WO2023212583A1 - Protéines immunitaires innées en tant que biomarqueurs pour lésion cérébrale traumatique chez des patients adultes et pédiatriques - Google Patents
Protéines immunitaires innées en tant que biomarqueurs pour lésion cérébrale traumatique chez des patients adultes et pédiatriques Download PDFInfo
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Definitions
- the present invention relates generally to the fields of neurology, immunology, and diagnostics.
- the present invention relates to the identification of biomarkers in biological samples which can predict the severity of neuronal injury, such as spinal cord and traumatic brain injury, in patients.
- the identified biomarkers may also be used in determining prognosis, directing therapeutic and rehabilitation efforts, and monitoring response to treatment for patients with a central nervous system injury.
- Nucleotide-binding oligomerization domain (NOD)-containing protein-like receptors (NLRs) are a recently discovered class of innate immune receptors that play a crucial role in initiating inflammatory responses following tissue injury in the central nervous system (CNS) (Abulafia et al., 2009, Silverman et al., 2009).
- NLRP1 also known as NAcht leucine-rich- repeat protein 1 (NALP-1)
- NALP-1 NAcht leucine-rich- repeat protein 1
- ASC caspase recruitment domain
- caspase-1 enzyme that orchestrate the early inflammatory processes after spinal cord injury (SCI) and traumatic brain injury (TBI) via IL- 1 13 activation (de Rivero Vaccari et al., 2008; 2009).
- inflammasomes are induced by physical damage to the plasma membrane, and by certain endogenous ligands referred to as danger associated molecular patterns (DAMPs) or exogenous ligands known as pathogen associated molecular patterns (PAMPs) (Bianchi, 2007, Wakefield et al., 2010).
- DAMPs danger associated molecular patterns
- PAMPs pathogen associated molecular patterns
- TLRs Toll-like receptors
- purinergic ATP-gated receptors which induce the transcription of pro- IL-1 ⁇ .
- Inflammasome-mediated IL-1 ⁇ overproduction is involved in the pathogenesis of type 2 diabetes, liver damage and muscular dystrophy (Kufer and Sansonetti, 2011).
- inflammasome activation could also drive adaptive immunity in types of dermatitis, skin related allergies and asthma (Kufer and Sansonetti, 2011).
- inflammasome components may be secreted into the extracellular milieu via a mechanism involving the exosome pathway (Bianchi, 2007). The inflammasome therefore has a complex connection with the control of adaptive immune responses that has become the subject of intense investigation. Whether inflammasomes are associated with tissue destructive inflammatory processes after SCI and TBI in humans has not been investigated.
- Traumatic brain injury has a complex, biphasic pathology that represents a significant public health concern in the United States and throughout the world. TBI affects an estimated 1.5 million people each year and causes one-third of injury-related deaths. Approximately 5.3 million Americans are living today with a permanent TBI-related disability representing an annual economic impact in excess of $56 billion. Predicting the severity and outcome of TBI and well as SCI is difficult, given the lack of objective, laboratory-based biomarkers.
- GCS Glasgow Coma Scale
- biomarkers S-10013, neuron-specific enolase, neurofilament light chain and glial fibrillary acidic protein are significantly increased in cases of SCI in experimental animal studies (Skouen et al., 1999, Ma et al., 2001, Nagy et al., 2002, Cornefjord et al., 2004, Loy et al., 2005, Cao et al., 2008, Pouw et aL, 2009). Although some biomarkers show promising results, these do not yet provide a sensitive prognostic tool. Quantitative standards for determining the extent of SCI and TBI during the acute phase must be developed and validated.
- DAMPS damage associated molecular patterns
- PAMPS pathogen associated molecular patterns
- TLR toll like receptors
- the inflammasome is a multi-protein complex that allows for the activation of caspase-1 using a scaffolding protein known as apoptosis-associated speck-like protein containing a caspase recruiting domain (ASC.) Oligomerization of ASC forms an ASC speck that binds to caspase-1, resulting in formation of the inflammasome complex and downstream activation of the pro-inflammatory cytokines interleukin (I L)-18 and IL-1 ⁇ , allowing for the cleavage and release of the inflammatory cytokines interleukin IL-1 ⁇ and IL-18, as well as the formation of a gasdermin-D pore as part of the programmed cell death mechanism of pyroptosis.
- I L interleukin
- IL-1 ⁇ pro-inflammatory cytokines
- Inflammasome formation can be triggered through numerous vectors and has been shown to be activated after TBI in rodents and humans. Increased levels of inflammasome proteins during the acute phase after TBI are associated with worsened functional outcomes. Chronic inflammatory activity is often seen months to years after injury, resulting in a secondary injury from chronically activated microglia and their subsequent release of inflammatory cytokines.
- CSF cerebrospinal fluid
- the present invention is based, in part, on the discovery that NLRP1 (NALP-1) inflammasome components are secreted into the cerebrospinal fluid (CSF) acutely after SCI and traumatic brain injury (TBI) in humans. Elevated inflammasome protein levels in the CSF following central nervous system (CNS) injury represent the degree of neuroinflammation in CNS tissue and reflect the extent of inflammatory-induced damage. The CSF levels of inflammasome protein following injury correlate with the degree of functional recovery in patients and thus, can be used as acute biomarkers to predict patient prognosis and direct therapeutic interventions. Accordingly, the present invention provides a method of assessing the severity of a CNS injury in a patient, including an accurate and predictable determination and diagnosis of traumatic brain injury (TBI).
- TBI traumatic brain injury
- the invention provides a method of evaluating a patient suspected of having a CNS injury comprising providing a biological sample from a patient presenting with clinical symptoms consistent with a CNS injury, measuring the level of at least one inflammasome protein in the biological sample, determining the presence or absence of a protein signature associated with a CNS injury or a more severe CNS injury, such as traumatic brain injury (TBI), wherein the protein signature comprises an elevated level of said at least one inflammasome protein, and selecting patients exhibiting the presence of the protein signature as having a CNS injury or a more severe CNS injury.
- said one or more inflammasome proteins are NLRP1 (NALP-1), ASC, or caspase-1.
- the diagnostic methods of the invention may further comprise administering a neuroprotective treatment to the patient based on the measured level of one or more inflammasome proteins, and following changes in the level of one or more inflammasome proteins as a mechanism to monitor response to treatment.
- the levels of one or more inflammasome proteins in the patient's sample can be used to prepare an inflammasome protein profile associated with CNS injury.
- the levels of inflammasome proteins in the profile may be determined relative to levels of the proteins in control samples or pre-determined reference values or ranges of reference values.
- the inflammasome protein profiles are, in some embodiments, indicative of the presence or severity of CNS injury in a patient.
- the inflammasome protein profiles are indicative of therapeutic efficacy of the neuroprotective treatment in the patient.
- the subject invention includes the discovery of cut-off values for certain immune proteins or inflammasome proteins which can determine whether the CNS injury is traumatic brain injury (TBI ) .
- the present invention also provides a method of determining a prognosis for a patient with a central nervous system injury.
- the method comprises providing a biological sample, such as cerebrospinal fluid, obtained from the patient shortly after injury (e.g., within a week of injury), and measuring the level of at least one inflammasome protein in the biological sample to prepare an inflammasome protein profile, wherein the inflammasome protein profile is indicative of the prognosis of the patient.
- an elevated level of at least one inflammasome protein relative to a pre-determined reference value or range of reference values is indicative of a poorer prognosis or unfavorable patient outcome.
- elevated inflammasome protein levels are predictive of the patient having a Glasgow Outcome Scale (GOS) score of 1 to 3 upon follow-up assessment.
- GOS Glasgow Outcome Scale
- a reduced level of at least one inflammasome protein relative to a pre-determined reference value or range of reference values is predictive of a favorable patient outcome (e.g., GOS score of 4 or 5 upon follow-up assessment).
- the method provides a prognosis for a patient with a spinal cord or traumatic brain injury.
- kits for preparing an inflammasome protein profile associated with CNS injury comprises a labeled-binding partner, such as labeled-antibody or fragment thereof, that specifically binds to one or more inflammasome proteins, wherein said one or more inflammasome proteins are selected from the group consisting of NLRP1, ASC, caspase-1, and combinations thereof.
- Inflammasome proteins and inflammatory cytokines were elevated after TBI, and that apoptosis-associated speck like protein containing a caspase recruitment domain (ASC), interleukin ( I L)-18, tumor necrosis factor (TNF)- ⁇ , IL-4 and IL-6 were the most promising biomarkers. Additionally, levels of these proteins correlated with known clinical indicators of pathological outcome, such as Glasgow coma scale (GCS). Our results show that inflammatory cytokines and inflammasome proteins are promising biomarkers for determining pathological outcomes after TBI. Additionally, levels of biomarkers could potentially be utilized to determine a patient's injury severity and subsequent pathological outcome.
- protective or therapeutic treatment of traumatic brain injury can be administered to a patient suffering from TBI, as determined by the disclosed method.
- a small molecule drug or large molecule such as a peptide, protein, antibody, or the like, binding to one or more of the specific immune protein or inflammasome biomarkers identified herein, presenting at levels meeting the cut-off value, or within 20% of the cut-off value for that biomarker, can be administered to the patient in an effective amount such that the immune protein or inflammasome biomarker, or its cascade production, is inactivated or disabled.
- NLRP1, ASC, and Caspase-1 are biomarkers that predict outcome after SCI.
- CSF samples were immunoblotted with antibodies against NLRP1, caspase-1 and ASC.
- CSF samples from uninjured patients were used as controls. Immunoblot analysis of 6 different cases of patients with SCI indicates that patients (2, 3 and 4) who express low levels of caspase-1 acutely after SCI have a better prognosis than subjects (1, 5 and 6) who have elevated levels of this protein in CSF.
- NLRP1 inflammasome proteins are expressed in cells of the CNS. Spinal cord sections were obtained from decedents that had injury to the spinal cord. Immunohistochemical analysis combined with light microscopy indicates that NLRP1 is expressed in neurons of the ventral horn (black arrows) myelinated axons (black arrow heads) and oligodendrocytes (yellow arrows) (top panel). Caspase-1 is expressed in swollen axons (spheroids, blue arrows), motor neurons (black arrows) and in oligodendrocytes (yellow arrows) (central panel).
- Figure 4 Box plots of expression of inflammasome proteins sorted by outcome category. The ends of the whiskers represent the lowest datum within 1.5 interquartile range of the lower quartile and the highest datum within 1.5 interquartile range of the upper quartile. The asterisks represent the outliers. Mann-Whitney U-tests indicate higher expression of ASC (A), caspase-1 (p20) (B), and NALP-1 (C) are significantly associated with an unfavorable outcome 5 months after injury (p ⁇ 0.0001). Representative immunoblots for each protein are shown. Samples were run on the same gel but were noncontiguous.
- FIG. Scatter plots and estimated linear regression of ASC (A), caspase-1 (p20) (B), and NALP-1 (C) expression in the CSF with GOS score. Probability values of the linear regression are shown in the top left of each graph. Expression of each protein correlated significantly with GOS score at 5 months post-injury. The p values on the x axis represent post hoc comparisons of a Kruskal-Wallis test. Representative immunoblots are shown. Samples were run on the same gel but were noncontiguous.
- FIG. Caspase-1 levels in CSF one, two, and three days following TBI in pediatric patients receiving hypothermia treatment or no treatment (normothermia).
- FIG. 7 Inflammatory cytokines and Inflammasome Proteins are Elevated afterTBI. Simple Plex Assay and MSD-VPLEX Inflammatory Panel of blood serum from TBI patients and age matched controls. Data were analyzed utilizing a two-tailed Mann-Whitney nonparametric test. Inflammatory cytokines and inflammasome proteins that showed statistically significant increase after TBI were plotted. Box and whisker plots show mean, quartiles, and outliers for each inflammatory protein of interest with respective p values listed above. Dots correspond to data points outside the 5th and 95th percent confidence interval.
- Results showed that A) Caspase-1: N: Control: 31, TBI: 78; B) ASC: N: Control: 28, TBI: 91; C) IL-18: N : Control: 31, TBI: 90; D) (TNF)- ⁇ : N: Control: 21, TBI: 51; E) IL-6: N: Control: 21, TBI: 46; and F) IL-4: N: Control: 20, TBI: 50; G) IL-10: N: Control: 19, TBI: 41; H) IL-8: N: Control: Control: 12, TBI: 52 were all significantly elevated in TBI patients when compared to controls. I) IL-2: N: Control: Control: 9, TBI: 19 was significantly decreased in TBI patients when compared to controls.
- FIG. 8 ROC of Inflammatory Biomarkers. ROC and AUC were calculated for each inflammatory cytokine and inflammasome proteins that were significantly different when comparing healthy uninjured controls and TBI patients.
- FIG. 9 ROC Comparison among Inflammatory Biomarkers.
- FIG. 10 Inflammatory biomarkers as predictors of TBI severity.
- FIG. 11 Inflammatory biomarkers as predictors of TBI outcome. Box and whisker plots showing the protein levels in pg/mL of caspasea-1 (A) and IL-10 (B) as a biomarker of outcome after TBI. Box and whiskers are shown for the 5th and 95th percentile. Dots correspond to data points outside the Sth and 95th percent confidence interval.
- FIG. 12 Residual Analysis. Residual analysis of the multivariate linear regression models used to explain GCS (A) and GOS-E (B) were performed to determine the goodness of fit of each of the models.
- FIG. 14 ROC of inflammasome protein biomarkers. ROC and AUC were calculated for each inflammasome protein that were significantly different when comparing healthy uninjured controls and pTBI patients.
- FIG. 15 Inflammatory biomarkers as predictors of TBI severity.
- FIG. 16 Inflammatory biomarkers as predictors of TBI outcome.
- A Box and whisker plots showing the protein levels in pg/mL of IL-18, 0 hr. after TBI at 12 months average. Box and whiskers are shown for the Sth and 95th percentile. Dots correspond to data points outside the Sth and 95th percent confidence interval.
- C Box and whisker plots showing the protein levels in pg/mL of IL-18, 48h after TBI at 12 months average.
- E Box and whisker plots showing the protein levels in pg/mL of IL-1 ⁇ , Oh after TBI at 12 months average.
- the present invention is based, in part, on the discovery that NLRP1 inflammasomes play an important role in inflammatory responses after SCI and TBI in humans.
- the present inventors have surprisingly found that nucleotide-binding leucine-rich repeat pyrin domain containing protein 1 (NLRP1), the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1 are secreted into the cerebrospinal fluid (CSF) of human patients following SCI and TBI.
- NLRP1 nucleotide-binding leucine-rich repeat pyrin domain containing protein 1
- ASC caspase recruitment domain
- caspase-1 caspase-1
- the present invention provides a method of assessing the severity of a central nervous system injury in a patient by measuring the level of at least one inflammasome protein in a biological sample obtained from the patient, wherein the measured level of said at least one inflammasome protein is indicative of the severity of the central nervous system injury in the patient.
- inflammasome refers to a multi-protein complex that activates caspase-1 activity, which in turn regulates IL-1 ⁇ , IL-18 and IL-33 processing and activation. See Arend et al. 2008; Li et al. 2008; and Martinon, et al. 2002, each of which is incorporated by reference in their entireties.
- an “inflammasome protein” is a protein component of inflammasome complexes and can include, but is not limited to, a nucleotide binding domain, leucine-rich repeat containing (NLR) family member (e.g., NLRP1), ASC, caspase-1, caspase-11 X-linked inhibitor of apoptosis protein (XTAP), and pannexin-1.
- NLR leucine-rich repeat containing
- ASC e.g., NLRP1
- caspase-1 caspase-11 X-linked inhibitor of apoptosis protein
- pannexin-1 pannexin-1.
- NLRP1 is also known as NAcht leucine-rich-repeat protein 1 (NALP-1).
- NALP-1 NAcht leucine-rich-repeat protein 1
- the method comprises measuring an inflammasome protein selected from the group consisting of NLRP1 (NALP-1), ASC, caspase-1, or combinations thereof.
- patient or “subject” are used interchangeably herein, and is meant a mammalian subject to be treated, with human patients being preferred.
- the patient is a pediatric padent.
- Pediatric patients include newborns (birth to 1 month of age), infants (1 month to 2 years of age), children (2 to 12 years of age), and adolescents (12-21 years of age).
- the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates.
- the present invention provides a method of evaluating a patient suspected of having a central nervous system (CNS) injury.
- the method comprises providing a biological sample from a patient presenting with clinical symptoms consistent with a CNS injury; measuring the level of at least one inflammasome protein in the biological sample; determining the presence or absence of a protein signature associated with a CNS injury or a more severe CNS injury, wherein the protein signature comprises an elevated level of said at least one inflammasome protein; and selecting patients exhibiting the presence of the protein signature as having a CNS injury or a more severe CNS injury.
- a patient may be suspected of having a CNS injury based on neurologic symptoms (motor, sensory, cognitive) and/or radiological evaluation (MRI, CT scan, X-ray) consistent with a CNS injury, e.g., after a physician's exam.
- a patient suspected of having a CNS injury, particularly a spinal cord injury may having a rating of A or Bon the American Spinal Cord Injury Association (ASIA) Impairment Scale.
- ASIA Impairment Scale is a standard diagnostic tool that assesses a patient's motor and sensory function. The classification ratings and accompanying descriptions of the ASTA Impairment Scale are as follows:
- a patient presenting with a classification rating of A or B on the ASIA Impairment Scale has no motor function below the level of the injury.
- a patient suspected of having a CNS injury may have a score of :S 12 (e.g., 3 to 12) on the Glasgow Coma Scale (GCS).
- GCS Glasgow Coma Scale
- the patient may have a GCS score of :S 8 (e.g., 3 to 8).
- the GCS is a neurological scale commonly used to assess the level of consciousness of patients after injury or trauma. The scale is composed of three tests (eye, verbal, and motor responses), each of which is assigned a value on a scale up to 6. The three values separately as well as their sum are considered. The lowest possible GCS score (the sum) is 3 (deep coma or death), while the highest is 15 (fully awake person).
- a GCS score ⁇ 9 is indicative of severe brain injury whereas a GCS score > 13 is indicative of minor brain injury.
- a GCS score between 9-12 is generally indicative of a moderate brain injury.
- a patient suspected of having a CNS injury may have one or more signs and symptoms of CNS injury, such as temporary loss of consciousness, confusion, disorientation, memory or concentration problems, headache, dizziness, loss of balance, nausea or vomiting, sensory disruptions (e.g., blurred vision, ringing in the ears, bad taste in the mouth, loss of sensation in limbs), loss of motor function, sensitivity to light or sound, mood changes or mood swings, depression or anxiety, fatigue, drowsiness, and sleep disturbances.
- the level, concentration, or abundance of one or more inflammasome proteins is measured in a biological sample obtained from a patient (e.g., a patient suspected of having or suffering from a CNS injury).
- the levels, concentrations, or abundance of one or more inflammasome proteins is indicative of the severity of CNS injury in the patient.
- a CNS injury includes, but is not limited to, a traumatic brain injury, a stroke- related injury, a cerebral aneurism-related injury, a spinal cord injury (e.g., contusions, compressions, lacerations), concussion-related injury (including post-concussion syndrome), cerebral ischemia, injury resulting from neurodegenerative diseases (including Parkinson's disease, Dementia Pugilistica, Huntington's disease, Alzheimer's disease, Creutzfeldt-Jakob disease), seizure-related injuries, multiple sclerosis, amyotrophic lateral sclerosis, and other CNS traumas.
- the levels, concentrations, or abundance of one or more inflammasome proteins is indicative of the severity of traumatic brain injury or spinal cord injury in the patient.
- biological sample refers to any bodily fluid or tissue obtained from a patient or subject.
- a biological sample can include, but is not limited to, whole blood, red blood cells, plasma, serum, peripheral blood mononuclear cells (PBMCs), urine, saliva, tears, buccal swabs, CSF, CNS microdialysate, and nerve tissue.
- the biological sample is CSF, saliva, serum, plasma, or urine.
- the biological sample is CSF.
- the measured level, concentration, or abundance of one or more inflammasome proteins in the biological sample is used to prepare an inflammasome protein profile, wherein the profile is indicative of the severity of a CNS injury in the patient or the patient's prognosis or recovery potential from a CNS injury.
- the inflammasome protein profile may comprise the level, abundance, or concentration of one or more inflammasome proteins measured in the patient's sample optionally in relation to a pre-determined value or range of reference values as described herein.
- the inflammasome proteins in the profile include NLRP1 (NALP-1), ASC, and/or caspase-1 (e.g., p20 subunit of caspase-1).
- the inflammasome protein profile comprises the level, abundance, or concentration of each of NLRP1 (NALP-1), ASC, and caspase-1 (e.g., p20 subunit of caspase-1).
- the method of evaluating a patient suspected of having a CNS injury comprises determining the presence or absence of a protein signature associated with a CNS injury or a more severe CNS injury based on the measured level, abundance, or concentration of one or more inflammasome proteins in the patient sample or on the inflammasome protein profile prepared from the patient's sample.
- the protein signature comprises an elevated level of at least one inflammasome protein. The level of said at least one inflammasome protein in the protein signature may be enhanced relative to the level of the protein in a control sample or relative to a pre-determined reference value or range of reference values as further described herein.
- the protein signature may, in certain embodiments, comprise an elevated level for each of caspase-1 (e.g., p20 subunit of caspase-1), NLRP1, and ASC. Patients who exhibit the protein signature may be selected or identified as having a CNS injury or a more severe CNS injury.
- caspase-1 e.g., p20 subunit of caspase-1
- NLRP1 e.g., NLRP1
- ASC a severe CNS injury.
- the level or concentration of at least one inflammasome protein can be assessed at a single time point (e.g., after a potential CNS injury) and compared to a pre-determined reference value or range of reference values or can be assessed at multiple time points (e.g., two, three, four, five or more) after a potential CNS injury and compared to a pre-determined reference value or to previously assessed values.
- a biological sample for measuring levels or concentrations of inflammasome proteins can be obtained from a patient within one hour of a potential CNS injury to two weeks following a potential CNS injury. In some embodiments, the biological sample is obtained within one day, two days, three days, four days, five days, six days, seven days, ten days, or twelve days of a CNS injury or potential injury.
- pre-determined reference value refers to a pre-determined value of the level or concentration of an inflammasome protein ascertained from a known sample.
- the pre-determined reference value can reflect the level or concentration of an inflammasome protein in a sample obtained from a control subject (i.e., an uninjured, healthy subject).
- the control subject may, in some embodiments, be age-matched to the patients being evaluated.
- the measured level or concentration of at least one inflammasome protein is compared or determined relative to the level or concentration of said at least one inflammasome protein in a control sample (i.e. obtained from an uninjured subject).
- the pre-determined reference value or range of reference values can reflect the level or concentration of an inflammasome protein in a sample obtained from a patient with a known severity of CNS injury as assessed by clinical measures or post-mortem analysis.
- a pre- determined reference value can also be a known amount or concentration of an inflammasome protein. Such a known amount or concentration of an inflammasome protein may correlate with an average level or concentration of the inflammasome protein from a population of control subjects or a population of patients with known levels of injury.
- the pre-determined reference value can be a range of values, which, for instance, can represent a mean plus or minus a standard deviation or confidence interval.
- a range of reference values can also refer to individual reference values for a particular inflammasome protein across various levels of CNS injury severity.
- an increase in the level of one or more inflammasome proteins e.g., NLRP1 (NALP-1), ASC, or caspase-1) relative to a pre-determined reference value or range of reference values is indicative of a more severe central nervous system injury.
- the method of assessing the severity of a CNS injury further comprises measuring the level or concentration of one or more proteins described in U.S. Patent Publication No. 2011/0177974, which is hereby incorporated by reference in its entirety, in addition to measuring the level or concentration of one or more inflammasome proteins.
- the method further comprises measuring the level or concentration of one or more proteins selected from ubiquitin C-terminal hydrolase LI; vesicular membrane protein p-24; synuclein; micro tubule-associated protein; synaptophysin; Vimentin; Synaptotagmin; Synaptojanin- 2; Synapsin2; CRMPI, 2; Amphiphysin-1; PSD95; PSD-93; Calmodulin dependent protein kinase TT (CAMPK)-alpha, beta, gamma; Myelin basic protein (M BP); Myelin proteolipid protein (PLP); Myelin Oligodendrocyte specific protein (MOSP); Myelin Oligodendrocyte glycoprotein (MOG); myelin associated protein (MAG); NF-H; NF-L; NF-M; Blll-tubulin-l or combinations thereof in the biological sample obtained from the patient in addition to measuring the level or concentration of one or more inflammasome proteins.
- proteins selected from ubiquitin
- the protein signature may comprise an elevated level of one or more of these proteins in addition to the elevated level of one or more inflammasome proteins.
- the method further comprises measuring the level or concentration of one or more proteins selected from S-10013, neuron-specific enolase, neurofilament light chain, glial fibrillary acidic protein (GFAP) or combinations thereof in the biological sample obtained from the patient in addition to measuring the level or concentration of one or more inflammasome proteins.
- GFAP glial fibrillary acidic protein
- the protein signature associated with a CNS injury, or a more severe CNS injury comprises an elevated level of one or more proteins selected from S-lOOp, neuron-specific enolase, neurofilament light chain, glial fibrillary acidic protein (GF AP) in addition to an elevated level of one or more inflammasome proteins (e.g., NLRP1 (NALP-1), ASC, or caspase-1).
- proteins selected from S-lOOp, neuron-specific enolase, neurofilament light chain, glial fibrillary acidic protein (GF AP) in addition to an elevated level of one or more inflammasome proteins (e.g., NLRP1 (NALP-1), ASC, or caspase-1).
- GF AP glial fibrillary acidic protein
- the methods of assessing the severity of a CNS injury in a patient or evaluating a patient suspected of having a CNS injury further comprise administering a neuroprotective treatment to the patient based on the measured level of said at least one inflammasome protein or when a protein signature associated with a CNS injury, or a more severe CNS injury, is identified.
- neuroprotective treatments include drugs that reduce excitotoxicity, oxidative stress, and inflammation.
- suitable neuroprotective treatments include, but are not limited to, methylprednisolone, 17 ⁇ -estradioll, 17 ⁇ -estradiol, ginsenoside, progesterone, simvastatin, deprenyl, minocycline, resveratrol, and other glutamate receptor antagonists (e.g., NMDA receptor antagonists) and antioxidants.
- the methods of evaluating a patient further comprise measuring the level of at least one inflammasome protein in a biological sample obtained from the patient following neuroprotective treatment, preparing a treatment protein signature associated with a positive response to the neuroprotective treatment, wherein the treatment protein signature comprises a reduced level of at least one inflammasome protein, and identifying patients exhibiting the presence of the treatment protein signature as responding positively to the neuroprotective treatment.
- a reduction in the level, abundance, or concentration of one or more inflammasome proteins is indicative of the efficacy of the neuroprotective treatment in the patient.
- the one or more inflammasome proteins measured in the sample obtained following treatment may be the same as or different than the inflammasome proteins measured in the sample obtained prior to treatment.
- the inflammasome protein levels may also be used to adjust dosage or frequency of a neuroprotective treatment.
- the present invention also provides a method of determining a prognosis for a patient with a central nervous system injury.
- the method comprises providing a biological sample obtained from the patient within a week of injury 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 is indicative of the prognosis of the patient.
- the biological sample is obtained from the patient within one week, within five days, or within three days of injury.
- an increase in the level of one or more inflammasome proteins (e.g., NLRP1, ASC, caspase-1, or combinations thereof) relative to a pre-determined reference value or range of reference values is indicative of a poorer prognosis. For instance, an increase of about 20% to about 300% in the level of one or more inflammasome proteins relative to a pre-determined reference value or range of reference values is indicative of a poorer prognosis.
- increased levels of caspase-1, particularly the p20 subunit of active caspase-1, relative to a pre-determined reference value or range of reference values acutely after injury (i.e., within a week of injury) is indicative of a poorer prognosis.
- an elevated level of at least one inflammasome protein relative to a pre-determined reference value or range of reference values is predictive of the patient's recovery potential or long-term outcome as assessed by the Glasgow Outcome Scale (GOS).
- GOS Glasgow Outcome Scale
- the GOS is a scale that allows for the objective assessment of a patient's recovery following brain injury.
- the scale is comprised of scores ranging from 1 to 5 with the following descriptions:
- an elevated level of at least one inflammasome protein relative to a pre- determined reference value or range of reference values is predictive of the patient having a GOS score of 1 to 3 upon follow-up assessment (i.e., the patient having an unfavorable outcome, such as death or severe disability).
- a reduced level of at least one inflammasome protein relative to a pre-determined reference value or range of reference values is predictive of the patient having a GOS score of 4 or 5 upon follow-up assessment (i.e., the patient having a favorable outcome, such as moderate to low disability).
- the inventors have found that the CSF levels of one or more inflammasome proteins within three days following a CNS injury are useful for predicting the long-term outcome or recovery potential of the patient. Elevated inflammasome proteins levels correlate with unfavorable outcomes for the patient, whereas reduced or low inflammasome protein levels correlate with favorable outcomes for the patient (Example 3).
- the inflammasome proteins of the invention and other marker proteins can be measured in a biological sample by various methods known to those skilled in the art.
- proteins can be measured by methods including, but not limited to, liquid chromatography, gas chromatography, mass spectrometry, radioimmunoassays, immunofluorescent assays, FRET-based assays, immunoblot, ELTSAs, or liquid chromatography followed by mass spectrometry (e.g., MALDI MS).
- mass spectrometry e.g., MALDI MS.
- immune proteins or inflammasomes, or inflammasome component proteins which are present at certain levels in a tissue sample of the patient, can establish whether a patient having a CNS injury is diagnosed as having traumatic brain injury (TBI). These levels are cut-off values for the determination of TBI, the diagnosis of which can be predictive of prognosis of the patient, and can be used to initiate protective or therapeutic intervention, such as administration of one or more drug (e.g., small molecule) or biologic (e.g., large molecule peptide, protein, or antibody) useful to protect the patient from further brain injury or reduce the symptoms of TBI.
- drug e.g., small molecule
- biologic e.g., large molecule peptide, protein, or antibody
- biomarkers and cut-off values which are useful in accordance with the invention to determine traumatic brain injury (TBI), include: Caspase-1: > 0.8150 pg/ml; ASC: > 284 pg/ml; IL-18: > 156 pg/ml; (TNF)- ⁇ : > 2.202 pg/ml; IL-6: > 6.443 pg/ml; IL-4: > 0.03868 pg/ml; IL-10: > 0.6527 pg/ml; IL-8: > 29.18 pg/ml, and IL-2: ⁇ 0.5145 pg/ml. A value within twenty percent ( ⁇ 20%) of any of the given value is considered equivalent to the given value.
- biomarkers and cut-off values which are useful in accordance with the invention to determine traumatic brain injury (TBI)
- TBI traumatic brain injury
- biomarkers and cut-off values include: Caspase- 1: > 2.51 pg/ml; ASC: > 469.5 pg/ml; IL-18: ⁇ 256 pg/ml; and IL-1 ⁇ : ⁇ 0.57 pg/ml.
- a value within twenty percent ( ⁇ 20%) of any of the given value is considered equivalent to the given value.
- neuroprotective treatments are neutralizing antibodies against an inflammasome protein or binding fragments thereof, such as those described in U.S. Patent Publication No. 2009/0104200, which is hereby incorporated by reference in its entirety.
- the neuroprotective treatment is an anti-ASC antibody or fragment thereof.
- Anti-ASC antibodies include antibodies that specifically bind to amino acid residues 178-193 of rat ASC (accession number BAC43754), e.g., amino acid sequence ALRQTQPYLVTDLEQS (SEQ ID NO:1), or antibodies that specifically bind to the amino acid sequence RESQSYLVEDLERS (SEQ. ID NO:2) of human ASC.
- the neuroprotective treatment is an anti-NLRPl antibody or fragment thereof.
- Suitable neutralizing anti-NLRPl antibodies or fragments thereof include antibodies that specifically bind to the amino acid sequence CEYYTEIREREREKSEKGR (SEQ ID NO:3) of human NLRP1 or the amino acid sequence MEESQSKEESNTEG (SEQ ID NO: 4) of rat NLRP1.
- the neutralizing antibodies or antibody fragments may be polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single-chain variable fragments (scFvs) and the like. Aptamers that specifically bind to an inflammasome protein or epitope thereof (e.g., SEQ ID NOs: 1-4) may also be suitable neuroprotective treatments.
- Neutralizing antibodies against an inflammasome protein or binding fragments thereof can include any one of, or two or more in combination, of the following: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ I D NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NQ:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:19; SEQ ID NQ:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:11; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; S
- compositions comprising one or more of the listed antibodies of SEQ ID NOs 1-41, one or more other antibody known or discovered to bind the biomarker molecule, or one or more small molecule drug known or discovered to inactivate the biomarker molecule, can be used in a pharmaceutically effective amount as an active component of the pharmaceutical composition and formulated for administration to a human or animal patient with one or more pharmaceutically acceptable solvent, excipient, or carrier.
- kits for preparing an inflammasome protein profile associated with CNS injury such as spinal cord injury or traumatic brain injury.
- the kits may include a reagent for measuring at least one inflammasome protein and instructions for measuring said at least one inflammasome protein for assessing the severity of a central nervous system injury in a patient.
- a "reagent" refers to the components necessary for detecting or quantitating one or more proteins by any one of the methods described herein.
- kits for measuring one or more inflammasome proteins can include reagents for performing liquid or gas chromatography, mass spectrometry, immunoassays, immunoblots, or electrophoresis to detect one or more inflammasome proteins as described herein.
- the kit includes reagents for measuring one or more inflammasome proteins selected from NLRP1, ASC, caspase-1, or combinations thereof.
- the kit comprises a labeled-binding partner that specifically binds to one or more inflammasome proteins, wherein said one or more inflammasome proteins are selected from the group consisting of NLRP1, ASC, caspase-1, and combinations thereof.
- Suitable binding partners for specifically binding to inflammasome proteins include, but are not limited to, antibodies and fragments thereof, aptamers, peptides, and the like.
- the binding partners for detecting NLPR I are antibodies or fragments thereof, aptamers, or peptides that specifically bind to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 of human NLRP1 and rat NLRP1, respectively.
- the binding partners for detecting ASC arc antibodies or fragments thereof, aptamers, or peptides that specifically bind to the amino acid sequence of SEQ ID NO: 1 or SEQ. ID NO: 2 of rat ASC and human ASC, respectively.
- Labels that can be conjugated to the binding partner 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).
- 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.
- enzyme labels e.g., alkaline phosphata
- the kit can include reagents for measuring one or more inflammasome proteins in CSF samples.
- the kits can include reagents for measuring one or more inflammasome proteins in other patient samples including nerve tissue, CNS microdialysate, blood, saliva, serum, plasma, or urine.
- the kits further comprise a set of reference values to which the measured level of one or more inflammasome proteins can be compared.
- Biomarkers are specific proteins that are used as indicators of the status of different physiological processes in an individual. Biomarkers are often used to determine the stage or severity of an underlying disease or injury. TBI presents as a multi-factorial series of events that affect a variety of cells within the central nervous system (CNS) including neurons, microglia, astrocytes, oligodendrocytes and endothelial cells. Thus, a variety of biomarkers need to be identified in order to have a better understanding of the different molecular events that affect different cell types after TBI in the clinical setting. To this point, two biomarkers have been approved by the FDA for the monitoring of TBI patients.
- CNS central nervous system
- biomarkers are Ubiquitin carboxy-terminal hydrolase (UCH-L1) and glial fibrillary acidic protein (GFAP).
- UCH-L1 is expressed in neurons, and it is highly upregulated after TBI, whereas GFAP is similarly expressed in astrocytes.
- GFAP is similarly expressed in astrocytes.
- these two FDA approved biomarkers for TBI offer clinicians an insight as to the degree of neuronal degradation and astroglial activation after brain injury.
- no approved fluid biomarker or series of biomarkers is available to determine the inflammatory response in the acute setting after TBI.
- inflammasome proteins are potentially effective indicators of TBI severity and pathological outcomes in TBI.
- Levels of ASC and caspase-1 are elevated in the blood of TBI patients with increased ASC levels correlating with more severe injury and worse outcomes.
- ASC and IL-18 were elevated in the cerebral spinal fluid (CSF) of patients after TBI, and caspase-1 correlated with increased intracranial pressure and poor outcomes.
- CSF cerebral spinal fluid
- IL-8 and IL-10 have been described to be elevated in the CSF and serum of patients with TBI
- protein levels of IL-6 in plasma have been shown to correlate with brain injury severity.
- CSF samples from seven patients with SCI or control patients were analyzed for levels of nucleotide-binding leucine-rich repeat pyrin domain containing protein 1 (NLRP1; also known as NAcht leucine-rich-repeat protein 1 (NALP-1)), apoptosis-associated speck- like protein containing a caspase recruitment domain (ASC), and caspase-1.
- NLRP1 nucleotide-binding leucine-rich repeat pyrin domain containing protein 1
- NALP-1 NAcht leucine-rich-repeat protein 1
- ASC caspase recruitment domain
- CSF samples were prepared with Laemali buffer. Immunoblot analysis was carried out with the Criterion system (Bio-Rad) as described previously (de Rivero Vaccari et al., 2008) using antibodies (1:1000 dilution) to NLRP1 (Bethyl Laboratories), Caspase-1 (Imgenex) and ASC (Santa Cruz).
- Proteins were resolved in 14-20% TGX Criterion precasted gels (Bio-Rad), transferred to polyvinylidene difluoride (PVDF) transfer membranes (Tropifluor - Applied Biosystems) and placed in blocking buffer (PBS, 0.1 % Tween-20, 0.4% 1-Block (Applied Biosystems) and then incubated for one hour with primary antibodies. Membranes were then incubated for one hour with anti-mouse, anti-rat, or anti-rabbit horseradish peroxidase (HRP)-linked antibodies. Signal visualization was performed by enhanced chemiluminescence.
- PVDF polyvinylidene difluoride transfer membranes
- Contusional injuries were characterized by an intact pia and relative preservation of the anatomical relations of various elements of the spinal cord, and variable degrees of injury ranging from involvement of the entire cross-sectional area to large usually asymmetric areas of tissue damage.
- Massive compression injuries were characterized by disruption of the pia and severe distortion and disruption of spinal cord parenchyma.
- Laceration injuries which by definition were perforating or penetrating injuries caused by weapons or projectiles, were associated with breaching of the pia and linear tearing of the cord tissue.
- tissue samples from the center of injury and at various distances above and below the injury were obtained.
- the data from tissue from the center of the lesion were used to compare the inflammatory responses between cases whereas those from the remote, uninjured segments of the spinal cord served as within-case controls. Between-case comparison of the remote samples was not possible because, for different cases, the distance of these samples from the lesion center was variable.
- NLRP1 is expressed in neurons of the ventral horn (black arrows), myelinated axons (arrow heads) and oligodendrocytes (yellow arrows) in injured spinal cords (Figure 2). Moreover, NLRP1 immunoreactivity in areas of the penumbra (C7) was higher than in areas distant to the epicenter (L2).
- DAB immunoreactivity for caspase-1 was detected in swollen axons (spheroids, blue arrows) ( Figure 2), and arterioles (not shown). At areas of the penumbra, caspase-1 staining is present in motor neurons (black arrows) of the ventral horn, and in the white matter in oligodendrocytes (yellow arrows). Caspase-1 immunoreactivity in oligodendrocytes (yellow arrows) was the same at all levels of the spinal cords examined, regardless of proximity to the epicenter. At areas distant to the epicenter (T12), caspase-1 was also present in motor neurons (black arrows) but with decreased immunoreactivity than the penumbra (C7). This finding indicates that caspase-1 immunoreactivity in neurons decreases as the distance to the epicenter increases, similarly to NLRP1.
- ASC At areas of the penumbra (C7) and distant to the epicenter (L2), neurons in the ventral horn (black arrows) and white matter oligodendrocytes (yellow arrow) showed ASC immunoreactivity. In addition, ASC was also present in macrophages/microglia at the epicenter (blue arrow heads). Moreover, ASC immunoreactivity was also detected in the substantia gelatinosa (dorsal horn) at C7 and L2 (not shown).
- the innate immune system senses microbial and viral pathogen-associated molecular patterns and danger signals released from damaged or stressed cells to trigger conserved intracellular signaling pathways that drive proinflammatory responses that are critical for productive innate and adaptive immunity. Excessive inflammatory responses become deleterious leading to tissue destruction.
- the results of this experiment provide evidence demonstrating that the NLRP1 inflammasome signaling system is activated in the innate immune response in damaged human spinal cord and brain tissue after trauma.
- inflammasome proteins may serve as biomarkers for other types of central nervous system injury.
- TBI traumatic brain injury
- GCS Glasgow Outcome Scale
- inflammasome proteins e.g., ASC, NALP-1, and caspase- 1
- inflammasome proteins are acutely elevated (e.g., within 72 hours) in the CSF of patients with TBI as compared with nontrauma controls. Elevation of these proteins likely reflects the extent of neuroinflammation, suggesting that inflammasome proteins can serve as acute biomarkers of CNS injury.
- CSF biomarkers are more specific indicators of neuropathology than serum biomarkers. Cerebrospinal fluid directly bathes the brain, closely reflecting the extracellular milieu and biochemical changes that are specific to the CNS.
- ASC active caspase-1
- NALP-1 NALP-1 in the CSF.
- therapeutic hypothermia attenuates the endogenous inflammatory response of the CNS to TBI by decreasing cytokine production and reducing activation of astrocytes and microglia (Aibiki, et al., 1999; Goss, et al., 1995; Kumar, et al., 1997; Truettner, et al., 2005), and cortical neurons exposed to moderate hypothermia in culture show a decrease in activation of the inflammasome (Tamura et al., in press).
- ASC, active caspase-1, and NALP-1 can serve as objective, biochemical indicators of treatment efficacy for patients with CNS injury.
- Example 4 Hypothermia Decreases Caspase-1 Activation after Traumatic Brain Injury in Pediatric Patients
- inflammasome proteins such as caspase-1
- CSF caspase-1 levels obtained from pediatric patients who received hypothermia treatment following TBT were compared to those obtained from pediatric patients who did not receive treatment following TBI.
- Cerebrospinal fluid of pediatric patients (ages 0.1 to 16 years) was obtained at different times after traumatic brain injury (day 1, 2 and 3). Patients were divided into those that received hypothermia treatment and those who did not (normothermia). As shown in Figure 6, the data indicate that within 24 hours after injury the levels of caspase-1 were lower in the hypothermia group when compared to the normothermia group.
- Exclusion criteria in this study consisted of patients with normal findings on the CT scan on admission, patients with a major extracranial trauma (defined as extracranial Injury Severity Score > 18 points), and patients with past medical history relevant to CNS pathology such as brain tumor, meningitis, cerebral vasculitis or stroke.
- Serum levels of the inflammatory cytokines (TNF)- ⁇ , IL-2, IL-4, IL-6, IL-8, IL-10, IL-13 and interferon (IFN)- ⁇ was measured utilizing the V-PLEX Proinflammatory Panel 1 (MSD) as in Scott, X.O., Stephens, M.E., Desir, M.C., Dietrich, W.D., Keane, R.W. and de Rivero Vaccari, J.P. (2020).
- Receiver operating characteristics were calculated to obtain the area under the curve (AUC) in order to obtain cut-off points and the respective specificity, sensitivity and likelihood ratio.
- the cut-off point for each analyte was chosen based on the highest likelihood ratio in the sensitivity vs. 1-specificity plot favoring a higher sensitivity than specificity values, to obtain assays with higher likelihood of reliability for each analyte.
- Positive and negative predictive values were also calculated along with overall assay accuracy.
- the p-value was determined using the following formula using Microsoft Excel (version 16.57): A Pearson correlation was carried out to obtain r in order to calculate the z-score to allow for comparison of ROC curves between analytes obtained from the same samples.
- TBI patients had significantly elevated levels of inflammasome signaling proteins caspase-1 (FIG. 7A), ASC (FIG. 7B) and IL-18 (FIG. 7C), as well as significantly elevated levels of cytokines (TNF)- ⁇ (FIG. 7D), IL-6 (FIG. 7E), IL-4 (FIG. 7F), IL-10 (FIG. 7G) and IL-8 (FIG. 7H).
- TBI inflammasome signaling proteins caspase-1
- ASC FIG. 7B
- IL-18 FIG. 7C
- TBI patients had significantly elevated levels of inflammasome signaling proteins caspase-1 (FIG. 7A), ASC (FIG. 7B) and IL-18 (FIG. 7C), as well as significantly elevated levels of cytokines (TNF)- ⁇ (FIG. 7D), IL-6 (FIG. 7E), IL-4 (FIG. 7F), IL-10 (FIG. 7G) and IL-8 (FIG. 7
- inflammasome proteins are potentially promising biomarkers for determining TBI pathological outcomes.
- inflammasome proteins caspase-1 (FIG. 8A), ASC (FIG. 8B) and IL-18 (FIG. 8C), and the inflammatory cytokines (TNF)- ⁇ (FIG. 8D), IL-6 (FIG. 8E), IL-4 (FIG. 8F), IL-10 (FIG. 8G), IL-8 (FIG. 8H) and IL-2 (FIG. 81) in the context of TBI, we plotted the ROC curve for each protein (Fig 8).
- IL-6 (Fig 8E) had the highest AUC 1.0 (Table 3) with a sensitivity of 100% and a specificity of 100% (Table 5).
- TNF- ⁇ had an AUC of 0.98 AUC (96% sensitivity, 95% specificity)
- IL-10 and IL-8 also presented high AUC values (0.97 and 0.95, respectively)
- IL-4 had an AUC of 0.79 (74% sensitivity, 75% specificity)
- IL-2 an AUC of 0.74 (95% sensitivity, 56% specificity).
- caspase-1 (FIG. 2A) had the highest AUC at 1.0 (Table 4) with a sensitivity of 100% and a specificity of 100%, followed by ASC with an AUC of 0.97 with a sensitivity of 92% and a specificity of 93% (Table 3).
- IL-18 presented an AUC of 0.81 (sensitivity of 83%, specificity of 74%).
- caspase-1 and IL-6 as useful inflammatory biomarkers superiorto all other biomarkers examined in this study; however, caspase-1 and IL-6 were not different from each other (FIG. 9B) .
- ASC and (TNF)- ⁇ were not different from each other but ASC was superior to IL-18 and IL-4.
- IL-10 and IL-8 were not different from each other, and IL-8 was superior to IL-2.
- the best model was chosen based on the AIC (14.37) by the stepwise method, and then the estimate (coefficients), standard error and p-values for each predictor and intercept (slope), as well as the BIC (35.52), residuals (FIG. 6A), RMSE (1.65), mean of residuals (-6.25E-17), confidence intervals, and the DW autocorrelation test for the best fit model were calculated.
- An adjusted R 2 value (0.78) was obtained for the model to determine the approximate contribution of the fitted model to the GCS (Table 6).
- the sensitivity and specificity of caspase-1 were 55 and 57%, respectively, with a PPV of 68% and a NPV of 44% with an accuracy of 56%.
- the sensitivity and specificity were 64 and 100%, respectively, with a PPV of 100% and a NPV of 61% with an accuracy of 77%.
- TBI is a significant medical condition which represents a major source of potential disability and economic strain.
- the effects of TBI are chronic with increased inflammatory activity and long- term learning and memory loss.
- non-invasive diagnostic tools are needed.
- the serum of TBI patients is an ideal source to measure the diagnostic and prognostic potential of signaling proteins that can provide clinicians with the biochemical status of patients as it pertains to neuronal damage, astrocyte activation, and the inflammatory response involving microglial neutrophils and other inflammatory cells.
- UCH- L1 has been approved by the FDA as a biomarker of neuronal damage after TBI, whereas GFAP has been approved as a biomarker of reactive astrogliosis.
- GFAP has been approved as a biomarker of reactive astrogliosis.
- no inflammatory marker in serum has been approved for the care of patients with TBI.
- IL-2 protein levels of the inflammatory cytokines (TNF)- ⁇ , IL-4, IL-6, IL8 and IL-10 were significantly elevated in the serum of TBI patients compared to controls, indicating a balance between pro-inflammatory ((TNF)- ⁇ , IL-6) and anti- inflammatory (IL-4, IL-10) cytokines at play acutely after TBI.
- IL-2 levels were elevated in the serum of healthy controls when compared to TBI patients, suggesting a loss of this cytokine acutely after injury and the potential loss of a mechanism responsible for keeping the pro- inflammatory environment at check.
- IL-2 is a growth factor that is secreted by activated T cells and plays a role in the generation of immunity through the proliferation and differentiation of B cells and T cells, and the amplification of NK cell activity.
- Our findings are consistent with the pro-inflammatory role that the absence of IL-2 results in and is similar to what is seen in like CNS injury, such as Traumatic Brain Injury (TBI).
- TBI Traumatic Brain Injury
- serum levels of IL-2 have been shown to be decreased in patients after ischemic stroke 32 .
- IL-2 or IL-2R elimination has been shown to result in systemic autoimmunity.
- caspase-1 and IL-6 with an AUC of 1.0 were the best inflammatory biomarkers of those examined in this study, followed by (TNF)- ⁇ and ASC with an AUC of 0.98 and 0.97, respectively, and IL-8 and IL-10 with an AUC of 0.97 and 0.96, respectively.
- the most sensitive inflammatory biomarkers were caspase-1 and IL-6, followed by (TNF)- ⁇ , IL-2, ASC and IL-8, whereas the most specific biomarkers were caspase-1, IL-6, IL-8.
- IL-10, (TNF)- ⁇ ASC the most sensitive inflammatory biomarkers
- caspase-1 and IL-6 presented an accuracy of 100%, followed by (TNF)- ⁇ with 96%, IL-8, 94% and ASC and IL-2 with an accuracy of 92%.
- caspase-1, ASC, (TNF)- ⁇ , IL- 6, IL-10 and IL-8 are the most reliable diagnostic inflammatory biomarkers of TBI, among those studied.
- caspase-1 and IL-10 as prognostic biomarkers further implicates the role that inflammation plays on patient outcomes.
- serum levels of (TNF)- ⁇ remain elevated for at least one-year post-injury.
- Serum levels of IL-6 have been shown to be more elevated in more severe cases of TBI, and that these elevated IL- 6 levels were associated with worsened outcomes.
- IL-8 is similar in that studies have also shown that increased IL-8 expression in serum or CSF of TBI patients is associated with increased chance for mortality and overall worsened pathological outcomes. This is thought to be due to its chemoattractant properties in which it is able to recruit and activate monocytes to the site of injury, increasing the overall inflammatory response after TBI or stroke.
- IL-4 however seems more ambiguous, with one study showing that administration of IL-4 after injury may offer some form of protection from the effects of TBI.
- IL-13 has been shown to have shared functionality with IL-4, and has also been shown to work with IL-2 to promote IFN production. Since there was a statistically significant difference for this analyte, we then determined whether IL-13 were a good biomarker of TBI severity. Our findings indicate that with an AUC of 0.75 and a cut-off point of 3.12 pg/mL, with a sensitivity of 71% and a of specificity 79%, IL-13 is a fair biomarker of TBI injury severity.
- a multivariate linear regression model consisting of IL13, IL-2 and IL-12 indicated that combined, these 3 biomarkers contribute to the GCS with an adjusted R 2 of 0.78; thus highlighting the importance of IL-13 and a key biomarker of injury severity.
- inflammasome proteins are reliable biomarkers of the inflammatory response in several conditions such as stroke, Alzheimer's disease, multiple sclerosis, age-related macular degeneration, psoriasis and non-alcoholic steatohepatitis, indicating that the inflammasome plays a major role in the pathophysiology of a variety of diseases affecting the CNS and the periphery. Moreover, those findings highlight the usefulness of inflammasome signaling proteins as biomarkers of injury and disease.
- inflammatory biomarker identification includes 1) measuring the levels of inflammatory problems in the serum of affected and unaffected individuals to determine if there are statistical differences between groups. 2) Determining the diagnostic biomarker characteristics (AUC, sensitivity, specificity, likelihood ratio, accuracy, PPV and NPV) of each inflammatory protein or analyte that was statistically significant when comparing the levels between affected and unaffected individuals. 3) Comparing the ROC among the different biomarkers to identify potential biomarker differences between groups.
- inflammatory biomarkers when combined with GFAP and UCH-L1, they offer the potential for clinicians to better understand not only the overall scope of injury but also a probable prognosis and potential for disability considering a variety of mechanisms contributing to the TBI pathology including neuronal damage ( UCH-L1), reactive astrogliosis (GFAP) and inflammation (caspase-1, ASC, (TNF)- ⁇ and IL-6).
- UCH-L1 neuronal damage
- GFAP reactive astrogliosis
- inflammation caspase-1, ASC, (TNF)- ⁇ and IL-6.
- TBI IN PEDIATRIC PATIENTS TBI IN PEDIATRIC PATIENTS
- the UF Institutional Review Board 01 (IRB201600237) approved this study with a modification of informed consent, allowing for participants or their authorized representatives to provide written informed consent within 72 hours of admission.
- Healthy controls (HC) were recruited in the Emergency Department. These HC were ineligible for enrollment if they had a history of TBI or were admitted with a traumatic injury or acute neurological process. Timed blood samples were collected for genetic and proteomic analysis from all participants at enrollment, 24, and 48 hours post injury.
- TBI-Common Data Elements TBI-Common Data Elements (TBI-CDE) Biospecimens and Biomarkers Working Group Consensus guidelines for plasma preparation were followed. 2 Blood sampling took place by venipuncture if required for routine clinical care, otherwise blood was collected via a catheter that was already in place as part of clinical care. A total of 2 cc/kg, up to max 5 ml (age 0-4 years); 10 ml (age 5-18 years). Blood samples were drawn into red top SST BD Vacutainer® Plus tubes. The analysis of this archive samples transferred from the University of Florida to the University of Miami was approved under the auspices of the University of Miami IRB (20210352: pTBIB).
- the GOS-E Peds was used as the primary tool to quantify long term outcomes following TBI.
- the GOS-E Peds is a validated clinical scale used to quantify rehabilitative success and identify permanent disability in a patient recovering from TBI. Measurements are designed to screen for onset of new cognitive or behavioral changes, including sleep disturbances, social disruptions, and learning impairments.
- the GOS-E Peds was conducted at subsequent pediatric neurology follow up visits or by phone if the patient was not scheduled for a visit or was lost to follow up at TBI clinic. All surveys were administered by a trained research associate and completed directly by the patient's caregiver.
- Receiver operating characteristics were calculated to obtain the area under the curve (AUC) in order to obtain cut-off points and the respective specificity, sensitivity and likelihood ratio.
- the cut-off point for each analyte was chosen based on the highest likelihood ratio in the sensitivity vs. 1-specificity plot , favoring a higher sensitivity than specificity values, to obtain assays with higher likelihood of reliability for each analyte.
- Positive, and negative predictive values were also calculated along with overall assay accuracy.
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Abstract
Un réseau de cytokines inflammatoires est utile en tant que biomarqueur utile pour la détection, le diagnostic et le traitement d'une lésion cérébrale traumatique. Des valeurs de coupure de cytokine inflammatoire ou de points de coupure, ROC, et d'autres caractéristiques ont été déterminées.
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