least one inflammasome protein is measured by an immunoassay using one or more antibodies against the at least one inflammasome protein. In some cases, the level of the at least one inflammasome protein is increased relative to the level of the at least one inflammasome protein in a control sample. In some cases, the level of the at least one inflammasome protein is increased relative to a predetermined reference value or range of reference values. In some cases, the at least one inflammasome protein is interleukin-18 (IL-18), apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof. In some cases, the at least one inflammasome protein is caspase-1, IL-18, and ASC. In some cases, the at least one inflammasome protein is ASC.In some cases, the antibody binds to a PYRIN-PAAD-DAPIN domain (PYD), a C-terminal caspase recruitment domain (CARD), or a portion of the PYD or CARD domain of the ASC protein. In some cases, the biological sample is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs).

[0015] In yet another aspect, the present invention provides a method for evaluating a patient suspected of having traumatic brain injury (TBI), comprising: measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with TBI, wherein the protein signature comprises an elevated level of the at least one inflammasome protein; and if the patient exhibits the presence of the protein signature, selecting the patient as having TBI. In some cases, the patient exhibits clinical symptoms consistent with TBI. In some cases, the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). In some cases, the level of the at least one inflammasome protein in the protein signature is measured by an immunoassay using one or more antibodies against the at least one inflammasome protein in the protein signature. In some cases, the at least one inflammasome protein is interleukin-18 (IL-18), IL-1β, apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof. In some cases, the at least one inflammasome protein includes caspase-1. In some cases, the at least one inflammasome protein includes ASC. In some cases, the antibody binds to a PYRIN-PAAD-DAPIN domain (PYD), a C-terminal caspase recruitment domain (CARD) domain, or a portion of the PYD or CARD domain of the ASC protein. In some cases, the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control.In some cases, at least one inflammasome protein comprises caspase-1, and the level of caspase-1 is at least 50% higher than the level of caspase-1 in the biological sample obtained from the control. In some cases, the at least one inflammasome protein comprises ASC, and the level of ASC is at least 50% higher than the level of ASC in the biological sample obtained from the control. In some cases, the biological sample obtained from the control is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). In some cases, the control is a healthy individual, and the healthy individual is an individual who does not exhibit clinical symptoms consistent with TBI. In some cases, the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values. In some cases, the biological sample obtained from the patient is serum, and the patient is selected as having TBI with at least 80%, 85%, 90%, 95%, 99%, or 100% sensitivity and at least 90% specificity. In some cases, the biological sample is serum, and the patient is selected as having TBI with at least 80%, 85%, 90%, 95%, 99%, or 100% specificity. In some cases, the biological sample is serum, and the patient is selected as having TBI with at least 90% sensitivity and at least 80% specificity. In some cases, the sensitivity and / or sensitivity are determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval. In some cases, the at least one inflammasome protein comprises ASC. In some cases, the cutoff value for determining the sensitivity, specificity, or both is selected from Table 11B, Table 12B, Table 14A, Table 16, Table 17, or Table 19. In some cases, the at least one inflammasome protein comprises caspase-1. In some cases, the cutoff value for determining the sensitivity, specificity, or both is selected from Table 11A or Table 15.

[0016] In yet another aspect, the present invention provides a method for evaluating a patient suspected of having brain injury, comprising: measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with brain injury, wherein the protein signature comprises an elevated level of the at least one inflammasome protein; and if the patient shows the presence of the protein signature, selecting the patient as having brain injury. In some cases, the patient exhibits clinical symptoms consistent with brain injury. In some cases, the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). In some cases, the level of the at least one inflammasome protein in the protein signature is measured by an immunoassay using one or more antibodies against the at least one inflammasome protein in the protein signature. In some cases, the at least one inflammasome protein is interleukin-18 (IL-18), IL-1β, apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof. In some cases, the at least one inflammasome protein includes ASC. In some cases, the antibody binds to a PYRIN-PAAD-DAPIN domain (PYD), a C-terminal caspase recruitment domain (CARD) domain, or a portion of the PYD or CARD domain of the ASC protein. In some cases, the at least one inflammasome protein includes caspase-1. In some cases, the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control. In some cases, the at least one inflammasome protein includes ASC, and the level of ASC is at least 50% higher than the level of ASC in the biological sample obtained from the control.In some cases, the at least one inflammasome protein comprises caspase-1, and the level of caspase-1 is at least 50% higher than the level of caspase-1 in the biological sample obtained from the control. In some cases, the biological sample obtained from the control is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). In some cases, the control is a healthy individual, and the healthy individual is an individual who does not exhibit clinical symptoms consistent with brain injury. In some cases, the brain injury is selected from traumatic brain injury, stroke, mild cognitive impairment, or multiple sclerosis. In some cases, the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values. In some cases, the brain injury is traumatic brain injury (TBI). In some cases, the biological sample obtained from the patient is serum, and the patient is selected as having TBI with at least 80%, 85%, 90%, 95%, 99%, or 100% sensitivity and at least 90% specificity. In some cases, the biological sample is serum, and the patient is selected as having TBI with at least 80%, 85%, 90%, 95%, 99%, or 100% specificity. In some cases, the biological sample is serum, and the patient is selected as having TBI with at least 90% sensitivity and at least 80% specificity. In some cases, the sensitivity and / or sensitivity are determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval. In some cases, the at least one inflammasome protein comprises ASC. In some cases, the cutoff value for determining the sensitivity, specificity, or both is selected from Table 11B, 12B, 14A, 16, 17, or 19. In some cases, the at least one inflammasome protein includes caspase-1. In some cases, the cutoff value for determining the sensitivity, specificity, or both is selected from Table 11A or 15. In some cases, the brain injury is mild cognitive impairment (MCI).In some cases, the biological sample obtained from the patient is serum, and the patient is selected as having MCI with at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sensitivity. In some cases, the biological sample is serum, and the patient is selected as having MCI with at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% specificity. In some cases, the biological sample is serum, and the patient is selected as having MCI with at least 90% sensitivity and at least 70% specificity. In some cases, the sensitivity and / or sensitivity are determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval. In some cases, the at least one inflammasome protein includes ASC. In some cases, the cutoff value for determining sensitivity, specificity, or both is selected from Table 22 or 23. In some cases, the at least one inflammasome protein comprises IL-18. In some cases, the cutoff value for determining sensitivity, specificity, or both is selected from Table 22 or 25. In some cases, the brain injury is multiple sclerosis (MS). In some cases, the biological sample obtained from the patient is serum, and the patient is selected as having MS with at least 80%, 85%, 90%, 95%, 99%, or 100% sensitivity and at least 90% specificity. In some cases, the biological sample is serum, and the patient is selected as having MS with at least 80%, 85%, 90%, 95%, 99%, or 100% specificity. In some cases, the biological sample is serum, and the patient is selected as having MS with at least 90% sensitivity and at least 80% specificity. In some cases, the at least one inflammasome protein comprises ASC. In some cases, the cutoff value for determining the sensitivity, specificity, or both is selected from Table 7. In some cases, the sensitivity and / or specificity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval. In some cases, the brain injury is a stroke.In some cases, the biological sample obtained from the patient is serum, and the patient is selected as having a stroke with at least 80%, 85%, 90%, 95%, 99%, or 100% sensitivity and at least 90% specificity. In some cases, the biological sample is serum, and the patient is selected as having a stroke with at least 80%, 85%, 90%, 95%, 99%, or 100% specificity. In some cases, the biological sample is serum, and the patient is selected as having a stroke with at least 100% sensitivity and at least 95% specificity. In some cases, the at least one inflammasome protein comprises ASC. In some cases, the cutoff value for determining the sensitivity, specificity, or both is selected from Table 8. In some cases, the biological sample obtained from the patient is serum-derived EVs, and the patient is selected as having a stroke with at least 80%, 85%, 90%, 95%, 99%, or 100% sensitivity and at least 90% specificity. In some cases, the biological sample is serum-derived EVs, and the patient is selected as having a stroke with at least 80%, 85%, 90%, 95%, 99%, or 100% specificity. In some cases, the biological sample is serum-derived EVs, and the patient is selected as having a stroke with at least 100% sensitivity and at least 100% specificity. In some cases, the at least one inflammasome protein comprises ASC. In some cases, the cutoff value for determining the sensitivity, specificity, or both is selected from Table 9. In some cases, the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval.

[0017] In yet another aspect, the present invention provides a method for evaluating a patient suspected of having mild cognitive impairment (MCI), comprising: measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with MCI, wherein the protein signature comprises an elevated level of the at least one inflammasome protein; and if the patient shows the presence of the protein signature, selecting the patient as having MCI. In some cases, the patient exhibits clinical symptoms consistent with MCI. In some cases, the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). In some cases, the level of the at least one inflammasome protein in the protein signature is measured by immunoassay using one or more antibodies against the at least one inflammasome protein in the protein signature. In some cases, the at least one inflammasome protein is interleukin-18 (IL-18), IL-1β, apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof. In some cases, the at least one inflammasome protein includes ASC. In some cases, the at least one inflammasome protein includes IL-18. In some cases, the antibody binds to a PYRIN-PAAD-DAPIN domain (PYD), a C-terminal caspase recruitment domain (CARD) domain, or a portion of the PYD or CARD domain of the ASC protein. In some cases, the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control.In some cases, the at least one inflammasome protein comprises ASC, and the level of ASC is at least 50% higher than the level of ASC in the biological sample obtained from the control. In some cases, the at least one inflammasome protein comprises IL-18, and the level of IL-18 is at least 25% higher than the level of IL-18 in the biological sample obtained from the control. In certain embodiments, for example, the following are provided: (Item 1) A method for evaluating a patient suspected of having multiple sclerosis (MS), comprising the steps of measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with MS, wherein the protein signature comprises an elevated level of the at least one inflammasome protein; and selecting the patient as having MS if the patient exhibits the presence of the protein signature. (Item 2) 2. The method of item 1, wherein the patient exhibits clinical symptoms consistent with MS. (Item 3) 3. The method according to item 1 or 2, wherein the MS is relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS) or progressive relapsing MS (PRMS). (Item 4) 2. The method of item 1, wherein the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). (Item 5) 2. The method of claim 1, wherein the level of the at least one inflammasome protein in the protein signature is measured by an immunoassay utilizing one or more antibodies against the at least one inflammasome protein in the protein signature. (Item 6) 2. The method of claim 1, wherein the at least one inflammasome protein is interleukin-18 (IL-18), IL-1 beta, apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof. (Item 7) 2. The method of claim 1, wherein the at least one inflammasome protein comprises each of caspase-1, IL-18, IL-1 beta, and ASC. (Item 8) Item 1, wherein the at least one inflammasome protein comprises ASC. (Item 9) 9. The method of claim 8, wherein the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD) domain, or a portion of the PYD or CARD domain of the ASC protein. (Item 10) 2. The method of claim 1, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control. (Item 11) 11. The method of claim 10, wherein the biological sample obtained from the subject is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). (Item 12) 11. The method of item 10, wherein the control is a healthy individual, wherein the healthy individual does not exhibit clinical symptoms consistent with MS. (Item 13) 11. The method of claim 10, wherein the at least one inflammasome protein comprises ASC, and the level of ASC is at least 50% higher than the level of ASC in the biological sample obtained from a control. (Item 14) Item 1, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values. (Item 15) 15. The method of claim 14, wherein the biological sample obtained from the patient is serum, and the patient is selected as having MS with a sensitivity of at least 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 90%. (Item 16) 15. The method of claim 14, wherein the biological sample is serum and the patient is selected as having MS with a specificity of at least 80%, 85%, 90%, 95%, 99% or 100%. (Item 17) 15. The method of claim 14, wherein the biological sample is serum and the patient is selected as having MS with a sensitivity of at least 90% and a specificity of at least 80%. (Item 18) Item 15. The method of item 14, wherein the at least one inflammasome protein comprises ASC. (Item 19) Item 19. The method of item 18, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 7. (Item 20) 16. The method of item 15, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval. (Item 21) A method for evaluating a patient suspected of having a stroke, comprising the steps of measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with stroke or stroke-related injury, wherein the protein signature comprises an elevated level of the at least one inflammasome protein; and selecting the patient as having suffered a stroke if the patient exhibits the presence of the protein signature. (Item 22) 22. The method of claim 21, wherein the patient presents with clinical symptoms consistent with stroke, and the stroke is an ischemic stroke, a transient ischemic stroke, or a hemorrhagic stroke. (Item 23) 22. The method of item 21, wherein the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). (Item 24) 22. The method of claim 21, wherein the level of the at least one inflammasome protein in the protein signature is measured by an immunoassay utilizing one or more antibodies against the at least one inflammasome protein in the protein signature. (Item 25) 22. The method of claim 21, wherein the at least one inflammasome protein is interleukin-18 (IL-18), IL-1 beta, apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof. (Item 26) 22. The method of claim 21, wherein the at least one inflammasome protein comprises each of caspase-1, IL-18, IL-1 beta, and ASC. (Item 27) 22. The method of claim 21, wherein the at least one inflammasome protein comprises ASC. (Item 28) 28. The method of claim 27, wherein the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD), or a portion of the PYD or CARD domain of the ASC protein. (Item 29) 22. The method of claim 21, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control. (Item 30) 30. The method of item 29, wherein the biological sample obtained from the subject is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). (Item 31) 30. The method of item 29, wherein the control is a healthy individual, wherein the healthy individual does not exhibit clinical symptoms consistent with MS. (Item 32) 30. The method of claim 29, wherein the at least one inflammasome protein comprises ASC, and the level of ASC in a serum sample obtained from the subject is at least 70% higher than the level of ASC in a serum sample obtained from a control. (Item 33) 30. The method of claim 29, wherein the at least one inflammasome protein comprises ASC, and the level of ASC in a serum-derived EV sample obtained from the subject is at least 110% higher than the level of ASC in a serum-derived EV sample obtained from a control. (Item 34) 22. The method of claim 21, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values. (Item 35) 35. The method of claim 34, wherein the biological sample obtained from the patient is serum, and the patient is selected as having suffered from stroke with a sensitivity of at least 80%, 85%, 90%, 95%, 99% or 100% and a specificity of at least 90%. (Item 36) 35. The method of claim 34, wherein the biological sample is serum and the patient is selected as having a stroke with a specificity of at least 80%, 85%, 90%, 95%, 99% or 100%. (Item 37) Item 38. The method of item 34, wherein the biological sample is serum, and the patient is selected as having a stroke with at least 100% sensitivity and at least 95% specificity. 36. The method of claim 35, wherein the at least one inflammasome protein comprises ASC. (Item 39) 39. The method of item 38, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 8. (Item 40) 35. The method of claim 34, wherein the biological sample obtained from the patient is serum-derived EVs, and the patient is selected as having a stroke with a sensitivity of at least 80%, 85%, 90%, 95%, 99% or 100% and a specificity of at least 90%. (Item 41) 35. The method of claim 34, wherein the biological sample is serum-derived EVs and the patient is selected as having stroke with a specificity of at least 80%, 85%, 90%, 95%, 99% or 100%. (Item 42) 35. The method of claim 34, wherein the biological sample is serum-derived EVs and the patient is selected as having a stroke with at least 100% sensitivity and at least 100% specificity. (Item 43) 41. The method of claim 40, wherein the at least one inflammasome protein comprises ASC. (Item 44) Item 44. The method of item 43, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 9. (Item 45) The method according to item 35 or 40, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval. (Item 46) A method of treating a patient diagnosed with multiple sclerosis (MS), comprising administering to the patient a standard of care treatment for MS, wherein the diagnosis of MS is made by detecting elevated levels of at least one inflammasome protein in a biological sample obtained from the patient. (Item 47) 47. The method of item 46, wherein the MS is relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS) or progressive relapsing MS (PRMS). (Item 48) 47. The method of item 46, wherein the standard of care treatment is selected from therapies directed to modifying disease outcome, managing recurrence, managing symptoms, or any combination thereof. (Item 49) 49. The method of item 48, wherein the treatment directed to modifying disease outcome is selected from beta-interferon, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, mitoxantrone, ocrelizumab, alemtuzumab, daclizumab, and natalizumab. (Item 50) A method of treating a patient diagnosed with stroke or stroke-related injury, comprising administering to the patient a standard of care treatment for stroke or stroke-related injury, wherein the diagnosis of stroke or stroke-related injury is made by detecting elevated levels of at least one inflammasome protein in a biological sample obtained from the patient. (Item 51) 51. The method of item 50, wherein the stroke is an ischemic stroke, a transient ischemic stroke, or a hemorrhagic stroke. (Item 52) 51. The method of item 50, wherein the stroke is an ischemic stroke or a transient ischemic stroke, and the standard of care treatment is selected from tissue plasminogen activator (tPA), an antiplatelet agent, an anticoagulant, carotid angioplasty, carotid endarterectomy, intra-arterial thrombolysis, and mechanical clot removal in cerebral ischemia (MERCI), or a combination thereof. (Item 53) 51. The method of claim 50, wherein the stroke is a hemorrhagic stroke and the standard of care treatment is aneurysm clipping, coil embolization, or arteriovenous malformation (AVM) repair. (Item 54) 54. The method of any one of items 46 to 53, wherein the increase in the level of the at least one inflammasome protein is measured by an immunoassay utilizing one or more antibodies against the at least one inflammasome protein. (Item 55) 55. The method of claim 54, wherein the level of the at least one inflammasome protein is increased relative to the level of the at least one inflammasome protein in a control sample. (Item 56) 55. The method of claim 54, wherein the level of the at least one inflammasome protein is increased relative to a predetermined reference value or range of reference values. (Item 57) 57. The method of claim 56, wherein the at least one inflammasome protein is interleukin-18 (IL-18), apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof. (Item 58) 57. The method of claim 56, wherein the at least one inflammasome protein is caspase-1, IL-18, and ASC. (Item 59) 57. The method of claim 56, wherein the at least one inflammasome protein is ASC. (Item 60) 60. The method of claim 59, wherein the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD), or a portion of the PYD or CARD domain of the ASC protein. (Item 61) 55. The method of item 54, wherein the biological sample is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). (Item 62) A method for evaluating a patient suspected of having a traumatic brain injury (TBI), comprising the steps of: measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with TBI, the protein signature comprising an elevated level of the at least one inflammasome protein; and selecting the patient as having TBI if the patient exhibits the presence of the protein signature. (Item 63) 63. The method of claim 62, wherein the patient exhibits clinical symptoms consistent with TBI. (Item 64) 63. The method of item 62, wherein the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). (Item 65) Item 63. The method of item 62, wherein the level of the at least one inflammasome protein in the protein signature is measured by an immunoassay utilizing one or more antibodies against the at least one inflammasome protein in the protein signature. (Item 66) 63. The method of claim 62, wherein the at least one inflammasome protein is interleukin-18 (IL-18), IL-1β, apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof. (Item 67) Item 63. The method of item 62, wherein the at least one inflammasome protein comprises ASC. (Item 68) Item 63. The method of item 62, wherein the at least one inflammasome protein comprises caspase-1. (Item 69) 68. The method of claim 67, wherein the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD) domain, or a portion of the PYD or CARD domain of the ASC protein. (Item 70) 63. The method of claim 62, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control. (Item 71) 71. The method of claim 70, wherein the at least one inflammasome protein comprises caspase-1, and the level of caspase-1 is at least 50% higher than the level of caspase-1 in the biological sample obtained from the control. (Item 72) 71. The method of claim 70, wherein the at least one inflammasome protein comprises ASC, and the level of ASC is at least 50% higher than the level of ASC in the biological sample obtained from the control. (Item 73) 71. The method of claim 70, wherein the biological sample obtained from the subject is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). (Item 74) 71. The method of claim 70, wherein the control is a healthy individual, wherein the healthy individual does not exhibit clinical symptoms consistent with TBI. (Item 75) 63. The method of claim 62, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values. (Item 76) 76. The method of claim 75, wherein the biological sample obtained from the patient is serum, and the patient is selected as having TBI with a sensitivity of at least 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 90%. (Item 77) 76. The method of claim 75, wherein the biological sample is serum and the patient is selected as having TBI with a specificity of at least 80%, 85%, 90%, 95%, 99% or 100%. (Item 78) 76. The method of claim 75, wherein the biological sample is serum and the patient is selected as having TBI with at least 90% sensitivity and at least 80% specificity. (Item 79) 77. The method of item 76, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval. (Item 80) 76. The method of claim 75, wherein the at least one inflammasome protein comprises ASC. (Item 81) 80. The method of item 79, wherein the cutoff value for determining the sensitivity, specificity or both is selected from Table 11B, Table 12B, Table 14A, Table 16, Table 17 or Table 19. (Item 82) 76. The method of claim 75, wherein the at least one inflammasome protein comprises caspase-1. (Item 83) 83. The method of item 82, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 11A or Table 15. (Item 84) A method for evaluating a patient suspected of having brain injury, comprising the steps of measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with brain injury, the protein signature comprising an elevated level of the at least one inflammasome protein; and selecting the patient as having brain injury if the patient exhibits the presence of the protein signature. (Item 85) 85. The method of claim 84, wherein the patient exhibits clinical symptoms consistent with brain injury. (Item 86) 85. The method of item 84, wherein the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). (Item 87) 85. The method of claim 84, wherein the level of the at least one inflammasome protein in the protein signature is measured by an immunoassay utilizing one or more antibodies against the at least one inflammasome protein in the protein signature. (Item 88) 85. The method of item 84, wherein the at least one inflammasome protein is interleukin-18 (IL-18), IL-1β, apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof. (Item 89) 85. The method of claim 84, wherein the at least one inflammasome protein comprises ASC. (Item 90) 89. The method of claim 88, wherein the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD) domain, or a portion of the PYD or CARD domain of the ASC protein. (Item 91) 85. The method of claim 84, wherein the at least one inflammasome protein comprises caspase-1. (Item 92) 85. The method of claim 84, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control. (Item 93) 93. The method of claim 92, wherein the at least one inflammasome protein comprises ASC, and the level of ASC is at least 50% higher than the level of ASC in the biological sample obtained from the control. (Item 94) Item 93. The method of item 92, wherein the at least one inflammasome protein comprises caspase-1, and the level of caspase-1 is at least 50% higher than the level of caspase-1 in the biological sample obtained from the control. (Item 95) 93. The method of claim 92, wherein the biological sample obtained from the subject is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). (Item 96) 93. The method of claim 92, wherein the control is a healthy individual, wherein the healthy individual does not exhibit clinical symptoms consistent with brain injury. (Item 97) 85. The method of claim 84, wherein the brain injury is selected from traumatic brain injury, stroke, mild cognitive impairment, or multiple sclerosis. (Item 98) 85. The method of claim 84, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values. (Item 99) 99. The method of claim 98, wherein the brain injury is a traumatic brain injury (TBI). (Item 100) 100. The method of claim 99, wherein the biological sample obtained from the patient is serum, and the patient is selected as having TBI with a sensitivity of at least 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 90%. (Item 101) 99. The method of claim 98, wherein the biological sample is serum and the patient is selected as having TBI with a specificity of at least 80%, 85%, 90%, 95%, 99%, or 100%. (Item 102) 100. The method of claim 99, wherein the biological sample is serum and the patient is selected as having TBI with at least 90% sensitivity and at least 80% specificity. (Item 103) 101. The method of claim 100, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval. (Item 104) Item 99. The method of item 99, wherein the at least one inflammasome protein comprises ASC. (Item 105) 105. The method of item 104, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 11B, 12B, 14A, 16, 17, or 19. (Item 106) Item 99. The method of item 99, wherein the at least one inflammasome protein comprises caspase-1. (Item 107) 107. The method of item 106, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 11A or 15. (Item 108) Item 99. The method of item 98, wherein the brain injury is multiple sclerosis (MS). (Item 109) 109. The method of claim 108, wherein the biological sample obtained from the patient is serum, and the patient is selected as having MS with a sensitivity of at least 80%, 85%, 90%, 95%, 99% or 100% and a specificity of at least 90%. (Item 110) 109. The method of claim 108, wherein the biological sample is serum and the patient is selected as having MS with a specificity of at least 80%, 85%, 90%, 95%, 99% or 100%. (Item 111) 109. The method of claim 108, wherein the biological sample is serum and the patient is selected as having MS with a sensitivity of at least 90% and a specificity of at least 80%. (Item 112) Item 109. The method of item 108, wherein the at least one inflammasome protein comprises ASC. (Item 113) 113. The method of item 112, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 7. (Item 114) 110. The method of claim 109, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval. (Item 115) 99. The method of claim 98, wherein the brain injury is a stroke. (Item 116) 116. The method of claim 115, wherein the biological sample obtained from the patient is serum, and the patient is selected as having a stroke with a sensitivity of at least 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 90%. (Item 117) 116. The method of claim 115, wherein the biological sample is serum and the patient is selected as having a stroke with a specificity of at least 80%, 85%, 90%, 95%, 99% or 100%. (Item 118) 116. The method of claim 115, wherein the biological sample is serum and the patient is selected as having a stroke with a sensitivity of at least 100% and a specificity of at least 95%. (Item 119) Item 117. The method of item 116, wherein the at least one inflammasome protein comprises ASC. (Item 120) 120. The method of item 119, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 8. (Item 121) 116. The method of claim 115, wherein the biological sample obtained from the patient is serum-derived EVs, and the patient is selected as having a stroke with a sensitivity of at least 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 90%. (Item 122) 116. The method of claim 115, wherein the biological sample is serum-derived EVs and the patient is selected as having stroke with a specificity of at least 80%, 85%, 90%, 95%, 99% or 100%. (Item 123) Item 115. The biological sample is serum-derived EVs, and the patient is selected as having stroke with at least 100% sensitivity and at least 100% specificity. The method described below. (Item 124) Item 122. The method of item 121, wherein the at least one inflammasome protein comprises ASC. (Item 125) 125. The method of item 124, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 9. (Item 126) 124. The method of any one of items 116-118 or 121-123, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval. (Item 127) A method for evaluating a patient suspected of having mild cognitive impairment (MCI), comprising the steps of measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with MCI, the protein signature comprising an elevated level of the at least one inflammasome protein; and selecting the patient as having MCI if the patient exhibits the presence of the protein signature. (Item 128) 128. The method of item 127, wherein the patient exhibits clinical symptoms consistent with MCI. (Item 129) Item 130. The method of Item 127, wherein the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). Item 128. The method of item 127, wherein the level of the at least one inflammasome protein in the protein signature is measured by an immunoassay utilizing one or more antibodies against the at least one inflammasome protein in the protein signature. (Item 131) 128. The method of claim 127, wherein the at least one inflammasome protein is interleukin-18 (IL-18), IL-1β, apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof. (Item 132) Item 128. The method of item 127, wherein the at least one inflammasome protein comprises ASC. (Item 133) 128. The method of claim 127, wherein the at least one inflammasome protein comprises IL-18. (Item 134) 132. The method of claim 131, wherein the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD), or a portion of the PYD or CARD domain of the ASC protein. (Item 135) 128. The method of claim 127, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control. (Item 136) Item 136. The method of item 135, wherein the at least one inflammasome protein comprises ASC, and the level of ASC is at least 50% higher than the level of ASC in the biological sample obtained from the control. (Item 137) 136. The method of claim 135, wherein the at least one inflammasome protein comprises IL-18, and the level of IL-18 is at least 25% higher than the level of IL-18 in the biological sample obtained from the control. (Item 138) Item 139. The method of Item 135, wherein the biological sample obtained from the subject is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs). 136. The method of item 135, wherein the control is a healthy individual, and the healthy individual is an individual who does not exhibit clinical symptoms consistent with MCI. (Item 140) 128. The method of claim 127, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values. (Item 141) 141. The method of claim 140, wherein the biological sample obtained from the patient is serum, and the patient is selected as having MCI with a sensitivity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 55%. (Item 142) 141. The method of claim 140, wherein the biological sample is serum and the patient is selected as having MCI with a sensitivity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. (Item 143) 141. The method of claim 140, wherein the biological sample is serum and the patient is selected as having MCI with a sensitivity of at least 70% and a specificity of at least 55%. (Item 144) 141. The method of claim 140, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval. (Item 145) Item 141. The method of item 140, wherein the at least one inflammasome protein comprises ASC. (Item 146) 146. The method of item 145, wherein the cutoff value for determining the sensitivity, specificity or both is selected from Table 22. (Item 147) Item 141. The method of item 140, wherein the at least one inflammasome protein comprises IL-18. (Item 148) 148. The method of item 147, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 22. [Brief explanation of the drawings]

[0018] [Figures 1A-1D] Figures 1A-1D illustrate that inflammasome proteins are elevated in the serum of MS patients. Protein levels (pg / ml) of caspase-1 (Figure 1A), ASC (Figure 1B), IL-1β (Figure 1C), and IL-18 (Figure 1D) in serum samples from patients with MS and healthy donors. p-values ​​for significance are shown in the respective box and whisker plots. Boxes and whiskers are shown for the 5th and 95th percentiles. Caspase-1: N = 9 controls and N = 19 MS patients; ASC: N = 115 controls and N = 32 MS patients; IL-1β: N = 21 controls and N = 8 MS patients; and IL-18: N = 119 controls and N = 32 MS patients.

[0019] [Figures 2A-2D] Figures 2A-2D illustrate the ROC curves for caspase-1 (Figure 2A), ASC (Figure 2B), IL-1β (Figure 2C), and IL-18 (Figure 2D) from serum samples of MS and healthy donors.

[0020] [Figure 3] Figure 3 illustrates serum inflammasome proteins as biomarkers for MS. ROC curves for caspase-1, ASC, IL-1 beta, and IL-18. Caspase-1: N=9 controls and N=19 MS; ASC: N=115 controls and N=32 MS; IL-1 beta: N=21 controls and N=8 MS; and IL-18: N=119 controls and N=32 MS.

[0021] [Figure 4]FIG. 4 illustrates a table containing characteristics of subjects with multiple sclerosis (MS) from Example 1.

[0022] [Figures 5A-5D] Figures 5A-5D illustrate that inflammasome proteins are elevated in the serum of stroke patients. Protein levels (pg / ml) of caspase-1 (Figure 5A), ASC (Figure 5B), IL-1 beta (Figure 5C), and IL-18 (Figure 5D) in serum samples from patients with stroke and healthy donors. p-values ​​for significance are shown in the respective box and whisker plots. Boxes and whiskers are shown for the 5th and 95th percentiles. NS = not significant. Caspase-1: N = 8 controls and N = 13 strokes; ASC: N = 75 controls and N = 16 strokes; IL-1 beta: N = 9 controls and N = 8 strokes; and IL-18: N = 79 controls and N = 15 strokes.

[0023] [Figure 6] Figure 6 illustrates serum inflammasome proteins as biomarkers of stroke. ROC curves for caspase-1, ASC, IL-1 beta, and IL-18. Caspase-1: N=8 controls and N=13 strokes; ASC: N=75 controls and N=16 strokes; IL-1 beta: N=9 controls and N=8 strokes; and IL-18: N=79 controls and N=15 strokes.

[0024] [Figure 7A-7C]Figure 7A illustrates a comparison of total protein levels from serum-derived extracellular vesicles (EVs). A Bradford assay was performed after isolation of EVs from serum to determine the total protein concentration in the isolates using the Invitrogen kit (INVTR) and the ExoQuick kit (EQ). Data are shown as mean + / - SEM. N = 6 per group. Figure 7B shows a representative image of the total protein loaded. An unstained image of serum-derived EV proteins. Equal amounts of protein lysate (10 ml) were loaded into each lane of the reference gel. Figure 7C shows a bar graph showing the quantification of the total lanes corresponding to the loaded EVs isolated using the Invitrogen kit (INVTR) and the ExoQuick kit (EQ).

[0025] [Figure 8A-B] Figures 8A-8F illustrate the characterization of EVs in serum from stroke patients. Figure 8A shows representative immunoblots of CD81- and NCAM-positive EVs isolated using the Invitrogen kit (IN) and the ExoQuick kit (EQ). + Control: positive control for isolated EVs. Quantification of CD81- (Figure 8B) and NCAM- (Figure 8C)-positive EVs isolated from serum using the Invitrogen kit (INV) and the ExoQuick kit (EQ). Figure 8D shows electron microscopy images of EVs isolated by the two different techniques. Bar = 100 nm. Nanoparticle tracking analysis / particle size distribution of isolated serum-derived EVs. Nanoparticle tracking analysis predicts the size distribution and concentration of particles in serum-derived EV samples isolated using the Invitrogen kit (Figure 8E) and the ExoQuick kit (Figure 8F). [Figure 8C-D]Figures 8A-8F illustrate the characterization of EVs in serum from stroke patients. Figure 8A shows representative immunoblots of CD81- and NCAM-positive EVs isolated using the Invitrogen kit (IN) and the ExoQuick kit (EQ). + Control: positive control for isolated EVs. Quantification of CD81- (Figure 8B) and NCAM- (Figure 8C)-positive EVs isolated from serum using the Invitrogen kit (INV) and the ExoQuick kit (EQ). Figure 8D shows electron microscopy images of EVs isolated by the two different techniques. Bar = 100 nm. Nanoparticle tracking analysis / particle size distribution of isolated serum-derived EVs. Nanoparticle tracking analysis predicts the size distribution and concentration of particles in serum-derived EV samples isolated using the Invitrogen kit (Figure 8E) and the ExoQuick kit (Figure 8F). [Figure 8E-F] Figures 8A-8F illustrate the characterization of EVs in serum from stroke patients. Figure 8A shows representative immunoblots of CD81- and NCAM-positive EVs isolated using the Invitrogen kit (IN) and the ExoQuick kit (EQ). + Control: positive control for isolated EVs. Quantification of CD81- (Figure 8B) and NCAM- (Figure 8C)-positive EVs isolated from serum using the Invitrogen kit (INV) and the ExoQuick kit (EQ). Figure 8D shows electron microscopy images of EVs isolated by the two different techniques. Bar = 100 nm. Nanoparticle tracking analysis / particle size distribution of isolated serum-derived EVs. Nanoparticle tracking analysis predicts the size distribution and concentration of particles in serum-derived EV samples isolated using the Invitrogen kit (Figure 8E) and the ExoQuick kit (Figure 8F).

[0026] [Figures 9A-9C]Figures 9A-9C illustrate that ASC is elevated in serum-derived EVs from stroke patients. Protein levels (pg / ml) of ASC (Figure 9A), IL-1 beta (Figure 9B), and IL-18 (Figure 9C) in serum-derived EVs from patients with stroke and healthy donors. p-values ​​for significance are shown in the respective boxplots. Boxes and whiskers are shown for the 5th and 95th percentiles. NS = not significant. ASC: N = 16 controls and N = 16 strokes; IL-1 beta: N = 10 controls and N = 9 strokes; and IL-18: N = 16 controls and N = 13 strokes.

[0027] [Figure 10] Figure 10 illustrates inflammasome proteins in serum-derived EVs as biomarkers of stroke. ROC curves for ASC, IL-1 beta, and IL-18. ASC: N=16 controls and N=16 strokes; IL-1 beta: N=10 controls and N=9 strokes; and IL-18: N=16 controls and N=13 strokes.

[0028] [Figure 11] FIG. 11 illustrates a table containing characteristics of subjects with stroke from Example 2.

[0029] [Figures 12A-12D] 12A-12D illustrate ROC curves for caspase-1 (FIG. 12A), ASC (FIG. 12B), IL-1 beta (FIG. 12C), and IL-18 (FIG. 12D) from serum samples of stroke and healthy donors.

[0030] [Figure 13A-B]Figures 13A-13F illustrate the characterization of inflammasome proteins in serum-derived EVs. Figure 13A shows a representative image of immunoblot analysis of inflammasome proteins in EVs from serum. Quantification of immunoblot analysis of (Figure 13B) NLRP3, (Figure 13C) caspase-1, (Figure 13D) ASC, (Figure 13E) IL-1 beta, and (Figure 13F) IL-18 in serum-derived EVs using the Invitrogen kit (IN) and the ExoQuick kit (EQ). Data are presented as mean + / - SEM. N=6 per group. *p<0.05. [Figure 13C-D] Figures 13A-13F illustrate the characterization of inflammasome proteins in serum-derived EVs. Figure 13A shows a representative image of immunoblot analysis of inflammasome proteins in EVs from serum. Quantification of immunoblot analysis of (Figure 13B) NLRP3, (Figure 13C) caspase-1, (Figure 13D) ASC, (Figure 13E) IL-1 beta, and (Figure 13F) IL-18 in serum-derived EVs using the Invitrogen kit (IN) and the ExoQuick kit (EQ). Data are presented as mean + / - SEM. N=6 per group. *p<0.05. [Figure 13E-F] Figures 13A-13F illustrate the characterization of inflammasome proteins in serum-derived EVs. Figure 13A shows a representative image of immunoblot analysis of inflammasome proteins in EVs from serum. Quantification of immunoblot analysis of (Figure 13B) NLRP3, (Figure 13C) caspase-1, (Figure 13D) ASC, (Figure 13E) IL-1 beta, and (Figure 13F) IL-18 in serum-derived EVs using the Invitrogen kit (IN) and the ExoQuick kit (EQ). Data are presented as mean + / - SEM. N=6 per group. *p<0.05.

[0031] [Figures 14A-14C] Figures 14A-14C illustrate ROC curves for ASC (Figure 14A), IL-1 beta (Figure 14B), and IL-18 (Figure 14C) from serum-derived extracellular vesicles of stroke and healthy donors.

[0032] [Figure 15A] Figures 15A-15D illustrate how inflammasome proteins are elevated in the serum of TBI patients. Protein levels (pg / ml) of ASC (Figure 15A), caspase-1 (Figure 15B), IL-18 (Figure 15C), and IL-1β (Figure 15D) in serum samples from patients with TBI and healthy donors (controls). ASC: N=120 controls and N=20 TBI. Caspase-1: N=11 controls and N=19 TBI. IL-18: N=120 controls and N=21 TBI. IL-1β: N=25 controls and N=10 TBI. Boxes and whiskers are shown for the 5th and 95th percentiles. *p<0.05. [Figure 15B] Figures 15A-15D illustrate how inflammasome proteins are elevated in the serum of TBI patients. Protein levels (pg / ml) of ASC (Figure 15A), caspase-1 (Figure 15B), IL-18 (Figure 15C), and IL-1β (Figure 15D) in serum samples from patients with TBI and healthy donors (controls). ASC: N=120 controls and N=20 TBI. Caspase-1: N=11 controls and N=19 TBI. IL-18: N=120 controls and N=21 TBI. IL-1β: N=25 controls and N=10 TBI. Boxes and whiskers are shown for the 5th and 95th percentiles. *p<0.05. [Figure 15C] Figures 15A-15D illustrate how inflammasome proteins are elevated in the serum of TBI patients. Protein levels (pg / ml) of ASC (Figure 15A), caspase-1 (Figure 15B), IL-18 (Figure 15C), and IL-1β (Figure 15D) in serum samples from patients with TBI and healthy donors (controls). ASC: N=120 controls and N=20 TBI. Caspase-1: N=11 controls and N=19 TBI. IL-18: N=120 controls and N=21 TBI. IL-1β: N=25 controls and N=10 TBI. Boxes and whiskers are shown for the 5th and 95th percentiles. *p<0.05. [Figure 15D]Figures 15A-15D illustrate how inflammasome proteins are elevated in the serum of TBI patients. Protein levels (pg / ml) of ASC (Figure 15A), caspase-1 (Figure 15B), IL-18 (Figure 15C), and IL-1β (Figure 15D) in serum samples from patients with TBI and healthy donors (controls). ASC: N=120 controls and N=20 TBI. Caspase-1: N=11 controls and N=19 TBI. IL-18: N=120 controls and N=21 TBI. IL-1β: N=25 controls and N=10 TBI. Boxes and whiskers are shown for the 5th and 95th percentiles. *p<0.05.

[0033] [Figures 16A-16D] 16A-16D illustrate ROC curves for caspase-1 (FIG. 16A), ASC (FIG. 16B), IL-1β (FIG. 16C), and IL-18 (FIG. 16D) from serum samples of TBI patients and healthy donors.

[0034] [Figure 17A] Figures 17A-17B illustrate how inflammasome proteins are elevated in the CSF of TBI patients. Protein levels (pg / ml) of ASC (Figure 17A) and IL-18 (Figure 17B) in CSF samples from patients with TBI and healthy donors (controls). ASC: N=21 controls and N=15 TBI. IL-18: N=24 controls and N=16 TBI. Boxes and whiskers are shown for the 5th and 95th percentiles. *p<0.05. [Figure 17B] Figures 17A-17B illustrate how inflammasome proteins are elevated in the CSF of TBI patients. Protein levels (pg / ml) of ASC (Figure 17A) and IL-18 (Figure 17B) in CSF samples from patients with TBI and healthy donors (controls). ASC: N=21 controls and N=15 TBI. IL-18: N=24 controls and N=16 TBI. Boxes and whiskers are shown for the 5th and 95th percentiles. *p<0.05.

[0035] [Figures 18A-18B] 18A-18B illustrate ROC curves for ASC (FIG. 18A) and IL-18 (FIG. 18B) from CSF samples of TBI patients and healthy donors.

[0036] [Figure 19A] Figures 19A-19C illustrate inflammasome proteins as prognostic biomarkers for TBI. Protein levels (pg / ml) of caspase-1 (Figure 19A), ASC (Figure 19B), and IL-18 (Figure 19C) in serum samples from patients with TBI. Groups were divided into favorable and unfavorable outcomes based on GOSE. Significance p-values ​​are shown in the respective box and whisker plots. Boxes and whiskers are shown for the 5th and 95th percentiles. Caspase-1: N=4 favorable and N=16 unfavorable; ASC: N=5 favorable and N=16 unfavorable; IL-18: N=5 favorable and N=16 unfavorable. [Figure 19B] Figures 19A-19C illustrate inflammasome proteins as prognostic biomarkers for TBI. Protein levels (pg / ml) of caspase-1 (Figure 19A), ASC (Figure 19B), and IL-18 (Figure 19C) in serum samples from patients with TBI. Groups were divided into favorable and unfavorable outcomes based on GOSE. Significance p-values ​​are shown in the respective box and whisker plots. Boxes and whiskers are shown for the 5th and 95th percentiles. Caspase-1: N=4 favorable and N=16 unfavorable; ASC: N=5 favorable and N=16 unfavorable; IL-18: N=5 favorable and N=16 unfavorable. [Figure 19C]Figures 19A-19C illustrate inflammasome proteins as prognostic biomarkers for TBI. Protein levels (pg / ml) of caspase-1 (Figure 19A), ASC (Figure 19B), and IL-18 (Figure 19C) in serum samples from patients with TBI. Groups were divided into favorable and unfavorable outcomes based on GOSE. Significance p-values ​​are shown in the respective box and whisker plots. Boxes and whiskers are shown for the 5th and 95th percentiles. Caspase-1: N=4 favorable and N=16 unfavorable; ASC: N=5 favorable and N=16 unfavorable; IL-18: N=5 favorable and N=16 unfavorable.

[0037] [Figures 20A-20B] 20A-20B illustrate the ROC curves for ASC outcome (favorable vs. unfavorable) for the second (FIG. 20A) and fourth (FIG. 20B) harvests.

[0038] [Figures 21A-21D] Figures 21A-21D illustrate that inflammasome proteins are elevated in the serum of MCI patients. Protein levels (pg / ml) of ASC (Figure 21A), caspase-1 (Figure 21B), IL-18 (Figure 21C), and IL-1 beta (Figure 21D) in serum samples from patients with MCI and age-matched healthy donors (controls). Significance p-values ​​are shown in the respective boxplots.

[0039] [Figures 22A-22D] Figures 22A-22D illustrate ROC curves for ASC (Figure 22A), caspase-1 (Figure 22B), IL-18 (Figure 22C), and IL-1beta (Figure 22D) from serum samples of MCI and age-matched healthy donors.

[0040] [Figure 23]Figure 23 illustrates inflammasome proteins in serum as biomarkers of MCI. ROC curves for caspase-1, ASC, IL-1 beta, and IL-18 from Figures 22A-22D are overlaid onto a single graph. DETAILED DESCRIPTION OF THE INVENTION

[0041] Detailed Description definition Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0042] As used herein, "protein" and "polypeptide" are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.

[0043] The terms "apoptosis-associated speck-like protein containing a caspase activation recruitment domain (CARD)" and "ASC" refer to the expression product of the ASC gene or an isoform thereof, or a protein having at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with ASC (e.g., NP_037390 (Q9ULZ3-1), NP_660183 (Q9ULZ3-2), or Q9ULZ3-3 in humans, or NP_758825 (BAC43754) in rats) and exhibiting the functional activity of ASC. The "functional activity" of a protein is any activity related to the physiological function of the protein. The functional activity of ASC includes, for example, activation of caspase-1 and recruitment of proteins to initiate cell death.

[0044] The term "ASC gene" or "ASC nucleic acid" refers to a nucleic acid sequence encoding a native ASC, a genomic sequence from which an ASC cDNA can be transcribed, and / or allelic variants and homologs of the foregoing. The term encompasses double-stranded DNA, single-stranded DNA, and RNA.

[0045] As used herein, the term "inflammasome" refers to a multiprotein (e.g., at least two protein) complex that activates caspase-1. Additionally, the term "inflammasome" can refer to a multiprotein complex that activates caspase-1 activity, which in turn controls the processing and activation of IL-1β, IL-18, and IL-33. See Arend et al. 2008; Li et al. 2008; and Martinon et al. 2002, each of which is incorporated by reference in its entirety. The terms "NLRP1 inflammasome," "NALP1 inflammasome," "NLRP2 inflammasome," "NALP2 inflammasome," "NLRP3 inflammasome," "NALP3 inflammasome," "NLRC4 inflammasome," "IPAF inflammasome," or "AIM2 inflammasome" refer to a protein complex of at least caspase-1 and one adaptor protein, such as ASC. For example, the terms "NLRP1 inflammasome" and "NALP1 inflammasome" can refer to a multiprotein complex containing NLRP1, ASC, caspase-1, caspase-11, XIAP, and pannexin-1 for activating caspase-1 and processing interleukin-1β, interleukin-18, and interleukin-33. The terms "NLRP2 inflammasome" and "NALP2 inflammasome" can refer to a multiprotein complex containing NLRP2 (also known as NALP2), ASC, and caspase-1, while the terms "NLRP3 inflammasome" and "NALP3 inflammasome" can refer to a multiprotein complex containing NLRP3 (also known as NALP3), ASC, and the terms "NLRC4 inflammasome" and "IPAF inflammasome" can refer to a multiprotein complex containing NLRC4 (also known as IPAF), ASC, and caspase-1. Additionally, the term "AIM2 inflammasome" can refer to a multiprotein complex containing AIM2, ASC, and caspase-1.

[0046] As used herein, the phrase "sequence identity" refers to the percentage of identical subunits at corresponding positions in two sequences (e.g., nucleic acid sequences, amino acid sequences) when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package from Accelrys CGC, San Diego, CA).

[0047] The phrases "therapeutically effective amount" and "effective dosage" refer to an amount sufficient to produce a therapeutically (e.g., clinically) desired result; the exact nature of this result will vary depending on the nature of the disorder being treated. For example, if the disorder being treated is SCI, the result may be improvement in motor skills and locomotor function, reduction in spinal cord pathology, and the like. The compositions described herein can be administered one or more times per day to one or more times per week. One of skill in the art will recognize that certain factors, including but not limited to, the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and the presence of other diseases, can affect the dosage and timing required to effectively treat a subject. Additionally, treatment of a subject with a therapeutically effective amount of a composition of the present invention can include a single treatment or a series of treatments.

[0048] As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent described herein or identified by the methods described herein to a patient, or to a tissue or cell system isolated from a patient having a disease, a symptom of a disease, or a predisposition to a disease, with the intent to treat, cure, alleviate, relieve, alter, correct, ameliorate, improve, or affect the disease, symptom of a disease, or predisposition to a disease.

[0049] The terms "patient," "subject," and "individual" are used interchangeably herein and refer to a mammalian subject being treated, such as, for example, a human patient. In some cases, the methods of the present invention find use in laboratory animals, veterinary applications, and the development of animal models for disease, including, but not limited to, rodents, including mice, rats, and hamsters, and primates.

[0050] As used interchangeably herein, "Absent in Melanoma2" and "AIM2" can refer to the expression product of the AIM2 gene or isoform; or a protein having at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to AIM2 (e.g., Accession Nos. NX_014862, NP004824, XP016858337, XP005245673, AAB81613, BAF84731, AAH10940) and exhibiting the functional activity of AIM2.

[0051] As used interchangeably herein, "NALP1" and "NLRP1" refer to the expression product of the NALP1 or NLRP1 gene or isoform; or a protein having at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to NALP1 (e.g., Accession Nos. AAH51787, NP_001028225, NP_127500, NP_127499, NP_127497, NP055737) and exhibiting the functional activity of NALP1.

[0052] As used interchangeably herein, "NALP2" and "NLRP2" refer to the expression product of the NALP2 or NLRP2 gene or isoform; or a protein having at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to NALP2 (e.g., Accession Nos. NP_001167552, NP_001167553, NP_001167554, or NP_060322) and exhibiting the functional activity of NALP2.

[0053] As used interchangeably herein, "NALP3" and "NLRP3" refer to the expression product of the NALP3 or NLRP3 gene or isoform; or a protein having at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to NALP3 (e.g., Accession Nos. NP_001073289, NP_001120933, NP_001120934, NP_001230062, NP_004886, NP_899632, XP_011542350, XP_016855670, XP_016855671, XP_016855672, or XP_016855673) and exhibiting the functional activity of NALP3.

[0054] As used interchangeably herein, "NLRC4" and "IPAF" refer to the expression product of the NLRC4 or IPAF gene or isoform; or a protein having at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to NLRC4 (e.g., Accession Nos. NP_001186067, NP001186068, NP_001289433, or NP_067032) and exhibiting the functional activity of NLRC4.

[0055] The terms "stroke" and "ischemic stroke" refer to when blood flow to a portion of the brain or spinal cord is interrupted. The terms "ischemic stroke" and "transient ischemic stroke" refer to when blood flow to a portion of the brain or spinal cord is interrupted by blockage of an artery that supplies oxygen-rich blood to the brain or spinal cord. The term "hemorrhagic stroke" refers to when blood flow to a portion of the brain or spinal cord is interrupted when an artery in the brain or spinal cord leaks blood or ruptures.

[0056] "Traumatic injury to the CNS" means any insult to the CNS from an external mechanical force, possibly resulting in permanent or transient impairment of the function of the CNS.

[0057] The term "antibody" is intended to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, anti-idiotypic (anti-Id) antibodies, antibodies that may be labeled in soluble or conjugated form, and fragments, regions, or derivatives thereof provided by any known method, including but not limited to, enzymatic cleavage, peptide synthesis, or recombinant techniques. Such anti-ASC and anti-NLRP1 antibodies of the present invention are capable of binding to portions of ASC and NLRP1, respectively, that interfere with the activation of caspase-1.

[0058] The method described herein comprises conventional molecular biology techniques.This type of technique is generally known in the art and is described in detail in methodological treatises such as Molecular Cloning: A Laboratory Manual, 3rd edition, vol.1-3, ed.Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; and Current Protocols in Molecular Biology, ed.Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (regularly updated). Immunological techniques are generally known in the art and are described in Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic Press, Burlington, MA, 2007; Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howard and Matthew R. Kaser, CRC Press, Boca Raton, FL, 2006; Medical Immunology, 6 thed., edited by Gabriel Virella, Informa Healthcare Press, London, England, 2007; and Harlow and Lane ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988.

[0059] Although compositions and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions and methods are described below.All publications, patent applications and patents listed herein are incorporated by reference in their entirety.In the case of conflict, the present specification, including definitions, will control.The specific embodiments discussed below are merely illustrative and are not intended to be limiting.

[0060] Overview Provided herein are compositions and methods for diagnosing or evaluating patients suspected of having brain injury. The method may include measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with brain injury, wherein the protein signature comprises an elevated level of at least one inflammasome protein; and selecting the patient as having brain injury if the patient exhibits the presence of the protein signature. The brain injury may be any insult to the patient's brain caused by trauma, degeneration, or congenital problems. The brain injury may be selected from multiple sclerosis (MS), stroke, Alzheimer's disease (AD), Parkinson's disease (PD), cognitive dysfunction (e.g., mild cognitive impairment (MCI)), or traumatic brain injury (TBI). In one embodiment, the brain injury is MS. In another embodiment, the brain injury is stroke. In yet another embodiment, the brain injury is TBI. In yet another embodiment, the brain injury is MCI.

[0061] In one embodiment, the present disclosure provides a method for diagnosing or evaluating patients with multiple sclerosis (MS), comprising the steps of: measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with MS, wherein the protein signature comprises an elevated level of at least one inflammasome protein; and if the patient shows the presence of the protein signature, selecting the patient as having MS. The patient may exhibit clinical symptoms consistent with MS. By using the methods and compositions provided herein, the patient may be diagnosed with any type of MS known in the art. MS may be relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS) or progressive relapsing MS (PRMS).

[0062] In another embodiment, the present specification provides a method for diagnosing or evaluating a patient suspected of suffering from stroke, comprising the steps of: measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with stroke or stroke-related injury, wherein the protein signature comprises an elevated level of at least one inflammasome protein; and if the patient shows the presence of the protein signature, selecting the patient as suffering from stroke.The patient can exhibit clinical symptoms that are known in the art to be consistent with stroke.The stroke can be ischemic stroke, transient ischemic stroke or hemorrhagic stroke.

[0063] In one embodiment, the present specification provides a method for diagnosing or evaluating patients with traumatic brain injury (TBI), comprising: measuring the level of at least one inflammasome protein in biological samples obtained from patients; determining the presence or absence of a protein signature associated with TBI, wherein the protein signature comprises an elevated level of at least one inflammasome protein; and if the patient shows the presence of the protein signature, selecting the patient as having TBI.Patient can exhibit clinical symptoms consistent with TBI.By using the method and composition provided herein, patient can be diagnosed with any kind of TBI known in the art.

[0064] In one embodiment, the present specification provides a method for diagnosing or evaluating patients with cognitive impairment.Cognitive impairment can be mild or severe.In one embodiment, cognitive impairment is mild cognitive impairment (MCI).This method includes the steps of: measuring the level of at least one inflammasome protein in biological samples obtained from patients; determining the presence or absence of a protein signature associated with cognitive impairment (for example, MCI), wherein the protein signature comprises an elevated level of at least one inflammasome protein; and if the patient shows the presence of the protein signature, selecting the patient as having cognitive impairment (for example, MCI).The patient can exhibit clinical symptoms consistent with cognitive impairment (for example, MCI).By using the methods and compositions provided herein, the patient can be diagnosed with any kind of cognitive impairment known in the art, such as MCI. Examples of symptoms often exhibited by subjects with MCI include forgetfulness (forgetting things more frequently and / or forgetting important events), difficulty concentrating (losing one's train of thought), feeling anxious or confused when making decisions, understanding instructions, or planning things, trouble navigating familiar environments, and / or impulsive and problematic judgment. Subjects with MCI may also experience depression, irritability, anxiety, or blunted affect.

[0065] In one aspect of the present invention, a method for diagnosing or evaluating a patient suspected of having a brain injury (e.g., MCI, TBI, stroke, or MS) comprises determining the presence or absence of a protein signature associated with brain injury based on the measured level, abundance, or concentration of one or more inflammasome proteins in a biological sample obtained from the patient, or an inflammasome protein profile prepared from the biological sample obtained from the patient. In certain embodiments, the protein signature comprises an elevated level of at least one inflammasome protein. The level of at least one inflammasome protein in the protein signature may be elevated relative to the level or percentage of the protein in a biological sample obtained from a control subject, or relative to a predetermined reference value or range of reference values ​​further described herein. The control subject may be a healthy individual. The healthy individual may be an individual who does not exhibit symptoms associated with brain injury (e.g., MCI, TBI, stroke, or MS). In certain embodiments, the protein signature may comprise an elevated level of at least one inflammasome protein. Patients exhibiting the protein signature can be selected or identified as having a brain injury (e.g., MCI, TBI, stroke, or MS).

[0066] In some embodiments, the measured level, concentration or abundance of one or more inflammasome proteins in biological samples is used to prepare an inflammasome protein profile, where the profile indicates the severity of brain injury (e.g., MCI, TBI, stroke or MS).The inflammasome protein profile can optionally include the level, abundance, percentage or concentration of one or more inflammasome proteins measured in patient biological samples relative to the level, abundance, percentage or concentration of one or more inflammasome proteins in biological samples obtained from control subjects, or relative to a predetermined value or reference value range described herein.The control subject can be a healthy individual.A healthy individual can be an individual who does not show symptoms related to brain injury (e.g., MCI, TBI, stroke or MS).

[0067] The level, percentage or concentration of at least one inflammasome protein can be assessed at a single time point and compared to a predetermined reference value or range of reference values, or can be assessed at multiple time points and compared to a predetermined reference value or previously assessed value.

[0068] As used herein, a "predetermined reference value" or a range of reference values ​​can refer to a predetermined value or a range of reference values ​​for the level or concentration of an inflammasome protein ascertained from a known sample. For example, the predetermined reference value or range of reference values ​​can reflect the level or concentration of an inflammasome protein in a biological sample obtained from a control subject (e.g., a healthy subject). In some embodiments, the control subject can be age-matched to the patient being evaluated. The biological samples obtained from the patient and the control subject can be the same type of sample (e.g., serum or serum-derived extracellular vesicles (EVs)). Thus, in certain embodiments, the measured level, percentage, or concentration of at least one inflammasome protein is compared or determined to the level, percentage, or concentration of the at least one inflammasome protein in a control sample (i.e., obtained from a healthy subject). The control or healthy subject can be a subject who does not exhibit symptoms associated with brain injury (e.g., MCI, TBI, stroke, or MS).

[0069] In other embodiments, the predetermined reference value or range of reference values ​​may reflect the level or concentration of an inflammasome protein in a sample obtained from a patient with a known severity of brain injury (e.g., MCI, TBI, stroke, or MS) assessed by clinical measurement or postmortem analysis. The predetermined reference value may also be a known amount or concentration of the inflammasome protein. Such a known amount or concentration of the inflammasome protein may be correlated with the average level or concentration of the inflammasome protein from a population of control subjects or a population of patients with a known level of the brain injury. In another embodiment, the predetermined reference value may be a range of values, which may represent, for example, the mean plus or minus the standard deviation or a confidence interval. The range of reference values ​​may also refer to individual reference values ​​for a particular inflammasome protein across various levels of severity of brain injury (e.g., MCI, TBI, stroke, or MS). In certain embodiments, an increased level of one or more inflammasome proteins (e.g., ASC, caspase-1, or IL-18) relative to the predetermined reference value or range of reference values ​​indicates more severe brain injury.

[0070] The at least one inflammasome protein detected or measured by any of the methods provided herein can be one or more inflammasome proteins. In one embodiment, the at least one inflammasome protein is a plurality of inflammasome proteins. A plurality can be at least or at most two, three, four, or five inflammasome proteins. The at least one inflammasome protein or a plurality of inflammasome proteins can be any inflammasome component known in the art, such as, for example, NAPL1 / NLRP1, NALP2 / NLRP2, NALP3 / NLRP3, IPAF / NLRC4, or AIM2 inflammasome. In one embodiment, the at least one inflammasome protein is apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, interleukin-18 (IL-18), or interleukin-1 beta (IL-1 beta). In one embodiment, the at least one inflammasome protein is apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain. In one embodiment, the at least one inflammasome protein is caspase- 1. In one embodiment, the at least one inflammasome protein is IL-18.

[0071] Inflammasome protein and other marker proteins of the methods provided herein can be measured in biological samples by various methods known to those skilled in the art.For example, protein can be measured by methods including but not limited to liquid chromatography, gas chromatography, mass spectrometry, immunoassay, radioimmunoassay, immunofluorescence assay, FRET-based assay, immunoblot, ELISA or liquid chromatography followed by mass spectrometry (for example, MALDI MS).Those skilled in the art can ascertain other suitable methods for measuring and quantifying any particular biomarker protein of the present invention.

[0072] In one embodiment, at least one inflammasome protein or multiple inflammasome proteins that are detected or measured by any method provided herein can be detected or measured by using immunoassay.Immunoassay can be any immunoassay known in the art.For example, immunoassay can be immunoblot, enzyme-linked immunosorbent assay (ELISA) or microfluidic immunoassay.An example of the microfluidic immunoassay that can be used in the method provided herein is Simple Plex™ platform (Protein Simple, San Jose, California).

[0073] Immunoassays for use in the methods provided herein can utilize antibodies directed against inflammasome proteins. The inflammasome component can be any inflammasome component known in the art, such as, for example, NAPL1, NALP2, NALP3, NLRC4, or AIM2 inflammasome. In one embodiment, the inflammasome protein is apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, interleukin-18 (IL-18), or interleukin-1 beta (IL-1 beta). In one embodiment, the inflammasome protein is apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain. In one embodiment, the inflammasome protein is caspase-1. In one embodiment, the inflammasome protein is IL-18. In one embodiment, the inflammasome protein is IL-1 beta.

[0074] Any suitable antibody that specifically binds to ASC can be used, for example, custom-made or commercially available ASC antibody can be used in the methods provided herein.Anti-ASC antibody can be, for example, an antibody that specifically binds to the domain or part of mammalian ASC protein, such as human or rat ASC protein.An example of anti-ASC antibody for use in the methods herein can be found in U.S. Patent No. 8,685,400, the contents of which are incorporated herein by reference in their entirety. Examples of commercially available anti-ASC antibodies for use in the methods provided herein include, but are not limited to, 04-147 anti-ASC from MilliporeSigma, clone 2EI-7 mouse monoclonal antibody, AB3607-anti-ASC antibody from MilliporeSigma, orb194021 anti-ASC from Biorbyt, LS-C331318-50 anti-ASC from LifeSpan Biosciences, AF3805 anti-ASC from R&D Systems, NBP1-78977 anti-ASC from Novus Biologicals, 600-401-Y67 anti-ASC from Rockland Immunochemicals, D086-3 anti-ASC from MBL International, AL177 anti-ASC from Adipogen, monoclonal anti-ASC (clone o93E9) antibody, anti-ASC antibody (F-9) from Santa Cruz Biotechnology, anti-ASC antibody (B-3) from Santa Cruz Biotechnology, Enzo Life Examples of suitable ASC antibodies include the ASC polyclonal antibody ADI-905-173 from Biosciences or the A161 anti-human ASC from Leinco Technologies. Human ASC proteins can be under accession numbers NP_037390.2 (Q9ULZ3-1), NP_660183 (Q9ULZ3-2) or Q9ULZ3-3. Rat ASC proteins can be under accession number NP_758825 (BAC43754). Mouse ASC proteins can be under accession number NP_075747.3.In one embodiment, the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD) of a mammalian ASC protein (e.g., human or rat ASC), or a portion or fragment thereof. In this embodiment, the antibody described herein specifically binds to an amino acid sequence having at least 65% (e.g., 65, 70, 75, 80, 85%) sequence identity with the PYD domain of human or rat ASC, or a fragment thereof. In one embodiment, the antibody binds to the C-terminal caspase recruitment domain (CARD) of a mammalian ASC protein (e.g., human or rat ASC), or a portion or fragment thereof. In this embodiment, the antibody described herein specifically binds to an amino acid sequence having at least 65% (e.g., 65, 70, 75, 80, 85%) sequence identity with the CARD domain of human or rat ASC, or a fragment thereof. In another embodiment, the antibody specifically binds to a region of rat ASC, e.g., the amino acid sequence of ALRQTQPYLVTDLEQS (SEQ ID NO: 1) (i.e., residues 178-193 of rat ASC with accession number BAC43754). In this embodiment, the antibody described herein specifically binds to an amino acid sequence that shares at least 65% (e.g., 65, 70, 75, 80, or 85%) sequence identity with the amino acid sequence of ALRQTQPYLVTDLEQS (SEQ ID NO: 1) of rat ASC. In another embodiment, the antibody specifically binds to a region of human ASC, e.g., the amino acid sequence of RESQSYLVEDLERS (SEQ ID NO: 2). In this embodiment, the antibody described herein specifically binds to an amino acid sequence that shares at least 65% (e.g., 65, 70, 75, 80, or 85%) sequence identity with the amino acid sequence of RESQSYLVEDLERS (SEQ ID NO: 2) of human ASC.

[0075] Any suitable anti-NLRP1 antibody (e.g., commercially available or custom-made) can be used in the methods provided herein.An example of an anti-NLRP1 antibody for use in the methods herein can be found in U.S. Patent No. 8,685,400, the contents of which are incorporated herein by reference in their entirety. Examples of commercially available anti-NLRP1 antibodies for use in the methods provided herein include, but are not limited to, human NLRP1 polyclonal antibody AF6788 from R&D Systems, rabbit polyclonal anti-NLRP1 ABF22 from EMD Millipore, rabbit polyclonal anti-NLRP1 NB100-56148 from Novus Biologicals, mouse polyclonal anti-NLRP1 SAB1407151 from Sigma-Aldrich, rabbit polyclonal anti-NLRP1 ab3683 from Abcam, rabbit polyclonal anti-NLRP1 orb325922 from Biorbyt, rabbit polyclonal anti-NLRP1 MBS7001225 from mybiosource, sheep polyclonal AF6788 from R&D systems, mouse monoclonal anti-NLRP1 oaed00344 from Aviva Systems, and rabbit polyclonal anti-NLRP1 from Aviva Systems. Examples of antibodies that may be used include ARO54478_P050, rabbit polyclonal anti-NLRP1 APO7775PU-N from Origene, rabbit polyclonal anti-NLRP1 ABIN768983 from Antibodies online, rabbit polyclonal anti-NLRP1 3037 from Prosci, rabbit polyclonal anti-NLRP1 12256-1-AP from Proteintech, mouse monoclonal anti-NLRP1 ALX-804-803-C100 from Enzo, mouse monoclonal anti-NLRP1 MA1-25842 from Invitrogen, mouse monoclonal anti-NLRP1 GTX16091 from GeneTex, rabbit polyclonal anti-NLRP1 200-401-CX5 from Rockland, and rabbit polyclonal anti-NLRP1 4990 from Cell Signaling Technology.The human NLRP1 protein may have accession numbers AAH51787, NP_001028225, NP_055737, NP_127497, NP_127499, or NP_127500. In one embodiment, the antibody binds to the pyrin, NACHT, LRR1-6, FIIND, or CARD domain of a mammalian NLRP1 protein (e.g., human NLRP1), or a portion or fragment thereof. In this embodiment, the antibody described herein specifically binds to an amino acid sequence that shares at least 65% (e.g., 65, 70, 75, 80, 85%) sequence identity with a specific domain of human NLRP1 (e.g., pyrin, NACHT, LRR1-6, FIIND, or CARD) or a fragment thereof. In one embodiment, a chicken anti-NLRP1 polyclonal antibody custom-designed and manufactured by Ayes Laboratories can be used. The antibody may be made against the following amino acid sequence of human NLRP1: CEYYTEIREREREKSEKGR (SEQ ID NO: 3). In one embodiment, the antibody specifically binds to an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0076] Any suitable antibody that specifically binds to caspase-1 can be used, for example, custom-made or commercially available, and can be used in the methods provided herein. Examples of commercially available anti-caspase-1 antibodies for use in the methods provided herein include R&D Systems: Catalog No. MAB6215 or Catalog No. AF6215; Cell Signaling: Catalog No. 3866, Catalog No. 225, or Catalog No. 4199; Novus Biologicals: Catalog No. NB100-56565, Catalog No. NBP1-45433, Catalog No. NB100-56564, Catalog No. MAB6215, Catalog No. AF6215, Catalog No. NBP2-67487, Catalog No. NBP2-15713, Catalog No. NBP2-15712, Catalog No. NBP1-87680, Catalog No. NB120-1872, Catalog No. NBP1-76605, or Catalog No. H00000834-M01.

[0077] Any suitable antibody that specifically binds to IL-18 can be used, for example, custom-made or commercially available, and can be used in the methods provided herein. Examples of commercially available anti-IL-18 antibodies for use in the methods provided herein include R&D Systems: Catalog No. D044-3, Catalog No. D045-3, Catalog No. MAB646, Catalog No. AF2548, Catalog No. D043-3, Catalog No. MAB2548, MAB9124, Catalog No. MAB91241, Catalog No. MAB91243, Catalog No. MAB91244, or Catalog No. MAB91242; Novus Biologicals: Catalog No. AF2548, Catalog No. D043-3, Catalog No. MAB2548, Catalog No. MAB9124, Catalog No. MAB91243, Catalog No. MAB91244, Catalog No. MAB91241, Catalog No. D045-3, Catalog No. MAB91242, or Catalog No. D044-3.

[0078] Any suitable antibody that specifically binds to IL-1 beta can be used, for example, custom-made or commercially available, and can be used in the methods provided herein. Examples of commercially available anti-IL-18 antibodies for use in the methods provided herein include R&D Systems: Catalog No. MAB601, Catalog No. MAB201, Catalog No. MAB6964, Catalog No. MAB601R, Catalog No. MAB8406, or Catalog No. MAB6215; Cell Signaling: Catalog No. 31202, Catalog No. 63124, Catalog No. 12426, or Catalog No. 12507; Novus Biologicals: Catalog number AF-201-NA, Catalog number NB600-633, Catalog number MAB201, Catalog number MAB601, Catalog number NBP1-19775, Catalog number NBP2-27345, Catalog number AB-201-NA, Catalog number NBP2-27342, Catalog number NBP2-67865, Catalog number NBP2-27343, Catalog number NBP2-27340, Catalog number NBP2-27340, Catalog number NB120-8319, Catalog number 23600002, Catalog number MAB8406, Catalog number NB100-73053, Catalog number NB120-10749, or Catalog number MAB601R.

[0079] Methods for determining the specificity and affinity of monoclonal antibodies by competitive inhibition can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993); and Muller, Meth. Enzymol. 92:589-601, 1983, which references are incorporated herein by reference in their entireties.

[0080] Anti-inflammasome (e.g., anti-ASC and anti-NLRP1) antibodies of the present invention can be routinely produced according to methods such as, but not limited to, inoculation of suitable animals with polypeptides or antigen fragments, in vitro stimulation of lymphocyte populations, synthetic methods, hybridomas, and / or recombinant cells expressing nucleic acids encoding such anti-ASC or anti-NLRP1 antibodies. Immunization of animals with purified recombinant ASC or a peptide fragment thereof, such as residues 178-193 (SEQ ID NO: 1) of rat ASC (e.g., accession number BAC43754) or SEQ ID NO: 2 of human ASC, is an example of a method for preparing anti-ASC antibodies. Similarly, immunization of animals with purified recombinant NLRP1 or a peptide fragment thereof, such as residues MEE SQS KEE SNT EG-Cys (SEQ ID NO: 4) of rat NARP1 or SEQ ID NO: 3 of human NALP1, is an example of a method for preparing anti-NLRP1 antibodies.

[0081] The monoclonal antibody that specifically binds to ASC or NLRP1 can be obtained by methods known to those skilled in the art.For example, see Kohler and Milstein, Nature 256:495-497, 1975; United States Patent No. 4,376,110; Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, NY, (1987, 1992); Harlow and Lane ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), the contents of which are incorporated herein by reference in their entirety.Such antibody can be any immunoglobulin class, including IgG, IgM, IgE, IgA, GILD, and any subclass thereof. A hybridoma producing a monoclonal antibody of the present invention may be cultivated in vitro, in situ or in vivo.

[0082] In any of the methods provided herein, a "biological sample" can refer to any bodily fluid or tissue obtained from a patient or subject. Biological samples can include, but are not limited to, whole blood, red blood cells, plasma, serum, peripheral blood mononuclear cells (PBMCs), urine, saliva, tears, buccal swabs, CSF, CNS microdialysis fluid, and neural tissue. In one embodiment, the biological sample is CSF, saliva, serum, plasma, or urine. In a specific embodiment, the biological sample is CSF. In another embodiment, the biological sample is serum-derived extracellular vesicles (EVs). EVs can be separated from serum by any method known in the art. It should be noted that the biological sample obtained from a patient or test subject can be the same type as the biological sample obtained from a control subject.

[0083] In some examples, the methods provided herein may be capable of diagnosing or detecting brain injury (e.g., MCI, stroke, MS or TBI) with at least about 70%, at least about 71%, at least about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, up to a 100% predictive success rate.

[0084] In some examples, the methods provided herein may have the ability to diagnose or detect brain injury (e.g., MCI, stroke, MS or TBI) with a sensitivity and / or specificity of at least about 70%, at least about 71%, at least about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to 100%.

[0085] In one embodiment, the brain injury is MS, such that detecting an elevated level of ASC in serum obtained from the patient, compared to a control provided herein (e.g., a predetermined reference value or range of reference values), determines that the patient has MS with at least 75%, 80%, 90%, 95%, 99%, or 100% sensitivity. In another embodiment, the brain injury is MS, such that detecting an elevated level of ASC in serum obtained from the patient, compared to a control provided herein (e.g., a predetermined reference value or range of reference values), determines that the patient has MS with at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% specificity. The predetermined reference value for these embodiments may be the cutoff value shown in Table 7. In yet another embodiment, the brain injury is MS, such that detecting an elevated level of ASC in serum obtained from the patient, compared to a control provided herein (e.g., a predetermined reference value or range of reference values), determines that the patient has MS with at least 90% sensitivity and at least 80% specificity. The predetermined reference values ​​for this embodiment may be the cutoff values ​​shown in Table 7. In some cases, the range of reference values ​​may be from about 300 pg / ml to about 340 pg / ml to reach a sensitivity of at least 90% and a specificity of at least 80%.

[0086] In one embodiment, the brain injury is a stroke, such that detecting an elevated level of ASC in serum obtained from the patient, compared to a control provided herein (e.g., a predetermined reference value or range of reference values), determines that the patient has a stroke with at least 75%, 80%, 90%, 95%, 99% or 100% sensitivity. In another embodiment, the brain injury is a stroke, such that detecting an elevated level of ASC in serum obtained from the patient, compared to a control provided herein (e.g., a predetermined reference value or range of reference values), determines that the patient has MS with at least 75%, 80%, 85%, 90%, 95%, 99% or 100% specificity. The predetermined reference values ​​for these embodiments may be the cut-off values ​​shown in Table 8. In another embodiment, the brain injury is a stroke, such that detecting an elevated level of ASC in serum obtained from the patient compared to a control (e.g., a predetermined reference value or range of reference values) provided herein determines that the patient has suffered a stroke with at least 100% sensitivity and at least 90% specificity. The predetermined reference value for this embodiment may be the cutoff value shown in Table 8. In some cases, the range of reference values ​​may be from about 380 pg / ml to about 405 pg / ml to achieve at least 100% sensitivity and at least 90% specificity. The stroke may be ischemic or hemorrhagic, as provided herein.

[0087] In one embodiment, the brain injury is a stroke such that detecting an elevated level of ASC in serum-derived EVs obtained from the patient, compared to a control (e.g., a predetermined reference value or range of reference values) provided herein, determines that the patient has a stroke with at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sensitivity. In another embodiment, the brain injury is a stroke such that detecting an elevated level of ASC in serum-derived EVs obtained from the patient, compared to a control (e.g., a predetermined reference value or range of reference values) provided herein, determines that the patient has MS with at least 75%, 80%, 90%, 95%, 99%, or 100% specificity. The predetermined reference values ​​for these embodiments may be the cutoff values ​​shown in Table 9. In another embodiment, the brain injury is a stroke, such that detecting an elevated level of ASC in serum-derived EVs obtained from the patient, compared to a control (e.g., a predetermined reference value or range of reference values) provided herein, determines with at least 100% sensitivity and at least 90% specificity that the patient has suffered a stroke. The predetermined reference value for this embodiment may be the cutoff value shown in Table 9. In some cases, the range of reference values ​​may be from about 70 pg / ml to about 90 pg / ml to reach at least 100% sensitivity and at least 90% specificity. The stroke may be ischemic or hemorrhagic, as provided herein.

[0088] In one embodiment, the brain injury is a TBI such that detecting an elevated level of ASC in serum obtained from the patient, compared to a control provided herein (e.g., a predetermined reference value or range of reference values), determines that the patient has a TBI with at least 75%, 80%, 90%, 95%, 99%, or 100% sensitivity. In another embodiment, the brain injury is a TBI such that detecting an elevated level of ASC in serum obtained from the patient, compared to a control provided herein (e.g., a predetermined reference value or range of reference values), determines that the patient has a TBI with at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% specificity. The predetermined reference values ​​for these embodiments may be the cutoff values ​​shown in Table 16. In yet another embodiment, the brain injury is a TBI such that detecting an elevated level of ASC in serum obtained from the patient, compared to a control provided herein (e.g., a predetermined reference value or range of reference values), determines that the patient has a TBI with at least 90% sensitivity and at least 80% specificity. The predetermined reference values ​​for this embodiment may be the cutoff values ​​shown in Table 16. In some cases, the range of reference values ​​may be from about 275 pg / ml to about 450 pg / ml to reach a sensitivity of at least 80% and a specificity of at least 70%.

[0089] In one embodiment, the brain injury is a TBI such that detecting an elevated level of caspase-1 in serum obtained from the patient compared to a control provided herein (e.g., a predetermined reference value or range of reference values) determines that the patient has a TBI with at least 75%, 80%, 90%, 95%, 99%, or 100% sensitivity. In another embodiment, the brain injury is a TBI such that detecting an elevated level of caspase-1 in serum obtained from the patient compared to a control provided herein (e.g., a predetermined reference value or range of reference values) determines that the patient has a TBI with at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% specificity. The predetermined reference values ​​for these embodiments may be the cutoff values ​​shown in Table 15. In yet another embodiment, the brain injury is a TBI such that detection of an elevated level of caspase-1 in serum obtained from the patient, compared to a control (e.g., a predetermined reference value or range of reference values) provided herein, determines that the patient has a TBI with at least 90% sensitivity and at least 80% specificity. The predetermined reference values ​​for this embodiment may be the cutoff values ​​shown in Table 15. In some cases, the range of reference values ​​may be from about 2.812 pg / ml to about 1.853 pg / ml to reach at least 70% sensitivity and at least 75% specificity.

[0090] In one embodiment, the brain injury is MCI, such that detecting an elevated level of ASC in serum obtained from the patient, compared to a control provided herein (e.g., a predetermined reference value or range of reference values), determines that the patient has MCI with at least 75%, 80%, 85%, 90%, 95%, 99% or 100% sensitivity. In another embodiment, the brain injury is MCI, such that detecting an elevated level of ASC in serum obtained from the patient, compared to a control provided herein (e.g., a predetermined reference value or range of reference values), determines that the patient has MCI with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% specificity. The predetermined reference values ​​for these embodiments may be the cut-off values ​​shown in Tables 22 and 23. In yet another embodiment, the brain injury is MCI, such that detecting an elevated level of ASC in serum obtained from the patient, compared to a control (e.g., a predetermined reference value or range of reference values) provided herein, determines that the patient has MCI with at least 90% sensitivity and at least 70% specificity. The predetermined reference values ​​for this embodiment may be the cutoff values ​​shown in Tables 22 and 23. In some cases, the range of reference values ​​may be from about 257 pg / ml to about 342 pg / ml to achieve at least 90% sensitivity and at least 70% specificity.

[0091] In one embodiment, the brain injury is MCI such that detecting an elevated level of IL-18 in serum obtained from the patient compared to a control provided herein (e.g., a predetermined reference value or range of reference values) determines that the patient has MCI with at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sensitivity. In another embodiment, the brain injury is MCI such that detecting an elevated level of IL-18 in serum obtained from the patient compared to a control provided herein (e.g., a predetermined reference value or range of reference values) determines that the patient has MCI with at least 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% specificity. The predetermined reference values ​​for these embodiments may be the cutoff values ​​shown in Tables 22 and 25. In yet another embodiment, the brain injury is MCI, such that detecting an elevated level of IL-18 in serum obtained from the patient, compared to a control (e.g., a predetermined reference value or range of reference values) provided herein, determines that the patient has MCI with at least 70% sensitivity and at least 55% specificity. The predetermined reference values ​​for this embodiment may be the cutoff values ​​shown in Tables 22 and 25. In some cases, the range of reference values ​​is from about 200 pg / ml to about 214 pg / ml to reach at least 70% sensitivity and at least 50% specificity.

[0092] In any of the methods provided herein, the sensitivity and / or specificity of an inflammasome protein (e.g., ASC) for predicting or diagnosing brain injury (e.g., MCI, stroke, MS, or TBI) is determined by calculating the area under the curve (AUC) value with a confidence interval (e.g., 95%). The area under the curve (AUC) can be determined from a receiver operating characteristic (ROC) curve with a 95% confidence interval.

[0093] In one embodiment, the brain injury is MS, such that detecting a level or concentration of at least one inflammasome protein in a biological sample obtained from a patient that is elevated by a predetermined percentage above the level of the same at least one inflammasome protein in a biological sample obtained from a control subject indicates that the patient has MS. The biological samples obtained from the patient and the control subject can be of the same type (e.g., serum or serum-derived EV). The predetermined percentage can be approximately, at most, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The at least one inflammasome protein can be selected from caspase-1, IL-18, IL-1 beta, and ASC. In one embodiment, the brain injury is MS, such that detection of a level or concentration of ASC in serum obtained from the patient that is at least 50% higher than the level of ASC in a serum sample obtained from a control subject indicates the patient as having MS.

[0094] In one embodiment, the brain injury is a stroke, such that detecting a level or concentration of at least one inflammasome protein in a biological sample obtained from a patient that is elevated by a predetermined percentage above the level of the same at least one inflammasome protein in a biological sample obtained from a control subject indicates that the patient has MS. The biological samples obtained from the patient and the control subject can be of the same type (e.g., serum or serum-derived EV). The predetermined percentage can be approximately, at most, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The at least one inflammasome protein can be selected from caspase-1, IL-18, IL-1 beta, and ASC. In one embodiment, the brain injury is a stroke, such that detecting a level or concentration of ASC in serum obtained from the patient that is at least 70% higher than the level of ASC in a serum sample obtained from a control subject indicates the patient has suffered a stroke. In one embodiment, the brain injury is a stroke, such that detecting a level or concentration of ASC in serum-derived EVs obtained from the patient that is at least 110% higher than the level of ASC in a serum-derived EV sample obtained from a control subject indicates the patient has suffered a stroke.

[0095] In one embodiment, the brain injury is a traumatic brain injury (TBI), such that detecting a level or concentration of at least one inflammasome protein in a biological sample obtained from a patient that is elevated by a predetermined percentage above the level of the same at least one inflammasome protein in a biological sample obtained from a control subject indicates that the patient has TBI. The biological samples obtained from the patient and the control subject can be of the same type (e.g., serum or serum-derived EV). The predetermined percentage can be approximately, at most, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The at least one inflammasome protein can be selected from caspase-1, IL-18, IL-1 beta, and ASC. In one embodiment, the brain injury is a TBI such that detection of a level or concentration of ASC in serum obtained from the patient that is at least 50% higher than the level of ASC in a serum sample obtained from a control subject indicates the patient as having a TBI.

[0096] In one embodiment, the brain injury is MCI, such that detecting a level or concentration of at least one inflammasome protein in a biological sample obtained from a patient that is elevated by a predetermined percentage above the level of the same at least one inflammasome protein in a biological sample obtained from a control subject indicates that the patient has MCI. The biological samples obtained from the patient and the control subject can be of the same type (e.g., serum or serum-derived EV). The predetermined percentage can be approximately, at most, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The at least one inflammasome protein can be selected from caspase-1, IL-18, IL-1 beta, and ASC. In one embodiment, the brain injury is MCI, such that detection of a level or concentration of ASC in serum obtained from the patient that is at least 50% higher than the level of ASC in a serum sample obtained from a control subject indicates the patient as having MCI.

[0097] The present invention also provides a method for determining a prognosis for a patient with a brain injury (e.g., MCI, stroke, MS, or TBI). In one embodiment, the method includes providing a biological sample obtained from the patient and measuring the level of at least one inflammasome protein in the biological sample to prepare an inflammasome protein profile as described above, wherein the inflammasome protein profile indicates the patient's prognosis. In some embodiments, an increase in the level of one or more inflammasome proteins (e.g., IL-18, NLRP1, ASC, caspase-1, or a combination thereof) relative to a predetermined reference value or range of reference values ​​indicates a worse prognosis. For example, an increase of about 20% to about 300% in the level of one or more inflammasome proteins relative to a predetermined reference value or range of reference values ​​indicates a worse prognosis. In some cases, the inflammasome protein is ASC, and the predetermined reference value can be from Tables 7-9, 16, 22, or 23.

[0098] Treatment method In other embodiments of the present invention, the method of diagnosing or evaluating a patient as having brain injury (e.g., MCI, stroke, MS, or TBI) further comprises administering a standard of care treatment for the brain injury (e.g., MCI, TBI, stroke, or MS) to the patient based on the measured level of the at least one inflammasome protein or if a protein signature associated with brain injury (e.g., MCI, stroke, or MS or TBI) is identified. The method of diagnosing or evaluating a patient as having brain injury (e.g., MCI, stroke, MS, or TBI) can be confirmed using the methods described herein. In some embodiments, the method of diagnosing or evaluating a patient as having brain injury further comprises administering a neuroprotective treatment to the patient based on the measured level of the at least one inflammasome protein or if a protein signature associated with brain injury, or if more severe brain injury, is identified. Such neuroprotective treatments include drugs that reduce excitotoxicity, oxidative stress, and inflammation. Thus, suitable neuroprotective treatments include, but are not limited to, methylprednisolone, 17alpha-estradiol, 17beta-estradiol, ginsenoside, progesterone, simvastatin, deprenyl, minocycline, resveratrol, and other glutamate receptor antagonists (e.g., NMDA receptor antagonists) and antioxidants. In some embodiments, the neuroprotective treatment is an antibody against an inflammasome protein, or a binding fragment thereof, such as an antibody against an inflammasome protein provided herein.

[0099] The success or response to standard treatment can also be monitored by measuring the level of at least one inflammasome protein. Thus, in some embodiments, a method for evaluating or diagnosing a patient with brain injury (e.g., MCI, stroke, MS, or TBI) further comprises measuring the level of at least one inflammasome protein in a biological sample obtained from the patient after treatment, preparing a treatment protein signature associated with a positive response to treatment, wherein the treatment protein signature comprises a reduction in the level of at least one inflammasome protein, and identifying patients who exhibit the presence of the treatment protein signature that responds positively to treatment. A reduction in the level, abundance, or concentration of one or more inflammasome proteins (e.g., ASC, IL-18, or caspase-1) indicates the effectiveness of the treatment in the patient. The one or more inflammasome proteins measured in the sample obtained after treatment can be the same or different from the inflammasome proteins measured in the sample obtained before treatment. The level of inflammasome proteins can also be used to adjust the dosage or frequency of treatment. Inflammasome protein levels can be ascertained using the methods and techniques provided herein.

[0100] In one embodiment, the brain injury (e.g., MCI, TBI, stroke, or MS) is MS, and the standard of care treatment is selected from therapies directed to modifying disease outcome, managing relapses, managing symptoms, or any combination thereof. Therapies directed to modifying disease outcome include beta-interferon, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, mitoxanthrone, ocrelizumab, alemtuzumab, daclizamide ... The agent may be selected from natalizumab and natalizumab, and the stroke is an ischemic stroke, transient ischemic stroke, or hemorrhagic stroke.

[0101] In another embodiment, the brain injury (e.g., MCI, TBI, stroke, or MS) is ischemic stroke or transient ischemic stroke, and the standard of care treatment is selected from tissue plasminogen activator (tPA), antiplatelet agents, anticoagulants, carotid angioplasty, carotid endarterectomy, intra-arterial thrombolysis, and mechanical clot removal in cerebral ischemia (MERCI), or a combination thereof. In yet another embodiment, the brain injury (e.g., TBI, stroke, or MS) is hemorrhagic stroke, and the standard of care treatment is aneurysm clipping, coil embolization, or arteriovenous malformation (AVM) repair.

[0102] In another embodiment, the brain injury (e.g., MCI, TBI, stroke, or MS) is TBI, and the standard of care treatment is selected from diuretics, anti-seizure medications, coma-inducing medications, surgery, and / or rehabilitation. Diuretics can be used to reduce the amount of fluid in tissues and increase urine output. Diuretics given intravenously to people with traumatic brain injury can help reduce pressure inside the brain. Anti-seizure medications can be given during the first week to avoid any additional brain damage that may be caused by seizures. Continuous anti-seizure treatment is used only if seizures occur. Coma-inducing medications can be used to put people into a temporary coma, as a comatose brain requires less oxygen to function. This can be particularly beneficial when increased pressure in the brain compresses blood vessels and prevents brain cells from receiving the normal amount of nutrients and oxygen. The severity of TBI can be assessed using the Glasgow Coma Scale. This 15-point test can help doctors or other emergency medical personnel assess the initial severity of brain injury by checking a person's ability to follow commands and move their eyes and limbs. Vocal coherence can also provide important clues. Ability is scored on the Glasgow Coma Scale, from 3 to 15. Higher scores indicate less severe injury.

[0103] In yet another embodiment, the brain injury (e.g., MCI, TBI, stroke, or MS) is MCI and the standard of care treatment is computerized cognitive training, grouped memory training, individual error-free learning sessions, family memory strategy intervention, DHA (docosahexaenoic acid), EPA (eicosapentaenoic acid), ginkgo biloba, donepezil, rivastigmine, triflusal, Huannao Yicong capsules, piribedil, nicotine Patches, vitamin E, vitamins B12 and B6, folic acid, rofecoxib, galantamine, cholinesterase inhibitors, memantine, lithium, Wuzi Yanzong granules, ginseng, and exercise.

[0104] kit Also provided herein is a kit for preparing an inflammasome protein profile associated with brain injury (e.g., MCI, stroke, MS, or TBI). The kit may include a reagent for measuring at least one inflammasome protein and instructions for measuring the at least one inflammasome protein to assess the severity of a patient's brain injury (e.g., MCI, stroke, MS, or TBI). As used herein, "reagent" refers to components necessary for detecting or quantifying one or more proteins by any one of the methods described herein. For example, in some embodiments, a kit for measuring one or more inflammasome proteins may include reagents for performing liquid or gas chromatography, mass spectrometry, immunoassay, immunoblot, or electrophoresis to detect one or more inflammasome proteins described herein. In some embodiments, the kit includes a reagent for measuring one or more inflammasome proteins selected from IL-18, ASC, caspase-1, or a combination thereof.

[0105] In one embodiment, the kit includes a labeled binding partner that specifically binds to one or more inflammasome proteins, wherein the one or more inflammasome proteins are selected from the group consisting of IL-18, ASC, caspase-1, and combinations thereof. Suitable binding partners for specific binding to inflammasome proteins include, but are not limited to, antibodies and fragments thereof, aptamers, peptides, etc. In certain embodiments, the binding partner for detecting ASC is an antibody or fragment thereof, and the antibody against ASC can be any antibody known in the art and / or commercially available. Examples of anti-ASC antibodies for use in the methods provided herein are described herein. In certain embodiments, the binding partner for detecting ASC is an antibody or fragment thereof, an aptamer, or a peptide that specifically binds to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of rat ASC and human ASC, respectively. In certain embodiments, the binding partner for detecting IL-18 is an antibody or fragment thereof. The antibody against IL-18 can be any antibody known in the art and / or commercially available, such as those provided herein. In certain embodiments, the binding partner for detecting caspase-1 is an antibody or a fragment thereof.The antibody to caspase-1 can be any antibody known in the art and / or commercially available, for example, as provided herein.In certain embodiments, the binding partner for detecting IL-1 beta is an antibody or a fragment thereof.The antibody to IL-1 beta can be any antibody known in the art and / or commercially available, for example, as provided herein.Labels that can be conjugated to binding partners include metal nanoparticles (e.g., gold, silver, copper, platinum, cadmium, and composite nanoparticles), fluorescent labels (e.g., fluorescein, Texas Red, green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, Alexa dye molecules, etc.), and enzyme labels (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, beta-lactamase, galactose oxidase, lactoperoxidase, luciferase, myeloperoxidase, and amylase). [Example]

[0106] The present invention is further illustrated by the following specific examples, which are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0107] Example 1 Examination of inflammasome proteins as biomarkers for multiple sclerosis (MS) Multiple sclerosis (MS) is an autoimmune disease that affects the brain and spinal cord. Critical to the care of patients with MS is the need for biomarkers that can predict disease onset, disease progression, and response to treatment. 1 .

[0108] Inflammasomes are key mediators of the innate immune response that were first described in the CNS to mediate inflammation after spinal cord injury 2 Inflammasomes are multiprotein complexes involved in the activation of caspase-1 and the processing of the pro-inflammatory cytokines IL-1β and IL-18. 3 .

[0109] In this example, the expression levels of inflammasome proteins in serum samples from patients with MS are determined. Furthermore, the sensitivity and specificity of inflammasome signaling proteins as biomarkers for MS are investigated.

[0110] material and method Participants: In this study, serum samples from 120 normal donors and 32 patients diagnosed with MS were analyzed. Samples were purchased from BioreclamationIVT. The normal donor group consisted of samples obtained from 60 male and 60 female donors, ranging in age from 20 to 70 years. The MS group consisted of samples obtained from patients ranging in age from 24 to 64 years (Figure 4).

[0111] Protein assay: The concentrations of serum inflammasome proteins ASC, IL-1β and IL-18 were analyzed using Simple Plex and Simple Plex Explorer software. The results shown correspond to the average of each sample performed in triplicate. It should be noted that any system / instrument known in the art can be used to measure the level of proteins (e.g., inflammasome proteins) in body fluids.

[0112] Biomarker analysis: Prism7 software (GraphPad) was used to analyze the data obtained from Simple Plex Explorer software. Comparisons between groups were performed after identifying outliers, followed by determining the area under the receiver operating characteristic (ROC) curve and 95% confidence interval (CI). A significance p-value of <0.05 was used. Sensitivity and specificity for each biomarker were obtained for a range of different cutoff points. Samples giving protein values ​​below the level of detection of the assay were not included in the analysis for that specimen.

[0113] ROC curves are summarized as the area under the curve (AUC). A perfect AUC value is 1.0, where 100% of subjects in a population are correctly classified as having or not having MS. In contrast, an AUC of 0.5 indicates that subjects are randomly classified as either positive or negative for MS, and has no clinical utility. It has been suggested that an AUC between 0.9 and 1.0 represents an excellent biomarker, an AUC between 0.8 and 0.9 represents a good biomarker, an AUC between 0.7 and 0.8 represents a promising biomarker, an AUC between 0.6 and 0.7 represents a poor biomarker, and an AUC between 0.5 and 0.6 represents a failed biomarker. 10 .

[0114] result Caspase-1, ASC, and IL-18 are elevated in the serum of MS patients Serum samples from MS patients were analyzed for protein expression of the inflammasome signaling proteins caspase-1, ASC, IL-1β, and IL-18 using a Simple Plex assay (Protein Simple) and compared with serum from healthy / control individuals (Figures 1A-1D). Protein levels of caspase-1, ASC, and IL-18 in the serum of MS patients were higher than in controls. However, IL-1β levels were lower in MS patients than in controls. These findings are consistent with previous reports demonstrating a role for the inflammasome in MS pathology. 6、8、11 .

[0115] ASC and caspase-1 are good serum biomarkers for MS To determine whether these inflammasome signaling proteins could be reliable biomarkers of MS pathology, we then determined the area under the curve (AUC) for caspase-1 (Figure 2A), ASC (Figure 2B), IL-1beta (Figure 2C), and IL-18 (Figure 2D). Of the three proteins measured, ASC was shown to be the best biomarker (Figure 3), with an AUC of 0.9448 and a CI between 0.9032 and 0.9864 (Table 1). In addition, caspase-1, with an AUC of 0.848 and a CI between 0.703 and 0.9929, is also a promising biomarker for MS.

[0116] [Table 1]

[0117] Furthermore, the cutoff point for ASC was 352.4 pg / ml with a sensitivity of 84% and a specificity of 90% (Table 2). For caspase-1, the cutoff point was 1.302 pg / ml with a sensitivity of 89% and a specificity of 56% (Table 2). We also found that for ASC, for 100% sensitivity, the cutoff point was 247.2 pg / ml with a specificity of 58.26%, and for 100% specificity, the cutoff point was 465.1 pg / ml and a sensitivity of 65.63%. In the case of caspase-1, for 100% sensitivity, the cutoff point was 1.111 pg / ml with a specificity of 44.44%. For 100% specificity, the cutoff point was 2.718 pg / ml with a sensitivity of 52.63%. Thus, these findings indicate that caspase-1 and ASC may be biomarkers for MS.

[0118] [Table 2]

[0119] conclusion In this study, statistically significantly higher levels of IL-18 were detected in the serum of MS patients compared with healthy controls. Additionally, the AUC for IL-18 in the patient cohort was 0.7075 with a CI between 0.6052 and 0.8097 and a sensitivity of 84%. However, the specificity was only 44% when the cutoff point was 190.1 pg / ml. When the cutoff point was 104.2 pg / ml, the sensitivity was 100%, but the specificity was only 6.723%. Similarly, when the cutoff point was 427.2 pg / ml, the specificity was 100%, but the sensitivity was only 15.63%.

[0120] Furthermore, IL-1β levels were significantly lower in the MS group than in the control group. The AUC was 0.7619 with a CI between 0.5806 and 0.9432. When the cutoff point was 0.825 with a specificity of 62%, the sensitivity was 100%.

[0121] Higher protein levels of caspase-1 were also found in the serum of MS patients. Importantly, the AUC for caspase-1 was 0.848 with a CI between 0.703 and 0.9929. At a cutoff point of 1.302 pg / ml, the sensitivity was 89% with a specificity of 56%. At a cutoff point of 1.111 pg / ml with a specificity of 44.44%, the sensitivity at 100% specificity was 52.63% at a cutoff point of 2.718 pg / ml.

[0122] Also in this example, ASC was the most promising biomarker, with an AUC of 0.9448 and a narrow CI between 0.9032 and 0.9864. A cutoff point of 352.4 pg / ml resulted in a sensitivity of 84% and a specificity of 90%. When the cutoff point was 247.2 pg / ml, the sensitivity was 100% and the specificity was 58%.

[0123] Thus, based on these findings, caspase-1 and ASC are promising biomarkers with high AUC values ​​and high sensitivity. Importantly, the combination of caspase-1 and ASC as biomarkers for MS with other diagnostic criteria may further increase the sensitivity of these biomarkers for MS beyond those described in this example. Some clinically used biomarkers, such as serum aquaporin 4 antibody (AQP4-IgG), used to distinguish between patients with MS and patients with neuromyelitis optica, have a median sensitivity of 62.3%, ranging from 12.5% ​​to 100%, depending on the assay used for measurement. 29 .

[0124] Since the 1960s, oligoclonal immunoglobulin (Ig) G bands (OCBs) have been used as a classic biomarker in the diagnosis of MS. 30 However, the specificity of IgG-OCB is only 61%, and as a result, other diagnostic criteria are required to clinically determine the diagnosis of MS. 31 Currently, CSF-limited IgG-OCB is a good predictor of conversion from CIS to CDMS, independent of MRI. 32 Similar results have been obtained when analyzing IgM-OCB. 33 Interestingly, IgG against measles, rubella, and varicella zoster (MRZ) is present in the CSF of MS patients, and thus MRZ-specific IgG has the potential to be used as a biomarker for MS diagnosis. 34 .

[0125] Importantly, in this study, caspase-1 and ASC were identified as potential biomarkers of MS pathology with high AUC values: 0.9448 and 0.848, respectively, with a sensitivity of over 80% and a specificity of ASC of 90%. Incorporation by Reference

[0126] The following references are incorporated by reference in their entirety for all purposes.

[0127] 1. Compston A. The pathogenesis and basis for treatment in multiple sclerosis. Clin Neurol Neurosurg. 2004;106:246-8.

[0128] 2. de Rivero Vaccari JP, Lotocki G, Marcillo AE, Dietrich WD and Keane RW. A molecular platform in neurons regulates inflammation after spinal cord injury. J Neurosci. 2008;28:3404-14.

[0129] 3. de Rivero Vaccari JP, Dietrich WD and Keane RW. Activation and regulation of cellular inflammasomes: gaps in our knowledge for central nervous system injury. J Cereb Blood Flow Metab. 2014;34:369-75.

[0130] 4. Ming X, Li W, Maeda Y, Blumberg B, Raval S, Cook SD and Dowling PC. Caspase-1 expression in multiple sclerosis plaques and cultured glial cells. J Neurol Sci. 2002;197:9-18.

[0131] 5. Cao Y, Goods BA, Raddassi K, Nepom GT, Kwok WW, Love JC and Hafler DA. Functional inflammatory profiles distinguish myelin-reactive T cells from patients with multiple sclerosis. Sci Transl Med. 2015;7:287ra74.

[0132] 6. Furlan R, Martino G, Galbiati F, Poliani PL, Smiroldo S, Bergami A, Desina G, Comi G, Flavell R, Su MS and Adorini L. Caspase-1 regulates the inflammatory process leading to autoimmune demyelination. J Immunol. 1999;163:2403-9.

[0133] 7. Inoue M, Williams KL, Gunn MD and Shinohara ML. NLRP3 inflammasome induces chemotactic immune cell migration to the CNS in experimental autoimmune encephalomyelitis. Proc Natl Acad Sci U S A. 2012;109:10480-5.

[0134] 8. Gris D, Ye Z, Iocca HA, Wen H, Craven RR, Gris P, Huang M, Schneider M, Miller SD and Ting JP. NLRP3 plays a critical role in the development of experimental autoimmune encephalomyelitis by mediating Th1 and Th17 responses. J Immunol. 2010;185:974-81.

[0135] 9. Brand FJ, 3rd, Forouzandeh M, Kaur H, Travascio F and de Rivero Vaccari JP. Acidification changes affect the inflammasome in human nucleus pulposus cells. J Inflamm (Lond). 2016;13:29.

[0136] 10. Xia J, Broadhurst DI, Wilson M and Wishart DS. Translational biomarker discovery in clinical metabolomics: an introductory tutorial. Metabolomics. 2013;9:280-299.

[0137] [ PubMed ] 11. Dumas A, Amiable N, de Rivero Vaccari JP, Chae JJ, Keane RW, Lacroix S and Vallieres L. The inflammasome pyrin contributes to pertussis toxin-induced IL-1beta synthesis, neutrophil intravascular crawling and autoimmune encephalomyelitis. PLoS Pathology. 2014;10:e1004150.

[0138] 12. Katsavos S and Anagnostouli M. Biomarkers in Multiple Sclerosis: An Up-to-Date Overview. Mult Scler Int. 2013;2013:340508.

[0139] [ PubMed ] 13. Kuhle J, Disanto G, Dobson R, Adiutori R, Bianchi L, Topping J, Bestwick JP, Meier UC, Marta M, Dalla Costa G, Runia T, Evdoshenko E, Lazareva N, Thouvenot E, Iaffaldano P, Direnzo V, Khademi M, Piehl F, Comabella M, Sombekke M, Killestein J, Hegen H, Rauch S, D'Alfonso S, Alvarez-Cermeno JC, Kleinova P, Horakova D, Roesler R, Lauda F, Loglu S, Uygun T, Uygun Uytasar A, Saip S, Menge T, Rajda C, Bergamaschi R, Moll N, Khalil M, Marignier R, Dujmovic I, Larsson H, Malmestrom C, Scarpini E, Fenoglio C, Wergeland S, Laroni A, Hannibali V, Romano S, Martinez AD, Carra A, Salvetti M, Uccelli A, Torkildsen O, Myhr KM, Galimberti D, Rejdak K, Lycke J, Frederiksen JL, Drulovic J, Confavreux C, Brassat D, Enzinger C, Fuchs S, Bosca I, Pelletier J, Picard C, Colombo E, Franciotta D, Derfuss T, Lindberg R, Yaldizli O, Vecsei L, Kieseier BC, Hartung HP, Villoslada P, Siva A, Saiz A, Toumani H, Havrdova E, Villar LM, Leone M, Barizzone N, Deisenhammer F, Teunissen C, Montalban X, Tintore M, Olsson T, Trojano M, Lehmann S, Castelnovo G, Lapin S, Hintzen R, Kappos L, Furlan R, Martinelli V, Comi G, Ramagopalan SV and Giovannoni G. Conversion from clinically isolated syndrome to multiple sclerosis: A large multicentre study. Mult Scler. 2015;21:1013-24.

[0140] 14. Lublin FD. New multiple sclerosis phenotypic classification. Eur Neurol. 2014;72 Suppl 1:1-5.

[0141] 15. Milo R and Miller A. Revised diagnostic criteria of multiple sclerosis. Autoimmun Rev. 2014;13:518-24.

[0142] 16. Inoue M, Chen PH, Siecinski S, Li QJ, Liu C, Steinman L, Gregory SG, Benner E and Shinohara ML. An interferon-beta-resistant and NLRP3 inflammasome-independent subtype of EAE with neuronal damage. Nat Neurosci. 2016;19:1599-1609.

[0143] 17. Inoue M, Williams KL, Oliver T, Vandenabeele P, Rajan JV, Miao EA and Shinohara ML. Interferon-beta therapy against EAE is effective only when development of the disease depends on the NLRP3 inflammasome. Sci Signal. 2012;5:ra38.

[0144] 18. Chen YC, Chen SD, Miao L, Liu ZG, Li W, Zhao ZX, Sun XJ, Jiang GX and Cheng Q. Serum levels of interleukin (IL)-18, IL-23 and IL-17 in Chinese patients with multiple sclerosis. J Neuroimmunol. 2012;243:56-60.

[0145] 19. Losy J and Niezgoda A. IL-18 in patients with multiple sclerosis. Acta Neurol Scand. 2001;104:171-3.

[0146] 20. Levesque SA, Pare A, Mailhot B, Bellver-Landete V, Kebir H, Lecuyer MA, Alvarez JI, Prat A, de Rivero Vaccari JP, Keane RW and Lacroix S. Myeloid cell transmigration across the CNS vasculature triggers IL-1beta-driven neuroinflammation during autoimmune encephalomyelitis in mice. J Exp Med. 2016;213:929-49.

[0147] 21. Dujmovic I, Mangano K, Pekmezovic T, Quattrocchi C, Mesaros S, Stojsavljevic N, Nicoletti F and Drulovic J. The analysis of IL-1 beta and its naturally occurring inhibitors in multiple sclerosis: The elevation of IL-1 receptor antagonist and IL-1 receptor type II after steroid therapy. J Neuroimmunol. 2009;207:101-6.

[0148] 22. Hauser SL, Doolittle TH, Lincoln R, Brown RH and Dinarello CA. Cytokine accumulations in CSF of multiple sclerosis patients: frequent detection of interleukin-1 and tumor necrosis factor but not interleukin-6. Neurology. 1990;40:1735-9.

[0149] 23. Maimone D, Gregory S, Arnason BG and Reder AT. Cytokine levels in the cerebrospinal fluid and serum of patients with multiple sclerosis. J Neuroimmunol. 1991;32:67-74.

[0150] 24. Tsukada N, Miyagi K, Matsuda M, Yanagisawa N and Yone K. Tumor necrosis factor and interleukin-1 in the CSF and sera of patients with multiple sclerosis. J Neurol Sci. 1991;104:230-4.

[0151] 25. Huang WX, Huang P and Hillert J. Increased expression of caspase-1 and interleukin-18 in peripheral blood mononuclear cells in patients with multiple sclerosis. Mult Scler. 2004;10:482-7.

[0152] 26. de Rivero Vaccari JP, Dietrich WD and Keane RW. Therapeutics targeting the inflammasome after central nervous system injury. Transl Res. 2016;167:35-45.

[0153] 27. de Rivero Vaccari JP, Lotocki G, Alonso OF, Bramlett HM, Dietrich WD and Keane RW. Therapeutic neutralization of the NLRP1 inflammasome reduces the innate immune response and improves histopathology after traumatic brain injury. J Cereb Blood Flow Metab. 2009;29:1251-61.

[0154] 28. Shaw PJ, Lukens JR, Burns S, Chi H, McGargill MA and Kanneganti TD. Cutting edge: critical role for PYCARD / ASC in the development of experimental autoimmune encephalomyelitis. J Immunol. 2010;184:4610-4.

[0155] 29. Jarius S and Wildemann B. Aquaporin-4 antibodies (NMO-IgG) as a serological marker of neuromyelitis optica: a critical review of the literature. Brain Pathol. 2013;23:661-83.

[0156] 30. Stangel M, Fredrikson S, Meinl E, Petzold A, Stuve O and Tumani H. The utility of cerebrospinal fluid analysis in patients with multiple sclerosis. Nat Rev Neurol. 2013;9:267-76.

[0157] 31. Teunissen CE, Malekzadeh A, Leurs C, Bridel C and Killestein J. Body fluid biomarkers for multiple sclerosis--the long road to clinical application. Nat Rev Neurol. 2015;11:585-96.

[0158] 32. Tintore M, Rovira A, Rio J, Tur C, Pelayo R, Nos C, Tellez N, Perkal H, Comabella M, Sastre-Garriga J and Montalban X. Do oligoclonal bands add information to MRI in first attacks of multiple sclerosis? Neurology. 2008;70:1079-83.

[0159] 33. Villar LM, Masjuan J, Gonzalez-Porque P, Plaza J, Sadaba MC, Roldan E, Bootello A and Alvarez-Cermeno JC. Intrathecal IgM synthesis predicts the onset of new relapses and a worse disease course in MS. Neurology. 2002;59:555-9.

[0160] 34. Brettschneider J, Tumani H, Kiechle U, Muche R, Richards G, Lehmensiek V, Ludolph AC and Otto M. IgG antibodies against measles, rubella, and varicella zoster virus predict conversion to multiple sclerosis in clinically isolated syndrome. PLoS One. 2009;4:e7638.

[0161] Example 2 Examination of inflammasome proteins as biomarkers for stroke Introduction A biomarker is a characteristic that can be objectively measured and evaluated as an indicator of a normal or pathological biological process.9 For this reason, in the context of stroke, biomarkers in blood or other body fluids can be used as indicators of stroke initiation. However, to date, no biomarkers are available that are routinely used in the diagnosis and management of stroke. To this end, cytokines such as IL-10 or tumor necrosis factor, and other inflammatory proteins such as C-reactive protein, high-mobility group box-1, or heat shock proteins, have been considered as possible candidates for further biomarker analysis in patients with stroke. 10~12 .

[0162] In this example, the Simple Plex assay (Protein Simple) was used to analyze serum and serum-derived EV samples from stroke patients and control donors for inflammasome protein levels: caspase-1, apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, and interleukin (IL)-1beta. Receiver-operating characteristic (ROC) curves and associated confidence intervals were calculated after analysis of serum and serum-derived EV samples from post-stroke patients and healthy, unaffected donors to measure the sensitivity and specificity of the inflammasome proteins and establish the potential of inflammasome signaling proteins as biomarkers for stroke.

[0163] method Participants: In this example, serum samples from 80 normal donors and 16 patients diagnosed with stroke were analyzed. Samples were purchased from Bioreclamation IVT. The normal donor group consisted of samples obtained from 40 male and 40 female donors, ranging in age from 46 to 70 years. The stroke group consisted of samples obtained from patients ranging in age from 46 to 87 years (Figure 11).

[0164] EV isolation: Total exosome isolation from serum kit (Invitrogen): Total exosome isolation from serum was used according to the manufacturer's instructions (Invitrogen). Briefly, 100 μl of each sample was centrifuged at 2000 × g for 30 minutes. The supernatant was then incubated with 20 μl of total exosome isolation reagent at 4 ° C for 30 minutes, followed by centrifugation at 10,000 × g for 10 minutes at room temperature. The supernatant was discarded, and the pellet was resuspended in 50 μl of PBS.

[0165] By ExoQuick: EVs were isolated from serum samples using ExoQuick (EQ, System Biosciences) as described in 6. Briefly, 100 μl of each sample was centrifuged at 3,000 × g for 15 minutes. The supernatant was then incubated with 24.23 μl of ExoQuick exosome precipitation solution (for serum) at 4 °C for 30 minutes, followed by centrifugation at 1,500 × g for 30 minutes. The supernatant was discarded, and the remaining EQ solution was centrifuged at 1,500 × g for 5 minutes. The pellet was then resuspended in 50 μl of PBS.

[0166] Protein assay: To determine the protein concentrations of caspase-1, ASC, IL-1β, and IL-18 in serum and serum-derived EVs, a Simple Plex assay was performed and analyzed using Simple Plex Explorer software. The results shown represent the average of triplicate samples. It should be noted that any system / instrument known in the art can be used to measure protein (e.g., inflammasome protein) levels in body fluids.

[0167] Protein quantification To quantify protein concentrations in isolated EVs, the Pierce Coomassie (Bradford) Protein Assay Kit (ThermoFisher Scientific, Inc.) was used according to the manufacturer's instructions. Serum-derived EVs were lysed (1:1 dilution) in lysis buffer as described. 6 .

[0168] Nanoparticle Tracking Analysis (NTA) EVs were analyzed using a NanoSight NS300 (Malvern Instruments Company, Nanosight, Malvern, United Kingdom). The isolated exosomes were diluted in PBS (1:1000) for analysis, and three 90-second videos were then recorded. Data were analyzed using Nanosight NTA 2.3 analysis software (Malvern Instruments Company), with a detection threshold optimized for each sample and a screening gain set to 10 to track as many particles as possible while maintaining minimal background. At least three independent measurements were performed for each isolated sample.

[0169] Immunoblotting For detection of inflammasome signaling proteins in isolated EVs, EVs were resuspended in protein lysis buffer. 15Immunoblotting was performed as described previously. Briefly, after dissolution of the pellet, proteins were resolved in 10-20% Criterion TGX stain-free precast gels (Bio-Rad) using antibodies (1:1000 dilution) against NLRP3 (Novus Biologicals), caspase-1 (Novus Biologicals), ASC (Santa Cruz), IL-1beta (Cell Signaling), IL-18 (Abcam), CD81 (Thermo Scientific), and NCAM (Sigma). Band density was quantified using UN-SCAN-IT gel 5.3 software (Silk Scientific Corporation). 10 μl of sample was loaded. The chemiluminescent substrate (LumiGlo, Cell Signaling) in the membrane was imaged using a ChemiDoc Touch imaging system (Bio-Rad).

[0170] Gel imaging Total protein in Criterion TGX stain-free precast gels was imaged using a ChemiDoc Touch imaging system (BioRad) by placing the gel in the ChemiDoc Touch tray after protein transfer, adjusting the image on the screen to show the entire gel, and running the stain-free blot settings in the application window.

[0171] statistical analysis Statistical comparisons between the Invitrogen and ExoQuick isolation procedures were performed using a two-tailed Student's t test.

[0172] Electron microscopy procedures EVs were loaded onto formvar-carbon-coated grids. A 10 μl drop of sample was then placed on clean Parafilm, and the grid was floated (face down) for 30 minutes. Subsequent steps were also performed by floating the grid on 10 μl of foam. The EV-loaded grid was then rinsed with 0.1 M Millonig's phosphate buffer (Electron Microscopy Sciences) for 5 minutes. Excess liquid was drained. The grid was then placed in 2% glutaraldehyde for 5 minutes. Subsequent washes were performed on seven different foams with 0.1 M Millonig's phosphate buffer for 5 minutes, followed by seven 2-minute rinses with distilled water to remove excess glutaraldehyde. The grid was then transferred to a 0.4% uranyl acetate solution for 5 minutes. The grid was then allowed to dry for imaging. Images were acquired using a Joel JEM-1400 transmission electron microscope and a digital Gatan camera at a voltage of 80 kV.

[0173] Biomarker analysis Data were analyzed using Prism7 software (GraphPad). Comparisons of protein levels between groups were performed by first identifying outliers, followed by one-tailed t-tests, and then determining the area under the ROC curve, as well as 95% confidence intervals and p-values ​​(p-values ​​of significance used were <0.05). Finally, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of each biomarker were obtained for a range of different cutoff points. Samples that gave protein values ​​below the level of detection of the assay were not included in the analysis for a particular specimen.

[0174] result Caspase-1, ASC, and IL-18 are elevated in the serum of stroke patients: To determine the protein levels of inflammasome proteins in serum from stroke patients and control donors, serum samples were analyzed using the Simple Plex system. Protein levels of caspase-1, ASC, and IL-18 were higher in the serum of stroke patients when compared to control samples, whereas levels of IL-1 were not significantly different (Figures 5A-5D). These findings confirm previous data indicating that inflammasomes are involved in the inflammatory response after stroke. 4、16 .

[0175] ASC as a serum biomarker for stroke: Higher levels of inflammasome proteins in serum from stroke patients may not be sufficient evidence to indicate that inflammasome proteins are good biomarkers for stroke. Therefore, ROC analysis was performed (Figure 6 and Figures 12A-12D) to determine the AUC. The AUC for ASC was 0.9975 with a confidence interval between 0.9914 and 1.004 (Table 3). The cutoff point for ASC was 404.8 pg / ml with 100% sensitivity and 96% specificity (Table 4). Thus, ASC appears to be a reliable biomarker for stroke.

[0176] [Table 3]

[0177] [Table 4]

[0178] Amount of protein loaded in EVs isolated from stroke patients: To calculate the amount of protein present in exosomes isolated from serum samples, a BCA assay was performed on isolates obtained by the Invitrogen and EQ methods. The data showed that the EQ method was able to isolate more protein than the Invitrogen method (Figures 7A-7C).

[0179] To visualize how much protein was loaded onto the gel during immunoblot analysis, we used the stain-free blot setting on the ChemiDoc Touch Imaging System. The representative image in Figure 7B shows that when 10 μl of serum-derived EVs resuspended in lysis buffer containing a protease inhibitor cocktail (Sigma) was loaded, the lane corresponding to the Invitrogen kit contained less protein than the lane corresponding to the EQ kit; however, there was no statistical significance between the groups.

[0180] The Invitrogen kit and EQ kit isolate CD81- and NCAM-positive EVs from the serum of stroke patients. To determine whether inflammasome proteins present in EVs are promising biomarkers for stroke, EVs from the serum of stroke patients were isolated. Two different EV isolation methods were used to identify the most appropriate method for isolating inflammasome-containing EVs. Additionally, the tetraspanin protein CD81, a marker for EVs {Andreu, 2014 #33}, and neural cell adhesion molecule (NCAM), a marker for neuron-derived EVs, were used to demonstrate that the isolated EVs were brain-derived {Vella, 2016 #36}. Thus, both methods, the Invitrogen kit and the EQ kit, were capable of isolating CD81- and NCAM-positive EVs (Figure 8A). However, although the EQ kit appears to isolate higher levels of these proteins, there was no statistically significant difference between the two groups (Figure 8B and Figure 8C). An EV-positive control isolation (System Biosciences) was also performed in parallel.

[0181] Electron microscopy was performed on EVs isolated by the two methods and found that the Invitrogen kit yielded more uniform, round vesicles (Figure 8D). Additionally, NTA analysis revealed that particle sizes ranged from 40 to 50 nm for both methods, with particle concentrations of EVs obtained by the Invitrogen method being 1.27e+009 particles / ml and 7.56e+008 particles / ml for the EQ method (Figure 8E and Figure 8F). Based on the particle size and uniformity of vesicles determined by electron microscopy, the Invitrogen method appears more suitable for isolating EVs.

[0182] Invitrogen's kit and EQ isolate inflammasome-positive EVs from serum of patients with stroke; inflammasome proteins have previously been shown to be present in EVs 6 The levels of inflammasome protein expression were compared between the two different methods, and no statistically significant differences were found in the levels of NLPR3, caspase-1, ASC, and IL-18 between the two methods. However, the EQ method was able to isolate EVs with higher levels of IL-1 beta than the Invitrogen method (see Figures 13A-13F).

[0183] ASC is elevated in EVs isolated from the serum of stroke patients: We isolated EVs from the serum of 16 age-matched donors and 16 stroke samples (Figure 11) and analyzed the levels of inflammasome proteins in these isolated EVs using Simple Plex technology. ASC protein levels remained elevated in serum-derived EVs from stroke samples compared to controls (Figures 9A-9C). However, while IL-1beta and IL-18 levels were not significantly different between the two groups, caspase-1 levels in these isolated EVs were below the detection limit of these assays for this specimen.

[0184] ASC in serum-derived EVs is a good biomarker for stroke: To determine whether inflammasome proteins in serum-derived EVs could be viable biomarkers for stroke, we performed ROC analysis (see Figures 14A-14C) and found that ASC was a reliable biomarker for stroke with an AUC of 1 (Table 5) and a cutoff point of 97.57 pg / ml (Table 6) (Figure 10).

[0185] [Table 5]

[0186] [Table 6]

[0187] conclusion In this example, we demonstrated that ASC is a reliable biomarker for the onset of stroke. The area under the curve (AUC) for ASC in serum was 0.9975 with a confidence interval between 0.9914 and 1.004. This AUC value is higher than that of other inflammasome signaling proteins analyzed in this study: caspase-1 (0.75), IL-1 beta (0.6111), and IL-18 (0.6675), indicating that ASC is a superior biomarker for the other inflammasome proteins examined in this study. The cutoff point for ASC was 404.8 pg / ml with 100% sensitivity and 96% specificity in the cohort of samples used. Importantly, the AUC increased to 1 when serum-derived EV samples from a small subset of patients were analyzed. Thus, the cutoff point for ASC in serum-derived EVs was found to be 97.57 pg / ml.

[0188] Although higher levels of protein isolation were obtained with the EQ kit in this study, the Invitrogen kit provided better quality EVs, as visualized by electron microscopy and NTA analysis of isolated vesicles. Importantly, both methods were effective in isolating EVs containing inflammasome proteins.

[0189] In conclusion, these studies highlight the potential of inflammasome proteins, particularly ASC, as biomarkers of stroke in serum and serum-derived EVs.

[0190] Incorporation by Reference The following references are incorporated by reference in their entirety for all purposes.

[0191] 1. Xu X and Jiang Y. The Yin and Yang of innate immunity in stroke. Biomed Res Int. 2014;2014:807978.

[0192] 2. Neumann S, Shields NJ, Balle T, Chebib M and Clarkson AN. Innate Immunity and Inflammation Post-Stroke: An alpha7-Nicotinic Agonist Perspective. Int J Mol Sci. 2015;16:29029-46.

[0193] 3. Brand FJ, 3rd, de Rivero Vaccari JC, Mejias NH, Alonso OF and de Rivero Vaccari JP. RIG-I contributes to the innate immune response after cerebral ischemia. J Inflamm (Lond). 2015;12:52.

[0194] 4. Abulafia DP, de Rivero Vaccari JP, Lozano JD, Lotocki G, Keane RW and Dietrich WD. Inhibition of the inflammasome complex reduces the inflammatory response after thromboembolic stroke in mice. J Cereb Blood Flow Metab. 2009;29:534-44.

[0195] 5. de Rivero Vaccari JP, Dietrich WD and Keane RW. Therapeutics targeting the inflammasome after central nervous system injury. Transl Res. 2016;167:35-45.

[0196] 6. de Rivero Vaccari JP, Brand F, 3rd, Adamczak S, Lee SW, Perez-Barcena J, Wang MY, Bullock MR, Dietrich WD and Keane RW. Exosome-mediated inflammasome signaling after central nervous system injury. J Neurochem. 2016;136 Suppl 1:39-48.

[0197] 7. Zhang ZG and Chopp M. Exosomes in stroke pathogenesis and therapy. J Clin Invest. 2016;126:1190-7.

[0198] 8. Ji Q, Ji Y, Peng J, Zhou X, Chen X, Zhao H, Xu T, Chen L and Xu Y. Increased Brain-Specific MiR-9 and MiR-124 in the Serum Exosomes of Acute Ischemic Stroke Patients. PLoS One. 2016;11:e0163645.

[0199] 9. Biomarkers Definitions Working G. Biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clin Pharmacol Ther. 2001;69:89-95.

[0200] 10. Whiteley W, Chong WL, Sengupta A and Sandercock P. Blood markers for the prognosis of ischemic stroke: a systematic review. Stroke. 2009;40:e380-9.

[0201] 11. Bustamante A, Simats A, Vilar-Bergua A, Garcia-Berrocoso T and Montaner J. Blood / Brain Biomarkers of Inflammation After Stroke and Their Association With Outcome: From C-Reactive Protein to Damage-Associated Molecular Patterns. Neurotherapeutics. 2016;13:671-684.

[0202] 12. Katan M and Elkind MS. Inflammatory and neuroendocrine biomarkers of prognosis after ischemic stroke. Expert Rev Neurother. 2011;11:225-39.

[0203] 13. Adamczak S, Dale G, de Rivero Vaccari JP, Bullock MR, Dietrich WD and Keane RW. Inflammasome proteins in cerebrospinal fluid of brain-injured patients as biomarkers of functional outcome: clinical article. J Neurosurg. 2012;117:1119-25.

[0204] 14. Brand FJ, 3rd, Forouzandeh M, Kaur H, Travascio F and de Rivero Vaccari JP. Acidification changes affect the inflammasome in human nucleus pulposus cells. J Inflamm (Lond). 2016;13:29.

[0205] 15. de Rivero Vaccari JC, Brand FJ, 3rd, Berti AF, Alonso OF, Bullock MR and de Rivero Vaccari JP. Mincle signaling in the innate immune response after traumatic brain injury. Journal of neurotrauma. 2015;32:228-36.

[0206] 16. de Rivero Vaccari JP, Patel HH, Brand FJ, 3rd, Perez-Pinzon MA, Bramlett HM and Raval AP. Estrogen receptor beta signaling alters cellular inflammasomes activity after global cerebral ischemia in reproductively senescence female rats. J Neurochem. 2016;136:492-6.

[0207] 17. de Rivero Vaccari JP, Dietrich WD and Keane RW. Activation and regulation of cellular inflammasomes: gaps in our knowledge for central nervous system injury. J Cereb Blood Flow Metab. 2014;34:369-75.

[0208] 18. de Rivero Vaccari JP, Dietrich WD and Keane RW. Therapeutics targeting the inflammasome after central nervous system injury. Translational research : the journal of laboratory and clinical medicine. 2015.

[0209] 19. de Rivero Vaccari JP, Lotocki G, Alonso OF, Bramlett HM, Dietrich WD and Keane RW. Therapeutic neutralization of the NLRP1 inflammasome reduces the innate immune response and improves histopathology after traumatic brain injury. J Cereb Blood Flow Metab. 2009;29:1251-61.

[0210] 20. de Rivero Vaccari JP, Lotocki G, Marcillo AE, Dietrich WD and Keane RW. A molecular platform in neurons regulates inflammation after spinal cord injury. J Neurosci. 2008;28:3404-14.

[0211] 21. Fann DY, Lee SY, Manzanero S, Tang SC, Gelderblom M, Chunduri P, Bernreuther C, Glatzel M, Cheng YL, Thundyil J, Widiapradja A, Lok KZ, Foo SL, Wang YC, Li YI, Drummond GR, Basta M, Magnus T, Jo DG, Mattson MP, Sobey CG and Arumugam TV. Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke. Cell Death Dis. 2013;4:e790.

[0212] 22. Minkiewicz J, de Rivero Vaccari JP and Keane RW. Human astrocytes express a novel NLRP2 inflammasome. Glia. 2013;61:1113-21.

[0213] 23. Sun X, Song X, Zhang L, Sun J, Wei X, Meng L and An J. NLRP2 is highly expressed in a mouse model of ischemic stroke. Biochem Biophys Res Commun. 2016;479:656-662.

[0214] 24. Ma Q, Chen S, Hu Q, Feng H, Zhang JH and Tang J. NLRP3 inflammasome contributes to inflammation after intracerebral hemorrhage. Ann Neurol. 2014;75:209-19.

[0215] 25. Fann DY, Lim YA, Cheng YL, Lok KZ, Chunduri P, Baik SH, Drummond GR, Dheen ST, Sobey CG, Jo DG, Chen CL and Arumugam TV. Evidence that NF-kappaB and MAPK Signaling Promotes NLRP Inflammasome Activation in Neurons Following Ischemic Stroke. Mol Neurobiol. 2017.

[0216] 26. Zhang N, Zhang X, Liu X, Wang H, Xue J, Yu J, Kang N and Wang X. Chrysophanol inhibits NALP3 inflammasome activation and ameliorates cerebral ischemia / reperfusion in mice. Mediators Inflamm. 2014;2014:370530.

[0217] 27. Mendis S, Davis S and Norrving B. Organizational update: the world health organization global status report on noncommunicable diseases 2014; one more landmark step in the combat against stroke and vascular disease. Stroke. 2015;46:e121-2.

[0218] 28. Esenwa CC and Elkind MS. Inflammatory risk factors, biomarkers and associated therapy in ischaemic stroke. Nat Rev Neurol. 2016;12:594-604.

[0219] 29. Ridker PM and Haughie P. Prospective studies of C-reactive protein as a risk factor for cardiovascular disease. J Investig Med. 1998;46:391-5.

[0220] 30. Rosenson RS and Stafforini DM. Modulation of oxidative stress, inflammation, and atherosclerosis by lipoprotein-associated phospholipase A2. J Lipid Res. 2012;53:1767-82.

[0221] 31. Oei HH, van der Meer IM, Hofman A, Koudstaal PJ, Stijnen T, Breteler MM and Witteman JC. Lipoprotein-associated phospholipase A2 activity is associated with risk of coronary heart disease and ischemic stroke: the Rotterdam Study. Circulation. 2005;111:570-5.

[0222] 33. Barber M, Langhorne P, Rumley A, Lowe GD and Stott DJ. Hemostatic function and progressing ischemic stroke: D-dimer predicts early clinical progression. Stroke. 2004;35:1421-5.

[0223] 34. Turaj W, Slowik A, Dziedzic T, Pulyk R, Adamski M, Strojny J and Szczudlik A. Increased plasma fibrinogen predicts one-year mortality in patients with acute ischemic stroke. J Neurol Sci. 2006;246:13-9.

[0224] 35. Mathivanan S, Ji H and Simpson RJ. Exosomes: extracellular organelles important in intercellular communication. J Proteomics. 2010;73:1907-20.

[0225] 36. Le Pecq JB. Dexosomes as a therapeutic cancer vaccine: from bench to bedside. Blood Cells Mol Dis. 2005;35:129-35.

[0226] 37. Kourembanas S. Exosomes: vehicles of intercellular signaling, biomarkers, and vectors of cell therapy. Annu Rev Physiol. 2015;77:13-27.

[0227] 38. Thery C, Zitvogel L and Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2002;2:569-79.

[0228] 39. Campos JH, Soares RP, Ribeiro K, Andrade AC, Batista WL and Torrecilhas AC. Extracellular Vesicles: Role in Inflammatory Responses and Potential Uses in Vaccination in Cancer and Infectious Diseases. J Immunol Res. 2015;2015:832057.

[0229] 40. Hurley JH, Boura E, Carlson LA and Rozycki B. Membrane budding. Cell. 2010;143:875-87.

[0230] 41. Thery C, Ostrowski M and Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 2009;9:581-93.

[0231] 42. Vella LJ, Sharples RA, Nisbet RM, Cappai R and Hill AF. The role of exosomes in the processing of proteins associated with neurodegenerative diseases. Eur Biophys J. 2008;37:323-32.

[0232] 43. Izquierdo-Useros N, Naranjo-Gomez M, Erkizia I, Puertas MC, Borras FE, Blanco J and Martinez-Picado J. HIV and mature dendritic cells: Trojan exosomes riding the Trojan horse? PLoS Pathog. 2010;6:e1000740.

[0233] 44. Luga V, Zhang L, Viloria-Petit AM, Ogunjimi AA, Inanlou MR, Chiu E, Buchanan M, Hosein AN, Basik M and Wrana JL. Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell. 2012;151:1542-56.

[0234] 45. Robbins PD and Morelli AE. Regulation of immune responses by extracellular vesicles. Nat Rev Immunol. 2014;14:195-208.

[0235] 46. Rekker K, Saare M, Roost AM, Kubo AL, Zarovni N, Chiesi A, Salumets A and Peters M. Comparison of serum exosome isolation methods for microRNA profiling. Clin Biochem. 2014;47:135-8.

[0236] 47. Taylor DD, Zacharias W and Gercel-Taylor C. Exosome isolation for proteomic analyses and RNA profiling. Methods Mol Biol. 2011;728:235-46.

[0237] 48. Caradec J, Kharmate G, Hosseini-Beheshti E, Adomat H, Gleave M and Guns E. Reproducibility and efficiency of serum-derived exosome extraction methods. Clin Biochem. 2014;47:1286-92.

[0238]

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

[0239]

Table 8-1

Table 8-2

Table 8-3

[0240]

Table 9-1

Table 9-2

[0241] (Example 3) Examination of inflammasome proteins as biomarkers for traumatic brain injury (TBI) As defined by the U.S. Centers for Disease Control ("CDC"), traumatic brain injury ("TBI") is "a disruption of the normal function of the brain that can be caused by a bump, blow, or impact to the head, or a penetrating head injury." Critical to the care of patients with TBI is the need for biomarkers that can predict onset, progression, and response to treatment. In addition, there is a need for minimally invasive methods of retrieving these biomarkers for analysis.

[0242] Inflammasomes are key mediators of the innate immune response that were first described in the CNS to mediate inflammation after spinal cord injury. 2 Inflammasomes are multiprotein complexes involved in the activation of caspase-1 and the processing of the pro-inflammatory cytokines IL-1β and IL-18. 3 .

[0243] In this example, the expression levels of inflammasome proteins in serum samples from patients with TBI are determined. Furthermore, the sensitivity and specificity of inflammasome signaling proteins as biomarkers for TBI are investigated.

[0244] material and method Participants: In this study, serum samples from 120 normal donors and 21 patients diagnosed with TBI were analyzed. Samples were purchased from BioreclamationIVT. The normal donor group consisted of samples obtained from 60 male and 60 female donors, ranging in age from 20 to 70 years. The TBI group consisted of samples obtained from patients ranging in age from 24 to 64 years. In addition, 21 control cerebrospinal fluid (CSF) samples were obtained from BioreclamationIVT, and 9 CSF samples were obtained from the patient cohort.

[0245] Protein assay: The concentrations of inflammasome proteins ASC, IL-1β, and IL-18 in serum and CSF were analyzed using Simple Plex and Simple Plex Explorer software. The results shown correspond to the average of each sample performed in triplicate. It should be noted that any system / instrument known in the art can be used to measure the level of proteins (e.g., inflammasome proteins) in body fluids. Samples were collected three times a day for the first five days after the patient arrived at the hospital. Samples were analyzed for the first and second collections (day 1), and the fourth and sixth collections (day 2).

[0246] Biomarker analysis: Prism7 software (GraphPad) was used to analyze the data obtained from Simple Plex Explorer software. Comparisons between groups were performed after identifying outliers, followed by determining the area under the receiver operating characteristic (ROC) curve and 95% confidence interval (CI). A significance p-value of <0.05 was used. Sensitivity and specificity for each biomarker were obtained for a range of different cutoff points. Samples giving protein values ​​below the level of detection of the assay were not included in the analysis for that specimen.

[0247] ROC curves are summarized as the area under the curve (AUC). A perfect AUC value is 1.0, where 100% of subjects in a population are correctly classified as having or not having TBI. In contrast, an AUC of 0.5 indicates that subjects are randomly classified as either positive or negative for TBI, and has no clinical utility. It has been suggested that an AUC between 0.9 and 1.0 represents an excellent biomarker, an AUC between 0.8 and 0.9 represents a good biomarker, an AUC between 0.7 and 0.8 represents a promising biomarker, an AUC between 0.6 and 0.7 represents a poor biomarker, and an AUC between 0.5 and 0.6 represents a failing biomarker. 5 .

[0248] result Caspase-1 and ASC are elevated in the serum of patients after TBI Serum samples from TBI patients were analyzed for protein expression of the inflammasome signaling proteins caspase-1, ASC, IL-1β, and IL-18 using a Simple Plex assay (Protein Simple) and compared with serum from healthy / control individuals (Figures 15A-15D). Protein levels of caspase-1, ASC, and IL-18 in serum from TBI patients were higher than in controls. However, levels of IL-1β were lower in TBI patients than in controls. ASC and caspase-1 are good serum biomarkers for TBI

[0249] To determine whether these inflammasome signaling proteins could be reliable biomarkers of TBI pathology, the areas under the curve (AUC) for caspase-1, ASC, IL-1β, and IL-18 (Figures 16A-D) were then determined. Of the proteins measured, caspase-1 and ASC proved to be the best biomarkers (Figures 16A and B), with AUCs of 0.93 (fourth harvest) and 0.90 (sixth harvest), respectively (Tables 10A-10D).

[0250] Table 10A-D: Results of ROC analysis for serum inflammasome signaling proteins caspase-1 (Table 10A), ASC (Table 10B), IL-1β (Table 10C) and IL-18 (Table 10D) including area, standard error (STD.ERROR), 95% confidence interval (CI) and p-value for the 1st, 2nd, 4th and 6th collections. [Table 10A] [Table 10B] [Table 10C] [Table 10D]

[0251] Furthermore, the cutoff point for caspase-1 was 1.943 pg / ml with 94% sensitivity and 89% specificity (Table 11A). For ASC, the cutoff point was 451.3 pg / ml with 85% sensitivity and 99% specificity (Table 11B). We also found that for caspase-1, for 100% sensitivity, the cutoff point was 1.679 pg / ml with 78% specificity. For ASC, the cutoff point was 153.4 pg / ml with 19% specificity (see Table 16 (fourth collection)). In the case of caspase-1, for 100% specificity, the cutoff point was 2.717 pg / ml with 78% sensitivity (see Table 15 (fourth collection)). For ASC at 100% specificity, the cutoff point was 462.4 pg / ml with a sensitivity of 85% (see Table 16 (4th collection)). Thus, these findings indicate that caspase-1 and ASC are reliable serum biomarkers for TBI.

[0252] Table 11A-B: Results of ROC analysis for caspase-1 (Table 11A) and ASC (Table 11B) in serum, including cutoff points (pg / ml), sensitivity and specificity, and positive and negative likelihood ratios (LR+ / LR-). [Table 11A] [Table 11B]

[0253] ASCs are elevated in the serum of patients with unfavorable outcomes after TBI TBI patients were divided into those with either a favorable or unfavorable outcome according to their clinical outcome based on the Glasgow Outcome Scale-Extended (GOSE), where patients with a score of 6 to 8 were considered to have a favorable outcome and those with a score of 1 to 4 were considered to have an unfavorable outcome (Tables 12A and 12B). Protein levels of ASC were higher in the serum of TBI patients with an unfavorable outcome compared with samples obtained from patients with a favorable outcome (Figure 19B), but the levels of caspase-1 (Figure 19A) and IL-18 (Figure 19C) were not found to be statistically different between the two groups.

[0254] ASC is a favorable prognostic biomarker for TBI in serum To determine whether ASC can be used as a prognostic biomarker for TBI, we determined the AUC for ASC at the second (Figure 20A) and fourth (Figure 20B) collections. The AUC for ASC was 0.9167 with a CI between 0.7914 and 1.042 at the fourth collection (Table 12A). Furthermore, the cutoff point was 547.6 pg / ml with a sensitivity of 86% and a specificity of 100% (Table 12B and Table 19 (fourth collection)). Thus, these findings indicated that ASC is a promising prognostic biomarker for TBI in serum.

[0255] Table 12A-B: Results of ROC analysis for ASC in serum for favorable outcome (Table 12A) versus unfavorable outcome (Table 12B), including area, standard error (STD.ERROR), 95% confidence interval (CI) and p-value (see Table 12A), cutoff point (pg / ml), sensitivity and specificity, and positive and negative likelihood ratios (LR+ / LR-) (see Table 12B) for the 1st, 2nd and 4th collections. [Table 12A] [Table 12B]

[0256] ASC and IL-18 are elevated in the CSF of patients after TBI CSF samples from TBI patients were analyzed and compared with CSF from healthy / control individuals for protein expression of the inflammasome signaling proteins ASC and IL-18 using a Simple Plex assay (Protein Simple) (Figures 17A and 17B). Protein levels of both ASC and IL-18 in the serum of TBI patients were higher than in the control group.

[0257] ASC and IL-18 are good CSF biomarkers of TBI To determine whether these inflammasome signaling proteins could be reliable biomarkers of TBI pathology, the areas under the curve (AUC) for ASC and IL-18 in CSF were then determined (Figures 18A and 18B). ASC and IL-18 were shown to be the best biomarkers (Figures 18A and 18B), with AUCs of 1.0 (6th harvest) and 0.84 (1st harvest), respectively (Tables 13A and 13B).

[0258] Tables 13A and 13B: Results of ROC analysis for ASC (Table 13A) and IL-18 (Table 13B) in CSF, including cutoff points (pg / ml), sensitivity and specificity, and positive and negative likelihood ratios (LR+ / LR-).

[0259] [Table 13A]

[0260] [Table 13B]

[0261] Furthermore, the cutoff point for ASC was 74.33 pg / ml with 100% sensitivity and 100% specificity (Tables 14A and 17). For IL-18, the cutoff point was 2.722 pg / ml with 80% sensitivity and 68% specificity (Tables 14B and 18). As shown in Table 18, in the case of IL-18, for 100% specificity, the cutoff point was 3.879 pg / ml with 60% sensitivity, and for 100% sensitivity, the cutoff point was 1.358 pg / ml with 16% specificity. Thus, these findings indicate that ASC and IL-18 are reliable serum biomarkers for TBI.

[0262] Table 14A-B: Results of ROC analysis for ASC (Table 14A) and IL-18 (Table 14B) in CSF, including cutoff points (pg / ml), sensitivity and specificity, and positive and negative likelihood ratios (LR+ / LR-).

[0263] [Table 14A]

[0264] [Table 14B]

[0265] conclusion In this study, statistically significantly higher levels of ASC and caspase-1 were detected in the serum of patients with TBI compared with healthy controls. In this study, we demonstrate that ASC and IL-18 are reliable biomarkers for TBI in CSF, with AUC values ​​of 1.0 and 0.84, respectively. Most importantly, obtaining CSF is a highly invasive procedure, so our findings in serum are even more applicable to a typical clinical setting. Accordingly, we found an AUC value of 0.90 for ASC and 0.93 for caspase-1. Thus, caspase-1 and ASC should be considered as biomarkers in the care of patients with brain injury.

[0266] The data also show that the AUC for ASC was 0.92 when comparing patients with chronically unfavorable outcomes to those with favorable outcomes after TBI, thus highlighting the utility of ASC as a serum TBI biomarker, in this case as a predictive biomarker of brain injury.

[0267] Thus, based on these findings, ASC and caspace-1 both Both ASC and IL-18 are promising biomarkers with high AUC values, high sensitivity, and high specificity in serum. In addition, based on these findings, both ASC and IL-18 are promising biomarkers with high AUC values, high sensitivity, and high specificity in CSF. Importantly, the combination of ASC and other diagnostic criteria as biomarkers for TBI may further increase the sensitivity of ASC as a biomarker for TBI beyond those described in this example.

[0268] Importantly, in this study, ASC was identified as a potential biomarker of TBI pathology with a high AUC value of 0.9448, as well as a sensitivity of over 80% and a specificity of over 90%.

[0269] Incorporation by Reference The following references are incorporated by reference in their entirety for all purposes.

[0270] 1. Adamczak, S., Dale, G., De Rivero Vaccari, JP, Bullock, MR, Dietrich, WD, and Keane, RW(2012). Inflammasome proteins in cerebrospinal fluid of brain-injured patients as biomarkers of functional outcome: clinical article. J Neurosurg 117, 1119-1125.

[0271] 2. Brand, FJ, 3rd, Forouzandeh, M., Kaur, H., Travascio, F., and De Rivero Vaccari, JP (2016). Acidification changes affect the inflammasome in human nucleus pulposus cells. J Inflamm (Lond) 13, 29.

[0272] 3. De Rivero Vaccari, JP, Brand, F., 3rd, Adamczak, S., Lee, SW, Perez-Barcena, J., Wang, MY, Bullock, MR, Dietrich, WD, and Keane, RW (2016). Exosome-mediated inflammasome signaling after central nervous system injury. J Neurochem 136 Suppl 1, 39-48.

[0273] 4. Keane, R.W., Dietrich, W.D., and De Rivero Vaccari, J.P. (2018). Inflammasome Proteins As Biomarkers of Multiple Sclerosis. Front Neurol 9, 135.

[0274] 5. Xia J, Broadhurst DI, Wilson M and Wishart DS. Translational biomarker discovery in clinical metabolomics: an introductory tutorial. Metabolomics. 2013;9:280-299.

[0275]

Table 15

[0276]

Table 16-1

Table 16-2

Table 16-3

Table 16-4

Table 16-5

Table 16-6

[0277]

Table 17-1

Table 17-2

[0278] [Table 18-1] [Table 18-2]

[0279] [Table 19-1] [Table 19-2]

[0280] Example 4 Examination of inflammasome proteins as biomarkers for mild cognitive impairment (MCI) Introduction A biomarker is a characteristic that can be objectively measured and evaluated as an indicator of a normal or pathological biological process. 1 Important to the care of patients with MCI is the need for biomarkers that can predict onset, deterioration, and response to treatment. In addition, there is a need for minimally invasive methods of retrieving these biomarkers for analysis.

[0281] method Participants: In this example, samples were purchased from BioIVT. Sample donors were enrolled in a study called "Prospective Collection of Samples for Research," supported by SeraTrials, LLC. under IRB number 20170439. Serum samples from 72 normal male and female donors ranging in age from 50 to 68 years, and serum samples from 32 male and female patients diagnosed with MCI ranging in age from 56 to 91 years (Table 20), were analyzed.

[0282] [Table 20-1] [Table 20-2] [Table 20-3] [Table 20-4] [Table 20-5] [Table 20-6] [Table 20-7] [Table 20-8] [Table 20-9] [Table 20-10]

[0283] Simple Plex Assay Analysis of inflammasome protein (caspase-1, ASC, IL-1β, and IL-18) concentrations in serum samples from MCI and age-matched controls. 2、3 This was done using the Ella System (Protein System) as described in.

[0284] Biomarker analysis The data obtained by the Simple Plex assay were analyzed using Prism7 software (GraphPad). First, outliers were removed and the receiver operating characteristic (ROC) was calculated, thus obtaining a 95% confidence interval, standard deviation, and p-value. A p-value of less than 0.05 was considered significant. Then, cutoff points were obtained for different specificities and sensitivities, as well as their respective likelihood ratios. 2、3 .

[0285] result ASC and IL-18 are elevated in the serum of patients with MCI Serum samples from MCI patients and age-matched healthy donors were analyzed for the protein expression levels of ASC (Fig. 21A), caspase-1 (Fig. 21B), IL-18 (Fig. 21C), and IL-1β (Fig. 21D). We found that the protein levels of ASC and IL-18 were significantly higher in the MCI group compared with the control group, suggesting the involvement of ASC and IL-18 in the pathology of MCI.

[0286] ASC is a promising serum biomarker for MCI To determine whether inflammasome signaling proteins can be used as biomarkers for MCI, the area under the curve (AUC) was determined for caspase-1 (Figure 22A), ASC (Figure 22B), IL-1β (Figure 22C), and IL-18 (Figure 22D). Figure 23 shows all ROC curves from Figures 22A-22D overlaid on each other. Of all proteins analyzed, ASC showed the highest AUC of 0.974 (p<0.0001), followed by IL-18 with an AUC of 0.6896 (p=0.0025) (Table 21). The cutoff point for ASC was 264.9 pg / ml with 100% sensitivity and 74% specificity (see Tables 22 and 23); IL-18 had a cutoff point of 213.9 pg / ml with 74% sensitivity and 58% specificity (Tables 22 and 25). In addition to Table 22, cutoff points and sensitivity / specificity data for caspase-1 and IL-1 beta can be found in Tables 24 and 26, respectively.

[0287] [Table 21]

[0288] [Table 22]

[0289] [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4] [Table 23-5]

[0290] [Table 24-1] [Table 24-2]

[0291] [Table 25-1] [Table 25-2] [Table 25-3] [Table 25-4] [Table 25-5] [Table 25-6]

[0292] [Table 26]

[0293] Incorporation by Reference The following references are incorporated by reference in their entirety for all purposes.

[0294] 1.) Biomarkers Definitions Working G. Biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clin Pharmacol Ther. 2001;69:89-95.

[0295] 2.) Brand FJ, 3rd, Forouzandeh M, Kaur H, Travascio F, & de Rivero Vaccari JP (2016) Acidification changes affect the inflammasome in human nucleus pulposus cells. J Inflamm (Lond) 13(1):29.

[0296] 3.) Keane RW, Dietrich WD, & de Rivero Vaccari JP (2018) Inflammasome Proteins As Biomarkers of Multiple Sclerosis. Front Neurol 9:135.

[0297] Numbered Embodiments of the Present Disclosure Other subject matter contemplated by this disclosure is presented in the following numbered embodiments.

[0298] Embodiment 1. A method of evaluating a patient suspected of having multiple sclerosis (MS), comprising: measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with MS, wherein the protein signature comprises an elevated level of the at least one inflammasome protein; and selecting the patient as having MS if the patient exhibits the presence of the protein signature.

[0299] Embodiment 2 The method of embodiment 1, wherein the patient exhibits clinical symptoms consistent with MS.

[0300] Embodiment 3. The method of embodiment 1 or 2, wherein the MS is relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS), or progressive relapsing MS (PRMS).

[0301] Embodiment 4. The method of any one of the above embodiments, wherein the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs).

[0302] Embodiment 5. The method of any one of the above embodiments, wherein the level of the at least one inflammasome protein in the protein signature is measured by an immunoassay utilizing one or more antibodies to the at least one inflammasome protein in the protein signature.

[0303] Embodiment 6. The method of any one of the preceding embodiments, wherein the at least one inflammasome protein is interleukin-18 (IL-18), IL-1 beta, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, or a combination thereof.

[0304] Embodiment 7. The method of any one of the preceding embodiments, wherein the at least one inflammasome protein comprises each of caspase-1, IL-18, IL-1beta, and ASC.

[0305] Embodiment 8. The method of any one of embodiments 1 to 6, wherein the at least one inflammasome protein comprises ASC.

[0306] Embodiment 9. The method of any one of embodiments 5 to 8, wherein the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD), or a portion of the PYD or CARD domain of the ASC protein.

[0307] Embodiment 10. The method of any one of the above embodiments, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control.

[0308] Embodiment 11. The method of embodiment 10, wherein the biological sample obtained from the subject is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs).

[0309] Embodiment 12 The method of embodiment 10 or 11, wherein the control is a healthy individual, wherein the healthy individual does not exhibit clinical symptoms consistent with MS.

[0310] Embodiment 13. The method of any one of embodiments 10 to 12, wherein the at least one inflammasome protein comprises ASC, and the level of ASC is at least 50% higher than the level of ASC in the biological sample obtained from a control.

[0311] Embodiment 14. The method of any one of embodiments 1 to 9, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values.

[0312] Embodiment 15. The method of embodiment 14, wherein the biological sample obtained from the patient is serum, and the patient is selected as having MS with a sensitivity of at least 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 90%.

[0313] Embodiment 16 The method of embodiment 14 or 15, wherein the biological sample is serum and the patient is selected as having MS with a specificity of at least 80%, 85%, 90%, 95%, 99%, or 100%.

[0314] Embodiment 17 The method of embodiment 14, wherein the biological sample is serum and the patient is selected as having MS with at least 90% sensitivity and at least 80% specificity.

[0315] Embodiment 18. The method of any one of embodiments 14 to 17, wherein the at least one inflammasome protein comprises ASC.

[0316] Embodiment 19 The method of embodiment 18, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 7.

[0317] Embodiment 20. The method of any one of embodiments 15-17, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval.

[0318] Embodiment 21. A method of evaluating a patient suspected of having a stroke, comprising the steps of measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with stroke or stroke-related injury, wherein the protein signature comprises an elevated level of the at least one inflammasome protein; and selecting the patient as having a stroke if the patient exhibits the presence of the protein signature.

[0319] Embodiment 22 The method of embodiment 21, wherein the patient presents with clinical symptoms consistent with stroke, and the stroke is an ischemic stroke, a transient ischemic stroke, or a hemorrhagic stroke.

[0320] Embodiment 23 The method of embodiment 21 or 22, wherein the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs).

[0321] Embodiment 24. The method of any one of embodiments 21 to 23, wherein the level of the at least one inflammasome protein in the protein signature is measured by an immunoassay utilizing one or more antibodies to the at least one inflammasome protein in the protein signature.

[0322] Embodiment 25. The method of any one of embodiments 21 to 24, wherein the at least one inflammasome protein is interleukin-18 (IL-18), IL-1 beta, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, or a combination thereof.

[0323] Embodiment 26 The method of any one of embodiments 21 to 25, wherein the at least one inflammasome protein comprises each of caspase-1, IL-18, IL-1beta, and ASC.

[0324] Embodiment 27 The method of any one of embodiments 21 to 25, wherein the at least one inflammasome protein comprises ASC.

[0325] Embodiment 28. The method of any one of embodiments 25 to 27, wherein the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD), or a portion of the PYD or CARD domain of the ASC protein.

[0326] Embodiment 29. The method of any one of embodiments 21 to 28, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control.

[0327] Embodiment 30. The method of embodiment 29, wherein the biological sample obtained from the subject is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs).

[0328] Embodiment 31 The method of embodiment 29 or 30, wherein the control is a healthy individual, and the healthy individual is an individual who does not exhibit clinical symptoms consistent with MS.

[0329] Embodiment 32. The method of any one of embodiments 29 to 31, wherein the at least one inflammasome protein comprises ASC, and the level of ASC in a serum sample obtained from the subject is at least 70% higher than the level of ASC in a serum sample obtained from a control.

[0330] Embodiment 33. The method of any one of embodiments 29 to 31, wherein the at least one inflammasome protein comprises ASC, and the level of ASC in a serum-derived EV sample obtained from the subject is at least 110% higher than the level of ASC in a serum-derived EV sample obtained from a control.

[0331] Embodiment 34 The method of any one of embodiments 21 to 28, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values.

[0332] Embodiment 35. The method of embodiment 34, wherein the biological sample obtained from the patient is serum, and the patient is selected as having a stroke with a sensitivity of at least 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 90%.

[0333] Embodiment 36 The method of embodiment 34 or 35, wherein the biological sample is serum and the patient is selected as having a stroke with a specificity of at least 80%, 85%, 90%, 95%, 99%, or 100%.

[0334] Embodiment 37 The method of embodiment 34, wherein the biological sample is serum and the patient is selected as having a stroke with at least 100% sensitivity and at least 95% specificity.

[0335] Embodiment 38 The method of any one of embodiments 35 to 37, wherein the at least one inflammasome protein comprises ASC.

[0336] Embodiment 39 The method of embodiment 38, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 8.

[0337] Embodiment 40. The method of embodiment 34, wherein the biological sample obtained from the patient is serum-derived EVs, and the patient is selected as having a stroke with a sensitivity of at least 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 90%.

[0338] Embodiment 41. The method of embodiment 34 or 40, wherein the biological sample is serum-derived EVs and the patient is selected as having a stroke with a specificity of at least 80%, 85%, 90%, 95%, 99%, or 100%.

[0339] Embodiment 42 The method of embodiment 34, wherein the biological sample is serum-derived EVs and the patient is selected as having a stroke with at least 100% sensitivity and at least 100% specificity.

[0340] Embodiment 43 The method of any one of embodiments 40 to 42, wherein the at least one inflammasome protein comprises ASC.

[0341] Embodiment 44 The method of embodiment 43, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 9.

[0342] Embodiment 45. The method of any one of embodiments 35-37 or 40-42, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval.

[0343] Embodiment 46. A method of treating a patient diagnosed with multiple sclerosis (MS), comprising administering to the patient a standard of care treatment for MS, wherein the diagnosis of MS is made by detecting an elevated level of at least one inflammasome protein in a biological sample obtained from the patient.

[0344] Embodiment 47 The method of embodiment 46, wherein the MS is relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS), or progressive relapsing MS (PRMS).

[0345] Embodiment 48 The method of embodiment 46 or 47, wherein the standard of care treatment is selected from therapies directed to modifying disease outcome, managing recurrence, managing symptoms, or any combination thereof.

[0346] Embodiment 49. The method of embodiment 48, wherein the treatment directed to modifying disease outcome is selected from beta-interferon, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, mitoxantrone, ocrelizumab, alemtuzumab, daclizumab, and natalizumab.

[0347] Embodiment 50. A method of treating a patient diagnosed with stroke or a stroke-related injury, comprising administering to the patient a standard of care treatment for stroke or stroke-related injury, wherein the diagnosis of stroke or stroke-related injury is made by detecting an elevated level of at least one inflammasome protein in a biological sample obtained from the patient.

[0348] Embodiment 51 The method of embodiment 50, wherein the stroke is an ischemic stroke, a transient ischemic stroke, or a hemorrhagic stroke.

[0349] Embodiment 52. The method of embodiment 50 or 51, wherein the stroke is an ischemic stroke or a transient ischemic stroke, and the standard of care treatment is selected from tissue plasminogen activator (tPA), an antiplatelet agent, an anticoagulant, carotid angioplasty, carotid endarterectomy, intra-arterial thrombolysis, and mechanical clot removal in cerebral ischemia (MERCI), or a combination thereof.

[0350] Embodiment 53 The method of embodiment 50 or 51, wherein the stroke is a hemorrhagic stroke and the standard of care treatment is aneurysm clipping, coil embolization, or arteriovenous malformation (AVM) repair.

[0351] Embodiment 54. The method of any one of embodiments 46 to 53, wherein the increased level of the at least one inflammasome protein is measured by an immunoassay utilizing one or more antibodies to the at least one inflammasome protein.

[0352] Embodiment 55. The method of any one of embodiments 46-54, wherein the level of the at least one inflammasome protein is increased relative to the level of the at least one inflammasome protein in a control sample.

[0353] Embodiment 56 The method of any one of embodiments 46 to 54, wherein the level of the at least one inflammasome protein is increased relative to a predetermined reference value or range of reference values.

[0354] Embodiment 57. The method of any one of embodiments 46 to 56, wherein the at least one inflammasome protein is interleukin-18 (IL-18), apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof.

[0355] Embodiment 58 The method of embodiment 56 or 57, wherein the at least one inflammasome protein is caspase-1, IL-18, and ASC.

[0356] Embodiment 59 The method of embodiment 56 or 57, wherein the at least one inflammasome protein is ASC.

[0357] Embodiment 60 The method of embodiment 59, wherein the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD), or a portion of the PYD or CARD domain of the ASC protein.

[0358] Embodiment 61. The method of any one of embodiments 46 to 60, wherein the biological sample is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs).

[0359] Embodiment 62. A method for evaluating a patient suspected of having a traumatic brain injury (TBI), comprising the steps of measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with TBI, wherein the protein signature comprises an elevated level of the at least one inflammasome protein; and selecting the patient as having TBI if the patient exhibits the presence of the protein signature.

[0360] Embodiment 63 The method of embodiment 62, wherein the patient exhibits clinical symptoms consistent with TBI.

[0361] Embodiment 64. The method of embodiment 62 or 63, wherein the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs).

[0362] Embodiment 65. The method of any one of embodiments 62 to 64, wherein the level of the at least one inflammasome protein in the protein signature is measured by an immunoassay utilizing one or more antibodies to the at least one inflammasome protein in the protein signature.

[0363] Embodiment 66. The method of any one of embodiments 62 to 65, wherein the at least one inflammasome protein is interleukin-18 (IL-18), IL-1β, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, or a combination thereof.

[0364] Embodiment 67. The method of any one of embodiments 61 to 66, wherein the at least one inflammasome protein comprises caspase-1.

[0365] The method of any one of embodiments 65 to 67, wherein at least one inflammasome protein comprises caspase-1, and the level of caspase-1 is at least 50% higher than the level of caspase-1 in a biological sample obtained from a control.

[0366] Embodiment 68 The method of any one of embodiments 61 to 66, wherein the at least one inflammasome protein comprises ASC.

[0367] Embodiment 69. The method of any one of embodiments 66 or 68, wherein the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD), or a portion of the PYD or CARD domain of the ASC protein.

[0368] Embodiment 70. The method of any one of embodiments 62 to 69, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control.

[0369] Embodiment 71. The method of embodiment 70, wherein the at least one inflammasome protein comprises caspase-1, and the level of caspase-1 is at least 50% higher than the level of caspase-1 in the biological sample obtained from the control.

[0370] Embodiment 72. The method of embodiment 70, wherein the at least one inflammasome protein comprises ASC, and the level of ASC is at least 50% higher than the level of ASC in the biological sample obtained from the control.

[0371] Embodiment 73. The method of any one of embodiments 70 to 72, wherein the biological sample obtained from the subject is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs).

[0372] Embodiment 74 The method of any one of embodiments 70 to 73, wherein the control is a healthy individual, and the healthy individual is an individual who does not exhibit clinical symptoms consistent with TBI.

[0373] Embodiment 75. The method of any one of embodiments 62 to 69, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values.

[0374] Embodiment 76. The method of embodiment 75, wherein the biological sample obtained from the patient is serum, and the patient is selected as having TBI with a sensitivity of at least 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 90%.

[0375] Embodiment 77. The method of embodiment 75 or 76, wherein the biological sample is serum and the patient is selected as having TBI with a specificity of at least 80%, 85%, 90%, 95%, 99%, or 100%.

[0376] Embodiment 78. The method of embodiment 75, wherein the biological sample is serum and the patient is selected as having TBI with at least 90% sensitivity and at least 80% specificity.

[0377] Embodiment 79. The method of any one of embodiments 76-76, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval.

[0378] Embodiment 80 The method of any one of embodiments 75 to 79, wherein the at least one inflammasome protein comprises ASC.

[0379] Embodiment 81 The method of embodiment 79, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 11B, Table 12B, Table 14A, Table 16, Table 17, or Table 19.

[0380] Embodiment 82 The method of any one of embodiments 75 to 79, wherein the at least one inflammasome protein comprises caspase-1.

[0381] Embodiment 83 The method of embodiment 82, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 11A or Table 15.

[0382] Embodiment 84. A method for evaluating a patient suspected of having a brain injury, comprising the steps of measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with brain injury, the protein signature comprising an elevated level of the at least one inflammasome protein; and selecting the patient as having a brain injury if the patient exhibits the presence of the protein signature.

[0383] Embodiment 85 The method of embodiment 84, wherein the patient exhibits clinical symptoms consistent with brain injury.

[0384] Embodiment 86 The method of embodiment 84 or 85, wherein the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs).

[0385] Embodiment 87. The method of any one of embodiments 84 to 86, wherein the level of the at least one inflammasome protein in the protein signature is measured by an immunoassay utilizing one or more antibodies to the at least one inflammasome protein in the protein signature.

[0386] Embodiment 88. The method of any one of embodiments 84 to 87, wherein the at least one inflammasome protein is interleukin-18 (IL-18), IL-1β, apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof.

[0387] Embodiment 89. The method of any one of embodiments 84 to 88, wherein the at least one inflammasome protein comprises ASC.

[0388] Embodiment 90 The method of embodiment 88 or 89, wherein the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD) domain, or a portion of the PYD or CARD domain of the ASC protein.

[0389] Embodiment 91 The method of any one of embodiments 84-88, wherein the at least one inflammasome protein comprises caspase-1.

[0390] Embodiment 92. The method of any one of embodiments 84 to 91, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control.

[0391] Embodiment 93 The method of embodiment 92, wherein the at least one inflammasome protein comprises ASC, and the level of ASC is at least 50% higher than the level of ASC in the biological sample obtained from the control.

[0392] Embodiment 94. The method of embodiment 92, wherein the at least one inflammasome protein comprises caspase-1, and the level of caspase-1 is at least 50% higher than the level of caspase-1 in the biological sample obtained from the control.

[0393] Embodiment 95. The method of any one of embodiments 92 to 94, wherein the biological sample obtained from the subject is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs).

[0394] Embodiment 96 The method of any one of embodiments 92-95, wherein the control is a healthy individual, and the healthy individual is an individual who does not exhibit clinical symptoms consistent with brain injury.

[0395] Embodiment 97. The method of any one of embodiments 84-96, wherein the brain injury is selected from traumatic brain injury, stroke, mild cognitive impairment, or multiple sclerosis.

[0396] Embodiment 98. The method of any one of embodiments 84 to 91, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values.

[0397] Embodiment 99. The method of embodiment 98, wherein the brain injury is a traumatic brain injury (TBI).

[0398] Embodiment 100. The method of embodiment 99, wherein the biological sample obtained from the patient is serum, and the patient is selected as having TBI with a sensitivity of at least 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 90%.

[0399] Embodiment 101. The method of embodiment 98 or 99, wherein the biological sample is serum and the patient is selected as having TBI with a specificity of at least 80%, 85%, 90%, 95%, 99%, or 100%.

[0400] Embodiment 102 The method of embodiment 99, wherein the biological sample is serum and the patient is selected as having TBI with at least 90% sensitivity and at least 80% specificity.

[0401] Embodiment 103. The method of any one of embodiments 100 to 102, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval.

[0402] Embodiment 104. The method of any one of embodiments 99 to 103, wherein the at least one inflammasome protein comprises ASC.

[0403] Embodiment 105 The method of embodiment 104, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Tables 11B, 12B, 14A, 16, 17, or 19.

[0404] Embodiment 106 The method of any one of embodiments 99 to 103, wherein the at least one inflammasome protein comprises caspase-1.

[0405] Embodiment 107 The method of embodiment 106, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 11A or 15.

[0406] Embodiment 108 The method of embodiment 98, wherein the brain injury is multiple sclerosis (MS).

[0407] Embodiment 109. The method of embodiment 108, wherein the biological sample obtained from the patient is serum, and the patient is selected as having MS with a sensitivity of at least 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 90%.

[0408] Embodiment 110. The method of embodiment 108 or 109, wherein the biological sample is serum and the patient is selected as having MS with a specificity of at least 80%, 85%, 90%, 95%, 99%, or 100%.

[0409] Embodiment 111 The method of embodiment 108, wherein the biological sample is serum and the patient is selected as having MS with at least 90% sensitivity and at least 80% specificity.

[0410] Embodiment 112. The method of any one of embodiments 108 to 111, wherein the at least one inflammasome protein comprises ASC.

[0411] Embodiment 113 The method of embodiment 112, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 7.

[0412] Embodiment 114. The method of any one of embodiments 109 to 113, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval.

[0413] Embodiment 115 The method of embodiment 98, wherein the brain injury is a stroke.

[0414] Embodiment 116. The method of embodiment 115, wherein the biological sample obtained from the patient is serum, and the patient is selected as having a stroke with a sensitivity of at least 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 90%.

[0415] Embodiment 117. The method of embodiment 115 or 116, wherein the biological sample is serum and the patient is selected as having a stroke with a specificity of at least 80%, 85%, 90%, 95%, 99%, or 100%.

[0416] Embodiment 118 The method of embodiment 115, wherein the biological sample is serum and the patient is selected as having a stroke with at least 100% sensitivity and at least 95% specificity.

[0417] Embodiment 119. The method of any one of embodiments 116 to 118, wherein the at least one inflammasome protein comprises ASC.

[0418] Embodiment 120 The method of embodiment 119, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 8.

[0419] Embodiment 121. The method of embodiment 115, wherein the biological sample obtained from the patient is serum-derived EVs, and the patient is selected as having a stroke with a sensitivity of at least 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 90%.

[0420] Embodiment 122. The method of embodiment 115 or 121, wherein the biological sample is serum-derived EVs and the patient is selected as having a stroke with a specificity of at least 80%, 85%, 90%, 95%, 99%, or 100%.

[0421] Embodiment 123 The method of embodiment 115, wherein the biological sample is serum-derived EVs and the patient is selected as having a stroke with at least 100% sensitivity and at least 100% specificity.

[0422] Embodiment 124. The method of any one of embodiments 121 to 123, wherein the at least one inflammasome protein comprises ASC.

[0423] Embodiment 125 The method of embodiment 124, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 9.

[0424] Embodiment 126. The method of any one of embodiments 116-118 or 121-123, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval.

[0425] Embodiment 127. A method for evaluating a patient suspected of having mild cognitive impairment (MCI), comprising the steps of measuring the level of at least one inflammasome protein in a biological sample obtained from the patient; determining the presence or absence of a protein signature associated with MCI, wherein the protein signature comprises an elevated level of the at least one inflammasome protein; and selecting the patient as having MCI if the patient exhibits the presence of the protein signature.

[0426] Embodiment 128 The method of embodiment 127, wherein the patient exhibits clinical symptoms consistent with MCI.

[0427] Embodiment 129. The method of embodiment 127 or 128, wherein the biological sample obtained from the patient is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs).

[0428] Embodiment 130. The method of any one of embodiments 127 to 129, wherein the level of the at least one inflammasome protein in the protein signature is measured by an immunoassay utilizing one or more antibodies to the at least one inflammasome protein in the protein signature.

[0429] Embodiment 131. The method of any one of embodiments 127 to 130, wherein the at least one inflammasome protein is interleukin-18 (IL-18), IL-1β, apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain, caspase-1, or a combination thereof.

[0430] Embodiment 132. The method of any one of embodiments 127 to 131, wherein the at least one inflammasome protein comprises ASC.

[0431] Embodiment 133. The method of any one of embodiments 127 to 131, wherein the at least one inflammasome protein comprises IL-18.

[0432] Embodiment 134. The method of any one of embodiments 131-132, wherein the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD), the C-terminal caspase recruitment domain (CARD), or a portion of the PYD or CARD domain of the ASC protein.

[0433] Embodiment 135. The method of any one of embodiments 127 to 134, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to the level of the at least one inflammasome protein in a biological sample obtained from a control.

[0434] Embodiment 136 The method of embodiment 135, wherein the at least one inflammasome protein comprises ASC, and the level of ASC is at least 50% higher than the level of ASC in the biological sample obtained from the control.

[0435] Embodiment 137. The method of embodiment 135, wherein the at least one inflammasome protein comprises IL-18, and the level of IL-18 is at least 25% higher than the level of IL-18 in the biological sample obtained from the control.

[0436] Embodiment 138. The method of any one of embodiments 135 to 137, wherein the biological sample obtained from the subject is cerebrospinal fluid (CSF), CNS microdialysate, saliva, serum, plasma, urine, or serum-derived extracellular vesicles (EVs).

[0437] Embodiment 139 The method of any one of embodiments 135-138, wherein the control is a healthy individual, and the healthy individual is an individual who does not exhibit clinical symptoms consistent with MCI.

[0438] Embodiment 140. The method of any one of embodiments 127 to 134, wherein the level of the at least one inflammasome protein in the protein signature is increased relative to a predetermined reference value or range of reference values.

[0439] Embodiment 141. The method of embodiment 140, wherein the biological sample obtained from the patient is serum, and the patient is selected as having MCI with a sensitivity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% and a specificity of at least 55%.

[0440] Embodiment 142. The method of embodiment 140 or 141, wherein the biological sample is serum and the patient is selected as having MCI with a sensitivity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.

[0441] Embodiment 143 The method of embodiment 140, wherein the biological sample is serum and the patient is selected as having MCI with a sensitivity of at least 70% and a specificity of at least 55%.

[0442] Embodiment 144. The method of any one of embodiments 140 to 143, wherein the sensitivity and / or sensitivity is determined using the area under the curve (AUC) from a receiver operating characteristic (ROC) curve with a 95% confidence interval.

[0443] Embodiment 145. The method of any one of embodiments 140 to 144, wherein the at least one inflammasome protein comprises ASC.

[0444] Embodiment 146 The method of embodiment 145, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 22.

[0445] Embodiment 147. The method of any one of embodiments 140 to 144, wherein the at least one inflammasome protein comprises IL-18.

[0446] Embodiment 148 The method of embodiment 147, wherein the cutoff value for determining the sensitivity, specificity, or both is selected from Table 22.

[0447] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to utilize concepts from the various patents, applications, and publications to provide still further embodiments.

[0448] These and other variations can be made to the embodiments in light of the above detailed description. Generally, in the following claims, the terms used should not be construed to limit the scope of the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments, along with the full range of equivalents to which such claims are entitled. Accordingly, the scope of the claims is not limited by the present disclosure.

Claims

1. A composition for use in a method of treating a patient having a stroke, the composition reducing ASC levels in the patient, the method comprising: (a) measuring the expression level of at least one inflammasome protein in a biological sample obtained from the patient, wherein the inflammasome protein is selected from the group consisting of NAPL1 / NLRP1, NALP2 / NLRP2, NALP3 / NLRP3, IPAF / NLRC4, AIM2, pryin, ASC, caspase-1, caspase-4, caspase-5, GSDM-D, IL-1β, and IL-18, and the expression level is measured by an immunoassay utilizing one or more antibodies that bind to at least one inflammasome protein; (b) comparing the expression level of the at least one inflammasome protein in the biological sample obtained from the patient with a control; (c) diagnosing the patient with stroke if the expression level of serum ASC in the biological sample obtained from the patient is greater than 300 picograms / ml, or if the expression level of serum-derived extracellular vesicle ASC in the biological sample obtained from the patient is greater than 70 picograms / ml; (d) administering a therapeutically effective amount of the composition, wherein the composition comprises an antibody having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; A composition comprising:

2. The composition described in claim 1, wherein the stroke is an ischemic stroke, a transient ischemic stroke, or a hemorrhagic stroke.

3. The composition described in claim 1, wherein the control is a predetermined reference value or range of reference values.

4. The composition described in claim 1, wherein the antibody binds to a PYRIN domain (PYD), a C-terminal caspase recruitment domain (CARD) domain, or a portion of the PYD or CARD domain of the ASC protein.

5. The composition described in claim 1, wherein the method further comprises administering to the patient a standard of care treatment for stroke or stroke-related injury.

6. The composition described in claim 5, wherein the stroke is an ischemic stroke or a transient ischemic stroke and the standard of care treatment is selected from tissue plasminogen activator (tPA), antiplatelet agents, anticoagulants, carotid angioplasty, carotid endarterectomy, intra-arterial thrombolysis and mechanical clot removal in cerebral ischemia (MERCI), or combinations thereof.

7. The composition described in claim 5, wherein the stroke is a hemorrhagic stroke and the standard care treatment is aneurysm clipping, coil embolization, or arteriovenous malformation (AVM) repair.

Images