BRAIN DAMAGE MARKER

The invention relates to a brain damage diagnostic method, carried out in vitro in samples from patients suspected of suffering from such damage. The method uses the detection of the chemokine CCL23 that allows deducting further prognostic information. The invention also relates to uses of means for the detection of this chemokine with the purpose of detecting the presence of brain damage caused by stroke, brain trauma, brain tumor, Alzheimer disease.

EXAMPLES

The following examples serve to illustrate the in vitro diagnostic method according to the invention.

All the tests were carried out with patients who arrived at the emergency department of the Vall D'Hebron University Hospital in Barcelona (Spain). The patients came to the Hospital with acute focal neurologic symptoms initiated in the previous 24 hours. Healthy individuals were used as controls (C). All the studies reported in the Examples were approved by the Ethics Committee of the Vall D'Hebron University Hospital in Barcelona, and all the subjects or their relatives gave their consent to participate in the study.

CCL23 Levels in Patients with Stroke (Cerebral Ictus) and Other Neurological Diseases

For this test, patients with stroke and patients afflicted with other neurological diseases who came to the Vall D'Hebron University Hospital Emergency Department in Barcelona (Spain) were studied. The patients came to the Hospital with acute neurological symptoms initiated during the 24 hours before their admission. Healthy individuals were used as controls (C).

Among the patients studied those whose symptoms or conditions were similar to those of the stroke, but without being so, were also included. Among the patients with neurological diseases which can be confused with the stroke patients with epilepsy, brain tumors, headache, hypoglycemia, syncope, damage to peripheral nerves and patients who had used toxic compounds (medicinal drugs, drugs of abuse, accidental poisoning, etc.) were identified.

Following the diagnostic protocols for this kind of cases a venous blood sample, which was introduced in tubes without anticoagulant, was taken from each patient at the time of the admission (baseline; B) within the first 24 hours from the beginning of the symptoms and before starting any treatment. The serum of the patients was separated by centrifugation at 3500 revolutions per minute (rpm) for 15 minutes at 4° C. and kept at −80° C. until analysis.

The levels of chemocine CCL23 were determined following the immunoassay called Custom Human Search Light Multiplex Immunoassay (Aushon Biosystems, Billerica, Mass.). This immunoassay is of ELISA type and is based on the detection of chemiluminescence of analytes the capture antibodies of which are arranged in arrays or 96 wells plates. The enzyme-substrate reaction produced a luminescent signal that was detected with a cooled CCD camera. The resulting image was analyzed with the software Array Analyst (Aushon Biosystems, Billerica, Mass.).

For the statistical analysis the package SPSS version 15.0 was employed. To determine the difference among independent samples the Mann-Whitney U or Kruskal Wallis tests were used.

As it can be seen fromFIG. 1(A) andFIG. 1(B), the control subjects (C) had CCL23 blood levels lower than those of the patients (n=146) (p<0.001) with the ischemic stroke (Ict), and lower than those of the patients with other diseases with symptoms of stroke (n=58), such as epilepsy (E) (n=15), tumors (T) (n=13), headache or migraine (M) (n=9), hypoglycemia (HypGlc) (n=3), syncope (Syn) (n=5), peripheral nerve injury (PNI) (n=10); and consumption of toxic chemicals (Tox) (n=3) (p<0.001).

InFIG. 1(A) the gray bars show the results of the measurement of the CCL23 levels in different patients who arrived at the Emergency Room of the hospital with different ailments, all of them related with a possible brain damage. The black line in each bar represents the average value of all the measurements and the black lines in each bar, the standard deviation of the mean. The control (C), healthy individuals, did not have elevated the CCL23 levels in blood.

Similarly, inFIG. 1(B), the bars represent the results of the measurement of the CCL23 levels, with an average value (horizontal black line) and the standard deviation (vertical lines). On this occasion, the CCL23 levels in patients who have suffered a stroke (Ict) and patients with symptoms similar to those of the stroke (mimics) are compared with respect to healthy individuals (C). Just as inFIG. 1(A), the levels both in stroke and mimics are higher than in the controls.

Replica. CCL23 Levels in Patients with Brain Damage of Various Etiologies

This example is a replica and served to validate the results of Example 1. For this, other commercial techniques, new groups of patients with stroke and new patients with also neurological diseases were used to determine the CCL23 blood levels.

Patients with acute ischemic stroke that were admitted within the first 3 hours after the onset of the symptoms and who received the tissue plasminogen activator (t-PA) in a standard dose of 0.9 mg/Kg (bolus at 10%, or continuous infusion at 90% for 1 hour) were assessed. A blood sample was extracted from each patient at the moment of the admission (baseline time) and before the beginning of any treatment.

Patients with symptoms or conditions similar to stroke (cerebral ictus), but without being it, and other acute neurological diseases were also included. All these patients were admitted within the first 24 hours after the beginning of the symptoms. Among the patients with other acute neurological diseases, patients with traumatic brain damage (TBI), epilepsy, hypoglycemia, migraine (cephalea), somatizations and brain tumors were included.

Following the internalization protocols for such cases, a sample of venous blood was taken from each patient at the moment of the admission (baseline; B) and within the first 24 hours from the beginning of the symptoms, before starting any treatment. The blood was collected in tubes without anticoagulant. The serum of the patients was separated by centrifugation at 3500 rpm for 15 minutes at 4° C. and keep at −80° C. until analysis.

The chemocine CCL23 levels were determined following the ELISA-type commercial immunoassay (RayBiotech, Inc.; Norcross Ga.). The tests were carried out following the instructions of the manufacturer and each sample was analyzed in duplicate, determining the mean of the two values. The inter-tests and intra-tests coefficients of variation (CV) were lower than 20%. The values are given in picograms per milliliter (pg/ml).

This ELISA-type immunoassay uses 96 wells plates coated with antibodies specific for human CCL23 chemocine. The standards and the samples were pipetted in the wells to determine the presence and amount of CCL23 in a sample through the immobilization by the antibody. The wells were washed and biotinylated anti-human CCL23 antibody was added. Once the unbound biotinylated antibody is removed, streptavidin conjugated to radish peroxidase (HPR-streptavidin) was pipetted. The wells were washed again and the substrate 3,3′,5,5′-Tetramethylbenzidine (TMB) was added until the coloration proportional to the amount of bound CCL23 developed. The TMB “stop solution” changed the color from blue to yellow and the color intensity was measured at 450 nm as indicated by the manufacturer's instructions.

The same as for Example 1, the SPSS package version 15.0 was used for the statistical analysis. To determine the difference among independent samples the Mann-Whitney U or Kruskal Wallis tests were used.

As it can be seen fromFIG. 2, the highest CCL23 levels were found in patients with traumatic brain damage, epilepsy and brain tumors.

This test allows to state that chemocine CCL23 is highly elevated in cases of brain damage from serious cause. With this, this molecule can be used as a distinctive marker for severe damages, such as a headache by possible brain tumor, for minor damages, such as a headache by tension cephalea. This is very advantageous because erroneous diagnoses are avoided and the evaluation of those subjects that might suffer a serious medical condition continues.

Likewise, for the cases of traumatic brain damage we can assess the severity of the trauma and thus be able to determine in a young patient (infants) or in a patient that cannot speak at the time of the exploration and admission to a hospital if the bump received on the head is serious or not. The determination of the severity of a bump that shows no external symptoms (bleeding, tissue damage, etc.) is of special importance because it allows discarding mild cases and even avoiding the unnecessary exposure to radiation (X-rays), or reducing the number of scanners (computerized tomography scans, magnetic resonance imaging, etc.) that are carried out in a hospital and which represent a high cost for the centre and the public health.

Time Profiles and Kinetic of the CCL23 Levels in Cases of Stroke (Cerebral Ictus)

Similarly than for Examples 1 and 2, in this case patients with cerebral ischemic stroke and with intracerebral hemorrhage were evaluated.

A sample of venous blood was extracted at the time of the admission (baseline) and before any treatment (in an average of 1 to 2 hours) from patients with acute ischemic stroke (Ict), who were admitted within the first three hours after the beginning of the symptoms and who received t-PA in a standard dose of 0.9 mg/Kg (bolus at 10%, or continuous infusion at 90% for 1 hour). The follow-up of these blood samples allowed to determine a profile at different times: baseline (B), 2 to 3.5 hours (Hr) after the beginning of the symptoms (already having initiated the treatment), and 12 hours (Hr) and 24 hours (Hr) after the beginning of the symptoms.

In another sub-group of patients a sample of venous blood was extracted at the time of the admission (baseline) and before any treatment (in an average of 1 to 2 hours). With the follow-up of these blood samples, a profile was determined at different times: baseline (B), 2 to 3.5 hours (Hr) after the beginning of the symptoms (already having initiated the treatment), at 12 hours (Hr), at the discharge (between 5 and 7 days) and at three months.

The serum was separated by centrifugation at 3500 rpm for 15 minutes at 4° C. and kept at −80° C. until analysis.

InFIG. 3(A) the results of the patients with ischemic stroke in the hyperacute stage of the stroke can be observed. From 24 hours onwards a clear increase in the levels of chemocine CCL23 was observed. According toFIG. 3(B), that illustrates the data in the hyperacute stage and in the sub-acute stage of the stroke, the patients had low levels at the time of the admission (baseline; B) in comparison with the levels at 12 and 24 hours. At 5-7 days and 3 months after the beginning of the symptoms the CCL23 blood values had reverted to the baseline values (B).

In relation with other clinical variables, there are no differences between risk factors and the CCL23 levels at the baseline time (arrival of the patient to the emergency room). Lower CCL23 levels, between 2-3.5 hours, were observed in patients who improved at 48 hours after admittance. This aspect allows giving the CCL23 levels a certain prognosis character.

These data allow determining at what point of the stroke the patient is, and this will allow the practitioner to start with safety or not the treatment with thrombolytic agents (t-PA). At the same time, chemocine CCL23 is a good marker for studying the evolution of a particular patient.

All the patients from this test were patients with spontaneous ICH (mostly due to rupture of a cerebral artery by arterial hypertension or cerebral amyloid angiopathy), i.e. those patients with secondary ICH and related with vascular malformation, altered clotting or the consumption of blood-thinning agents, traumatic brain damage, hemorrhagic infarction and tumor bleeding were excluded from the study. Patients with tumor bleeding and those that had been subjected to a surgical process were also excluded.

Once the blood samples were taken as in Examples 1 and 2, the serum was separated by centrifugation at 3500 rpm for 15 minutes at 4° C. and keep at −80° C. until analysis.

Also as in the other Examples 1 and 2, the controls (C) were healthy subjects.

Following the protocol of Example 2, the levels of chemocine CCL23 were determined with the ELISA-type commercial immunoassay (RayBiotech, Inc; Norcross Ga.).

The statistical analysis was conducted with the SPSS package, version 15.0. To determine the difference among related samples (e.g.: consecutive time points) the Friedman and Wilcoxon tests were used.

The analyzed samples were those taken at baseline (B), at 24 hours from admittance and at 48 hours from admittance (hyperacute stage of stroke).

InFIG. 4, showing the results obtained with ICH patients in hyperacute stage, it can be seen that the highest levels of CCL23 occur between 24 and 48 hours with respect to the baseline (B). The data inFIG. 4showed along the bars correspond to the values of the samples from hyperacute phase of all the analysed patients. The horizontal black line is the mean of the samples. These results are also interesting for determining at what point the patient is when entering the emergency room and if he/she may be subjected to a type of treatment or to another.

Comparative Test with Detection of CCL-2 (MCP-1)

The inventors also assessed the levels of another chemocine, the chemocine ligand 2 (with C-C motif) (CCL-2), also known as chemotactic protein-1 (MCP-1) in patients with ischemic stroke (Ict; n=9) and in patients with other diseases classified as stroke mimetics, also called Mimics (Mim; n=2) and they compared them with the levels of healthy controls (C; n=4). For this, they used the SearchLight Array technique as it has been defined in Example 1 with an antibody specific of this chemocine.

As it can be seen fromFIG. 5, there are no significant differences in the levels of chemocine CCL-2 among healthy controls, mimics and stroke.

At the same time, the trend or evolution over time of the CCL-2 levels was also tested in patients with ischemic stroke (ICT; n=17), regarding healthy subjects as controls (C; n=10), and that the time profile in stroke does not make any kind of peak and stays stable over time unlike CCL23. The CCL-2 analysis was carried out with a commercial array for the detection of chemocines (Chemocines Array).

The results can be viewed inFIG. 6, where the dashed lines show the reference levels of the controls; and the different bars show the levels of the patients at the time of admission (baseline, B) and at 1, 3, 7 days and 3 months. There are no differences in the CCL-2 (MCP-1) profile in the acute (baseline at 3 days) or sub-acute (up to 3 months) stage of the stroke.

The data reflected for CCL-2 in this example allow affirming that the determination of CCL23 according to the invention is really advantageous because, on the one hand with CCL23 there are significant differences between patients and healthy controls (see Examples 1 and 2). On the other hand, the CCL23 time profile does have variations (contrary to the stability of the CCL-2 levels) over time (see Example 3). These variations of the CCL23 levels not observed for CCL-2 allow knowing at what point the stroke started and at the same time its severity, two information values of great interest in this type of disorder, as indicated above.

Replication Test in Patients with Traumatic Brain Injury or Damage (TBI)

Considering the importance of this kind of brain damage cause, two (n=2) new cases of TBI admitted at the Hospital were studied.

Thus, following the internalization protocols for such cases, and as indicated in Example 2, a sample of venous blood was taken from each patient at the moment of the admission (baseline; B) and within the first 24 hours from the beginning of the symptoms, before starting any treatment. The blood was collected in tubes without anticoagulant. The serum of the patients was separated by centrifugation at 3500 rpm for 15 minutes at 4° C. and keep at −80° C. until analysis.

The chemocine CCL23 levels were determined following the ELISA-type commercial immunoassay (RayBiotech, Inc.; Norcross Ga.). The tests were carried out following the instructions of the manufacturer and each sample was analyzed in duplicate, determining the mean of the two values. The inter-tests and intra-tests coefficients of variation (CV) were lower than 20%. The values are given in picograms per milliliter (pg/ml).

The mean of the samples was calculated and as can be deduced fromFIG. 7(black horizontal line in the bars), the subjects with TBI had high levels of CCL-23 (at 24 h from the beginning of the symptoms, before starting any treatment) with respect of the controls (C), which were healthy subjects. Thus, this assay serves for corroborating the previous data depicted in Example 2.

CCL23 as Diagnostic Marker of Brain Damage in Alzheimer Disease

The inventors have also detected high levels of the chemocine CCL23 in patients suffering from Alzheimer Disease (AD).

From 36 patients diagnosed of suffering Alzheimer following the diagnostic protocols, also a venous blood sample, which was introduced in tubes without anticoagulant, was taken from each patient. The serum of the patients was separated by centrifugation at 3500 revolutions per minute (rpm) for 15 minutes at 4° C. and kept at −80° C. until analysis.

FIG. 8shows the levels of CCL23 detected in blood (mean of the 36 samples) of patients with AD in respect of healthy subjects or controls (C, n=17).FIG. 8clearly shows that in AD patients CCL23 levels were higher than those of the controls. The data depicted in thisFIG. 8correspond to the values detected in samples and corrected considering the age factor, since it has been detected that the levels of CCL23 increase with age also in healthy subjects, although they do not reach the AD levels.

These represent noteworthy results, because the levels of CCL23 in AD patients are meaningful high, so that they represent a clear marker for the disease.

In addition and also of special interest is the fact that among AD patients, the greater the levels of CCL23 in blood, the greater the cognitive impairment of the subject. The cognitive impairment is usually determined using the parameter known as minimental (MMSE)

“The mini-mental state examination (MMSE) is a brief 30-point questionnaire test that is used to screen for cognitive impairment. It is commonly used in medicine to screen for dementia. It is also used to estimate the severity of cognitive impairment and to follow the course of cognitive changes in an individual over time, thus making it an effective way to document an individual's response to treatment. The standard MMSE form which is currently published by Psychological Assessment Resources is based on its original 1975 conceptualization, with minor subsequent modifications by the authors.

Any score greater than or equal to 25 points (out of 30) indicates a normal cognition. Below this, scores can indicate severe (9 points), moderate (10-20 points) or mild (21-24 points) cognitive impairment.

These data of CCL23 levels in relation to the MMSE parameter are depicted inFIG. 9, wherein it can be seen that at lower MMSE (which means greater cognitive impairment) a higher amount of CCL23 (pg/ml) in blood is detected. The evolution of AD leads to an increase in neuronal damage associated with neurodegenerative processes, as well as brain atrophy, among other manifestations of brain damage.

Therefore, taken altogether these data allow concluding that determining CCL23 levels not only serves for diagnosing that the patient is suffering from AD, but also in which stage of the disease is, or what the extension of the damage is. This makes easy, in turn, the prescription of a more accurate or adequate medical regimen, and also the determination of the most likely prognosis of the disease.

CCL23 as Distinctive Marker of Tumors Vs. Chronic Migraines

In order to have an in-depth analysis of the levels of CCL23 in patients with migraine, the inventors also performed an analysis of the levels of this chemocine along time with these type of subjects.

Thus, patient finally diagnosed of suffering from migraine were followed (n=19) along time. With this aim blood samples processed as in Example 1 were extracted at basal time (admittance at Hospital), at 24 hours from the initial of the onset of the headache, and at 72 hours from the onset. The levels of the CCL23 increased during the migraine attack to finally decrease at 72 hours (data not shown). These data indicate that, using the levels of CCL23, migraines or major headaches can be differentiated from other minor headaches (tensional headache), in which the levels of the chemocine were not increased in respect of the controls.

Anyway, and more interestingly, is the fact that the blood levels of CCL23 in chronic phases of the migraines are lower than the levels observed in patients with brain tumors.

These data derive from a comparative assay between patients finally diagnosed of suffering from brain tumors (n=11) and from patients with migraine of more than one week (n=19). The results of this additional test between these two types of subjects are depicted inFIG. 10. As can be seen inFIG. 10, the mean (horizontal black line in the bars) of the tested samples of subjects suffering from migraine showed lower levels of CCL23 than the mean of the tested samples of subjects finally diagnosed of having a brain tumor causing headache.

These results altogether allow concluding that, although in an acute phase of a migraine the levels of CCL23 can resemble those of a brain tumor patient, the levels will be lowered in case of a chronic migraine (headache of more than one week). Therefore, the pathology that could be initially classified as tumor, can be easily classified as a simple migraine only determining along time the levels of CCL23. This is important because tedious and/or expensive further diagnostic methods (biopsies, scanners, etc.) can be avoided.

CCL23 Levels in Different Stroke Subtypes

The Oxford Community Stroke Project classification (OCSP) relies primarily on the initial stroke symptoms. Based on the extent of the symptoms, the stroke episode is classified as total anterior circulation infarct (TACI), partial anterior circulation infarct (PACI), lacunar infarct (LACI) or posterior circulation infarct (POCI). These four entities predict the extent of the stroke. OCSP clinical syndrome usually correlates well with the site and size of ischemic tissue lesion and the likely arterial lesion, also the OCSP clinical syndrome diagnosed in the hyperacute phase of stroke correlates with clinical outcome, and, when applied within 48 hours of stroke, with the underlying computed tomographic (CT) lesion.

The levels or mean of the amount of CCL23 (pg/ml) were measured in blood samples obtained within 24 h of stroke onset among patients suffering from strokes and classified according to Oxford Community Stroke Project classification (OCSP).

Based on the extent of the symptoms patients were classified as total anterior circulation infarct (TACI, n=10), partial anterior circulation infarct (PACI, n=10), and lacunar infarct (LACI, n=10).

Levels of CCL23 were determined as in the Examples before and using the ELISA technique (Technique 2, RayBiotech). The higher level of CCL23 was found among larger strokes (TACI), followed by PACI and LACI (p=0.14).

As can be seen inFIG. 11, the difference between groups trend to be significant when comparing the values of TACI+PACI (together) versus LACI (p=0.07) and it is significant for TACI versus LACI (p=0.04) patients.

These data indicate that the determination of the CCL23 levels is also useful to verify a diagnosed stroke episode, and even to diagnose and categorize said episode by an alternative way.

REFERENCES CITED IN THE APPLICATION