RU2491291C2 - Modified peptide and using it for cns cancer screening and therapeutic effectiveness tests - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention describes a chemically modified peptide that is a fragment of human protein S100β consisting of a sequence of 15 amino acids corresponding to the amino acids in position 18-32 of the amino acid sequence of protein S100β. The peptide is covalently bound to a spacer (Ahx) consisting of 2-aminoheptane acid or a similar compound, and covalently bound to the spacer by a biotin molecule (Bio). The peptide is described by general formula: A-Tyr¹-Ser²-Gly³-Arg⁴-Glu⁵-Gly⁶-Asp⁷-Lys⁸-His⁹-Lys¹⁰-Leu¹¹-Lys¹²-Lys¹³-Ser¹⁴-Glu¹⁵-E, wherein A represents Bio-Ahx, Bio-Acp, Bio or 0, and B represents 0 or -NH₂. Bio means biotin; Ahx means a residue of 2-aminoheptane acid; Acp means a residue of 6-aminocapronic acid, 0 means the absence of any amino acid. A diagnostic technique and a method for prediction of melanoma provides measuring blood natural antibodies (nAB) to the peptide according to the invention with the stages of melanoma diagnosed by the antibody concentration as compared to the reference. The clinical outcome and therapeutic effectiveness in a craniocereberal injury are predicted by measuring the nAB to the peptide according to the invention. If observing the blood nAB level in the patients lower as compared to the standard reference, the favourable clinical outcome is diagnosed, and the therapy is stated to be effective.

EFFECT: invention may be used effectively as instant diagnosing of the disturbed blood-brain barrier and monitoring of the clinical effectiveness in CCIs and some cancers.

3 cl, 9 dwg, 1 tbl, 5 ex

Description

The invention relates to the field of:

- medicine, namely, the diagnosis of violations of the blood-brain barrier (BBB) and monitoring the effectiveness of the therapy;

- immuno-diagnostics, namely, to determine the level of content of natural antibodies in blood serum;

- peptide chemistry, namely, the construction and modification of synthetic peptide fragments for use as an antigen for determining natural antibodies to natural proteins of the human body, in particular for determining natural antibodies to human S100β protein.

Traumatic brain damage in TBI includes two main mechanisms: primary brain damage as a result of direct exposure to mechanical energy, and its secondary damage resulting from complex and diverse mechanisms that are triggered from the moment of injury.

Speaking of primary damage to the brain, they mean direct damage to blood vessels, axons, neurons and glia that occurs when exposed to mechanical energy and leads to numerous forms of brain damage (diffuse axonal injuries; bruises and crushings of the brain; some types of primary intracranial hemorrhage; multiple common intracerebral hemorrhages; concussions and ruptures of the brain stem). Most authors believe that the early detection and elimination of intracranial and extracranial factors is fundamental in the treatment of patients with traumatic brain injury [Gaitur E.I. et al., 1996; Potapov A.A. et al., 1996; Chesnut R. M., e.a., 1993; Pietropaoli JA, e.a., 1992].

Cerebral ischemia is the most important factor accompanying secondary brain damage, and often contributes to adverse TBI outcomes. Some authors believe that the basis of post-traumatic brain disease can be immunological mechanisms, which in some cases facilitate the course of brain injury, and in others cause additional damage to brain tissue, contributing to the development of cerebral edema [Archelos J.J., Hartung H-P., 2000; Gannushkina I.V., 1974]. The most important in post-traumatic brain injuries, according to the author, is the loss of the body's ability to restore the integrity of the BBB in time. A similar situation is observed with the development of brain tumors. If in the early stages of tumor growth the BBB function remains close to normal, then, as the tumor grows, its permeability increases, which leads to the infiltration of nervous tissue by immune cells, on the one hand, and the penetration of brain antigens into the blood, on the other hand [Fenstermaker RA and Ciesielski MJ, 2004].

In this regard, the search for biochemical markers of brain damage and assessment of the state of the BBB integrity are important tasks of modern neurobiology.

An effective marker in assessing brain damage is the S-100β protein [Kruijk et al, 2001; Ingebrigtsen, Romner, 2003]. S-100 is a specific protein capable of binding calcium. The protein got its name due to the property of remaining in a dissolved state in a saturated solution of ammonium sulfate. The S-100 protein family consists of more than 18 tissue-specific monomers, two of which α and β with a molecular weight of 10.4 KD and 10.5 KD, form homo- and heterodimers, which are present in high concentration in the cells of the nervous system. S-100 (ββ) is found in high concentrations in astrocytes and Schwann cells; the heterodimer S-100 (αβ) is present in astrocytes. S-100 (β) and S-100 (α) are represented as homo and heterodimers in some tissues of malignant tumors (melanoma, glioma and some soft tissue tumors). S-100 protein is metabolized in the kidney, and its half-life is two hours. Protein S-100β is a biochemical marker of brain cell damage. An increase in the content of this protein in blood serum and cerebrospinal fluid was noted in many diseases accompanied by an increase in membrane permeability and / or death of glial cells due to necrosis and apoptosis, as well as a violation of the integrity of the blood-brain barrier structure. An increase in the concentration of S-100β protein in biological fluids, designated in some studies as S-100 protein, was found in acute ischemic stroke [Wunderekhen et al., 1999; Herrman, Vos, Wunderlich, 2000], perinatal hypoxic-ischemic damage to the central nervous system in newborns, malignant melanoma [Henze et al., 1999; Hauschild A. et al., 1997] and a number of other diseases [Golosnaya et al., 2004].

Protein S-100 is involved in the differentiation of cytoskeletal structures and Ca ^+2- dependent cellular information processes. Astrocyte necrosis, in case of brain damage due to trauma or malignant tumors, causing the S-100β protein to escape from the cytosol into the extracellular space, as well as integral membrane damage in the shadow zone of ischemic lesions, leading to cytotoxic and vasogenic edema, can cause a significant increase in the content S-100β protein in blood serum.

The determination of the S-100β protein itself in serum is currently widely used in neurology and partly in oncology. Diagnostic kits are available on the market, for example: CanAg S100 EIA (CanAg Diagnostics Sweden), Human S100B ELISA Kit (Bio Vendor Laboratory Medicine, Czech Republic), Human S100B ELISA Kit (Abnova Corporation, Taiwan), S-100 Beta EIA Kit (Alpco Diagnostics , CUIA), Human S100 beta ELISA Kit (Cosmo Bio Co., Ltd. Japan). Despite the existing variety of diagnostic kits, their high price significantly limits their use in the Russian market. In addition, the determination of the S-100β protein in the blood is a rather crude indicator, which, although it makes it possible to diagnose acute brain damage, does not make it possible to predict the effectiveness of treatment and the possible outcome of the disease.

Known methods for determining the level of antibodies, including protein S100β, for example, described in RF patent No. 2107913 "Method for screening women of childbearing age using the ELI-P test system to predict the development of the embryo / fetus and the birth of a healthy or abnormal baby" ( Poletaev A.B .; Vabishchevich N.K .; Morozov S.G.). The closest to the claimed solution can be considered an invention protected by RF patent No. 2259567 "Method for screening detection of individuals at risk of developing pathology of the nervous system and monitoring the condition of patients suffering from neuropsychiatric diseases" (Poletaev AB, Ambrosimova AA, Sokolov M.A.).

The considered methods have a number of significant differences and disadvantages:

- the S100 protein isolated from the brain of animals and / or whole or fragmented idiotypic antibodies to this protein are used as a component of test systems. The use of denatured proteins and antibodies makes it difficult to standardize the analysis performed, as

- protein denaturation reduces shelf life and accuracy of the test system.

- the approach has never been investigated as a diagnostic tool for assessing disorders of the BBB permeability and monitoring the effectiveness of TBI therapy,

- the described biomarker has never been used for the diagnosis of cancer, such as malignant melanoma, anaplastic astrocytoma and glioblastoma multiforme.

The objective of this technical solution is to increase the effectiveness of testing neurological and oncological diseases of the nervous system, as well as diagnosing the prognosis of the outcome and the effectiveness of the treatment of traumatic brain injury, by the method of determining specific antibodies to the protein. Efficiency is expressed in increasing specificity, reducing the variability of the test, reducing the amount of sorbed antigen, lowering the cost of testing compared to using the S100β protein, by creating a modified peptide.

To accomplish this task, a chemically modified peptide was developed - a fragment of the human protein S100β, which is used as follows: the S100β peptide (18-32) is used as an antigen for the immunological diagnostic method, for which it is immobilized on standard types of immunological sorbents (polystyrene well plate, nitrocellulose (nylon and magnetic or nano-particles), a patient blood sample is taken from which the serum is released, and the level of NAT to S100β is determined in it.

Using the proposed modified synthetic peptide, it is possible to analyze and / or monitor the NAT for S100β protein in human and animal blood serum samples and optimize testing of neurological and oncological diseases of the nervous system by increasing the specificity of the test, reducing the variability of test results by 2–3 times, reduce the amount of adsorbed antigen from 12 to 50 times.

The essence of this technical solution consists in the fact that a modified peptide is proposed, consisting of a sequence of 15 amino acids (18-32) belonging to the human protein S100β, a spacer covalently linked to it (Ahx), consisting of 2-aminoheptanoic acid or a similar compound, and covalently linked to the spacer of a biotin molecule (Bio) according to the formula (Table 1): A-Tyr ¹ -Ser ² -Gly ³ -Arg ⁴ -Glu ⁵ -Gly ⁶ -Asp ⁷ -Lys ⁸ -His ⁹ -Lys ¹⁰ -Leu ^11- Lys ¹² -Lys ¹³ -Ser ¹⁴ -Glu ¹⁵ -B, where A is Bio-Ahx, Bio-Acp, Bio or O, and B is O or -NH ₂ (SEQ ID NO 1-5) ( Bio stands for b otin, Ahx denotes the residue of 2-aminoheptanoic acid, Acp - denotes a 6-aminokaproinovoy acid, O indicates the absence of amino acids).

The sequence of the human S100β protein is known: Met-Ser-Glu-Leu-Glu-Lys-Ala-Met-Val-Ala-Leu-Ile-Asp-Val-Phe-His-Gln-Tyr-Ser-Gly-Arg-Glu- Gly-Asp-Lys-His-Lys-Leu-Lys-Lys-Ser-Glu-Leu-Lys-Glu-Leu-Ile-Asn-Asn-Glu-Leu-Ser-His-Phe-Leu-Glu-Glu- Ile-Lys-Glu-Gln-Glu-Val-Val-Asp-Lys-Val-Met-Glu-Thr-Leu-Asp-Asn-Asp-Gly-Asp-Gly-Glu-Cys-Asp-Phe-Gln- Glu-Phe-Met-Ala-Phe-Val-Ala-Met-Val-Thr-Thr-Ala-Cys-His-Glu-Phe-Phe-Glu-His-Glu.

The calculation for the sequence of the peptide fragment with the most pronounced immunological properties was performed by computer analysis using the BSITE1 (Antigenic Sites Analysis Program, using the consensus scale parameters) and Peptide Companion (calculation using the Hopp and Woods hydrophilicity scale data). An analysis of the data, taking into account the given fragment size (10-15 amino acid residues), the stability of the preparations, and the characteristics of the solid-phase peptide synthesis, allowed us to choose the sequence 18-32 as the antigenic determinant:

Tyr-Ser-Gly-Arg-Glu-Gly-Asp-Lys-His-Lys-Leu-Lys-Lys-Ser-Glu.

The corresponding peptide was synthesized in the form of:

Bio-Ahx-Tyr-Ser-Gly-Arg-Glu-Gly-Asp-Lys-His-Lys-Leu-Lys-Lys-Ser-Glu-NH2

The calculated structure was modified by the addition of a “spacer” in the form of 2-aminoheptanoic acid. Adding a “spacer” optimizes the steric position of the peptide after sorption on the surface with an immunological plate. A biotin molecule was covalently attached to the "spacer" to ensure optimal affinity binding to the surface of immunological plates or other sorbents coated with streptavidin suitable for use in immunological analysis. Such a formula can increase the specificity of the analysis when using 10-15 times less antigen than when using the protein antigen S100β. This approach not only optimizes the analysis, but also leads to its significant reduction in cost.

Example 1. The advantages of using a modified peptide in the determination of antibodies to S100β.

The effectiveness of using the modified S100β peptide (18-32) as an antigen in the system of enzyme-linked immunosorbent assay (ELISA) in comparison with the natural protein S100β was studied. In the experiment, the blood serum of rats with experimental traumatic brain injury (n = 9) was used in comparison with the group of control animals (n = 10). The study showed that the use of the modified S100β peptide (18-32) led to a decrease in variability when determining the NAT for S100β: 19% for the protein antigen and 6% for the peptide. In this case, the same signal (optical density) value was achieved using 12 times less peptide antigen: 0.5 μg per cell of the immunological tablet for the protein antigen 0.04 μg per cell for the peptide. Thus, the use of the modified S100β peptide (18-32) optimized the analysis. Figure 1 shows the results of an enzyme-linked immunosorbent assay of rat serum with experimental traumatic brain injury (TBI) using S100β protein (0.5 μg / cell) and modified S100β (18-32) peptide (0.04 μg / cell) in as an antigen. The ordinate is the optical density of the solution after analysis at a wavelength of 450 nm.

Studies have also shown that the modified S100β peptide has a 100-1000-fold higher binding efficiency with immunosorbents in various immunodiagnostics compared to the S100β protein. The binding efficiency is achieved due to the high-affinity interaction of biotin, which is part of the structure of the modified peptide. and immunosorbent-coating protein Avidin. Thus, a high binding constant allows one to achieve almost complete binding of the antigen to the sorbent, which leads to 100-1000 fold antigen savings when using a modified peptide. When using the S100β protein, most of the antigen remains in solution.

Example 2. The relationship between the effectiveness of therapy during the recovery period after traumatic brain injury in rats and the level of NAT to the modified S100β peptide (18-32).

Studies have been conducted on the relationship between the level of NAT to the modified S100β peptide (18-32) and the effectiveness of experimental therapy. The study was conducted on male albino rats (n = 9) weighing 160-220 g. In animals, a conditioned reflex of active swimming avoidance was previously developed. TBI modeling was carried out using the weight-drop method. Experimental therapy was started 0.5 hours after mechanical exposure. The well-known peptide neuroprotector Cortexin (0.15 mg / kg) (Geropharm, Russia) and the cerebrolysin preparation (0.2 ml / kg) (EbwePharma, Austria) were used. The drugs were administered intraperitoneally once a day. The control group received physiological saline in the volume of drug administration. The course of treatment continued throughout the observation period - 7-10 days. Assessment of the restoration of pre-formed conditioned reflexes was carried out starting from the first day after the application of a head injury, and continued daily throughout the observation period (14 days). On the 14th day of the experiment, blood samples were taken from animals. The serum NAT level for S100β was determined using the modified S100β peptide (18-32) as an antigen in the enzyme-linked immunosorbent assay (ELISA). The results of the study showed that there is a difference in this indicator between the control group (animals without TBI) and groups of animals with TBI with treatment (cortexin, cerebrolysin) and without treatment. The level of NAT to S100β in animals with injury exceeded the control by 1.5-2.5 times depending on the type of treatment. The dynamics of the recovery of the conditioned reflex of active swimming avoidance during experimental treatment of the effects of traumatic brain injury in rats is shown in FIG. 2.

Figure 3 shows the level of NAT to S100β in rat blood on day 14 of experimental treatment of the effects of traumatic brain injury in rats. Groups: 1 - TBI + Cortexin, 2 - TBI + cerebrolysin, 3 - TBI control (without treatment), 4 - control (without injury) (n = 9; P≤0.05).

The presence of a pronounced correlation between the rate of restoration of the conditioned reflex in animals (R = -0.78; p = 0.02) and the resistance of neurological disorders (R = 0.68; p = 0.05) and the level of NAT to S100β was revealed. The experiment showed that the experimental TBI leads to a significant increase in the level of NAT to S100β by day 14 after injury. In addition, the study demonstrated the existence of a relationship between the rate of recovery of central nervous system functions after head injury and the level of nAT to S100β. At the same time, the success of experimental therapy and the rapid restoration of CNS functions were associated with a lower level of NAT to S100β.

Example 3. Monitoring the level of NAT to S100β in children in the recovery process after head injury in the background of standard therapy.

The blood serum of 114 children in the dynamics in the first days after a head injury was studied. At the same time, the content of S100β protein (using the CanAg S100 EIA test) (CanAg Diagnostics Sweden) and NAT to the modified S100β peptide (18-32) using the modified S100β peptide (18-32) as an antigen in the enzyme-linked immunosorbent assay (ELISA) were evaluated . Figure 4 shows the performance in severe head injury with subsequent good recovery. Diagnosis: Combined severe head injury, brain contusion. Figure 5 - data for disability resulting from severe head injury. Diagnosis: Open TBI, fracture of the base of the skull.

The results obtained allow us to draw the following conclusions:

1. In most patients, an increase in the content of NAT to S100β (18-32) is observed simultaneously with an increase in the concentration of S100β protein in the blood (see Figure 4).

2. In children with a good restoration of cerebral functions after a head injury and a good response to the therapy, the level of NAT to S100β (18-32) in the blood (n = 54) (Figure 6) was significantly lower than in patients with severe consequences and few effective treatment (n = 45) (Fig.7). The dynamics of the level of S100β (18-32) nAT was characterized by a decrease in the level (Figure 4).

3. It was revealed that in some children with severe head injury, there is an increase in the content of NAT to S100β (18-32). The dynamics of the level of NAT to S100β was characterized by an increase in the level (Fig. 7).

4. Fig. 8 shows that in a patient in an extremely difficult vegetative state with constant resuscitation measures (complete disability due to drowning - severe hypoxia), an increased content of NAT to S100β (18-32) was observed for a long time, moreover , with a worsening clinical condition, a secondary increase in NAT to S100β was noted (18-32). AT to S100B are increased without increasing the S100B protein itself. In this case, it is the increase in AT to S100B that serves as an indicator of damage to barrier mechanisms, which suggests that early damage to the BBB is an unfavorable sign.

From this we can conclude that in these cases, NAT to S100β (18-32) are a sensitive biomarker of brain damage and BBB in the delayed period after a head injury. The fundamental and main differences in the use of the level of NAT to S100β (18-32) from the level of S100β protein in the blood are the following:

1. Despite the fact that an increase above the norm of both indicators occurs simultaneously after a head injury, their further dynamics are significantly different. So, the level of S100β protein decreases to normal by 3-4 days after a head injury regardless of the severity, characteristics of the course and outcome of the disease, while the dynamics of NAT to S100β (18-32) depends on these indicators (Figs. 4, 5 and 8).

2. With a favorable outcome of severe head injury and head injury of moderate severity, there is a slight increase in NAT to S100β (18-32), followed by a decrease by the 7-10th day (Figure 4). In the event of an unfavorable outcome of TBI with subsequent disability of the patient, there is a progressive increase in NAT to S100β (18-32) and their achievement of higher absolute values (Figs. 5 and 7).

3. In case of mild TBI with a favorable outcome, the dynamics of the levels of S-100β protein and NAT to S100β (18-32) coincide: a slight rapid increase in the indicator followed by normalization within 7-10 days (Figures 4 and 6).

4. In vegetative conditions (coma) resulting from a severe head injury, there is a complex dynamics of the level of NAT to S100β (18-32) with periodic drops and rises against the background of a normal level of S-100β protein (Fig. 8).

5. An extreme case is a fatal outcome. In this case, the dynamics of both indicators are identical: a significant rise, reaching its peak 10-15 days after a head injury, is replaced by a gradual decrease approaching the norm at the time of death.

Thus, on the basis of the studies, it follows that the determination of the level of natural antibodies (nAT) to the modified peptide is a reliable way to diagnose the prognosis of the outcome and the effectiveness of the treatment of traumatic brain injury. When the level of NAT in the blood of patients from 110 to 500% in relation to the control standard sample, the possibility of complications during the disease is diagnosed. In a series of sequential studies, a further increase in the level of NAT in the blood of patients from 110 to 500% in relation to the control standard sample is an indicator of the development of complications during the disease and an unfavorable prognosis, and when the level of NAT is reduced from 110% to 50% in relation to the control the standard sample is a criterion for a favorable course of the disease and prognosis.

The level of NAT to the modified S100β peptide (18-32) is a unique, much more specific and informative indicator than the previously used S100β protein level. It allows not only to ascertain the fact of injury and damage to the BBB, but also to evaluate the dynamics of the course of the disease, the effectiveness of therapy and make predictions of the outcome of the disease.

Example 4. The level of NAT to S100β in patients with malignant melanoma

Malignant melanoma is one of the most dangerous types of skin cancer. However, early diagnosis of the disease is an important condition for the effectiveness of therapy and reduction of mortality. The most important task is the early detection of metastases. To identify the diagnostic potential of the level of NAT to S100β (18-32), an analysis of the blood serum of patients at different stages of the disease was performed (Table 1). The results of the analysis demonstrate a significant increase in the level of NAT to S100β (18-32), starting from stage III of the disease. The sharpest increase in the level was noted at the stage of metastasis. The data indicate the promise of using NAT to S100β (18-32) as a diagnostic and, possibly, prognostic criterion for malignant melanoma.

Table 1. The level of NAT to S100β in the blood of patients at various stages of malignant melanoma. AJCC melanoma stages Number of patients NAT level S100β (18-32) (% relative to control) I (localized) 9 99 IIA (localized) 5 112 III (metastases to the region) eleven 120 * IV (distant metastases) 12 180 ** * P≤0.05, ** P≤0.01

Example 5. The relationship of the level of NAT to S100β (18-32) with tumor volume in patients with gliomas

In 14 patients with a diagnosis of anaplastic astrocytoma (8) and glioblastoma multiforme (6), an analysis of the level of NAT to S100β (18-32) in the blood was performed. Figure 9 shows the data individually for each patient.

In preparation for surgery, all patients underwent MRI (magnetic resonance imaging) to determine the volume and location of the tumor. Tumor volume was calculated using appropriate software. Analysis of NAT to S100β (18-32) was performed 3 days before surgery. Tumor volume was compared with the level of NAT to S100β (18-32). Direct proportionality between the tumor volume and the level of NAT to S100β was revealed (Fig. 9).

Thus, the present invention has a number of fundamental differences:

- it includes a specially designed stable synthetic peptide fragment of the human S100β protein S100β (18-32);

- this peptide fragment in a special way through a spacer is covalently linked to a biotin molecule, which can significantly improve the sorption of antigen due to high affinity binding to strept-avidin coated immuno-sorbent;

- the present invention with high efficiency is applicable for rapid diagnosis of BBB lesions and monitoring the effectiveness of TBI therapy, as well as an independent prognostic criterion for the course of the disease.

- the present invention is applicable for the diagnosis of a number of oncological diseases such as melanoma and glioma, and for monitoring the effectiveness of the therapy.

Claims (3)

1. A modified peptide for diagnosing and predicting the outcome and effectiveness of the treatment of diseases and brain injuries, comprising a sequence of 15 amino acids corresponding to amino acids at positions 18-32 of the amino acid sequence of the human S100β protein, and having the formula:
A-Tyr ^l -Ser ² -Gly ³ -Arg ⁴ -Glu ⁵ -Gly ⁶ -Asp ⁷ -Lys ⁸ -His ⁹ -Lys ^l0 -Leu ^ll -Lys ¹² -Lys ^l3 -Ser ^l4 -Glu ¹⁵ -B, where A is Bio-Ahx, Bio-Acp, Bio or 0, and B is 0 or -NH ₂ shown in SEQ ID NO 1-5, where Bio is biotin, Ahx is the residue of 2-aminoheptanoic acid, Acp is the remainder of 6-aminocaproic acid, 0 indicates the absence of an amino acid. 2. A method for diagnosing and predicting melanoma by determining the level of natural antibodies (NAT) in the blood to the peptide according to claim 1, and when the level of NAT in the blood to the control sample is increased to 99%, stage I melanoma according to AJCC classification is diagnosed with an increase in the level of NAT up to 112% - stage II, with an increase in the level of NAT to 120% - stage III and with an increase in the level of NAT to 180% - stage IV of melanoma. 3. A method for predicting the outcome and effectiveness of the treatment of traumatic brain injury, characterized in that they determine the level of natural antibodies (nAT) to the peptide according to claim 1 by determining the level of NAT in relation to the control standard sample and at the level of NAT in the blood of patients within from 50 and up to 110% with respect to the control standard sample, a favorable outcome of the disease and treatment efficiency are diagnosed, when the level of NAT in the blood of patients from 110 and up to 500% with respect to the control standard sample is diagnosed with the incidence of complications during the disease, during a series of sequential studies, a further increase in the level of NAT in the blood of patients from 110 to 500% in relation to the control standard sample is an indicator of the development of complications during the disease and an unfavorable prognosis, and when the level of NAT is decreased from 110% to 50% in relation to the control standard sample is a criterion for a favorable course of the disease and prognosis.

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