RU2414228C1 - Method of patient-specific immunotherapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to clinical immunology and aims at patient-specific immunotherapy. That is ensured by the use in a nidus zone of supernatant of stimulated autologous leukocytes with the concentration of neutrophilic antimicrobial peptides in supernatant not less than 2000 pg/ml. Supernatant is administered in applications and/or subcutaneously, or submucosally, or in instillations for 5-10 days.

EFFECT: method provides prevention of secondary immunodeficiency, reduced inflammation, wound decontamination, stimulated repair, allows lower dosages and period of administering antibiotics, due to joint action of a complex of cytokines and antimicrobial neutrophilic peptides produced by autologous leukocytes.

6 cl, 3 tbl, 4 ex

Description

The invention relates to medicine, namely to clinical immunology, and is intended for personified immunotherapy.

Currently, the selection of optimal methods of treatment of the wound process is an urgent task for doctors of various specialties.

The features of modern infection are both the presence of microorganism strains resistant to antibacterial therapy and the occurrence of an immunosuppressive state, which leads to complications of the inflammatory process.

The urgent need for a patient-centered approach has become apparent thanks to the latest advances in science about the individual characteristics of susceptibility to diseases and their treatment. It is known that most drugs affect less than half of patients, and side effects can be worse than the diseases themselves.

One of the directions of personified immunotherapy is associated with the use of autologous cytokines. A known method of personalized immunotherapy in the treatment of purulent wounds using autologous cytokines, in which the supernatant obtained by isolating lymphocytes from the patient’s blood and culturing them in the presence of phytohemagglutinin at a dose of 8-10 μg / ml for 1.5-3 hours is applied to the wound subsequent washing and re-culturing for 18-24 hours. Daily application of the obtained product to the wound allows significantly reducing the inflammatory reaction, exudation, size of the wound defect on the first day of treatment, to the early appearance of granulations. This reduces the treatment time, in particular in the hospital (SU 1498498, 06/19/86). This method is taken as the closest analogue. However, this method is not without drawbacks, namely, the producers of cytokines in this method are peripheral blood mononuclear cells isolated using expensive imported reagents. The number of secreted cells is small, and only cytokines are the main active principle.

The proposed personalized method of immunotherapy is based on the local use in the area of the pathological focus of cytokines and antimicrobial peptides obtained by culturing stimulated leukocytes of the patient. The use of a complex of cytokines and antimicrobial neutrophilic peptides that are autologous to the patient as a therapeutic agent does not cause the development of allergic reactions and reduces the time for the use of antibiotics. Local use of peptides provides a quick directed action of autologous cytokines and antimicrobial neutrophilic peptides on the pathological process in the damage zone, which manifests itself in an anti-inflammatory, antimicrobial, wound healing effect. Obtaining from the patient’s blood a larger amount of an autologous drug, which, along with anti-inflammatory and wound healing effects, has direct antiviral and antibacterial activity, and local use of an autologous drug can reduce the time for using antibiotics and even cancel them.

The objective of the proposed method is to develop an improved personalized method of immunotherapy.

The technical result of the proposed method is the prevention of secondary immunodeficiency, a fairly quick relief of the inflammatory process with simultaneous simplification and cheapening of the method.

The technical result is achieved by reducing or eliminating the use of antibiotics through the simultaneous action of a complex of natural cytokines and antimicrobial neutrophilic peptides (endogenous antibiotics) produced by autologous leukocytes with antimicrobial activity in the supernatant of at least 2000 pg / ml.

The preparation of autologous immunopeptides (antimicrobial neutrophilic peptides) is obtained by isolating leukocytes from peripheral blood, stimulating them with a mitogen (e.g. phytohemagglutinin), culturing for 20-24 hours, obtaining an autologous supernatant, sterilizing it and evaluating biological activity. For this, 20-40 ml of blood is taken from a cubital vein from a patient in a sterile tube containing an anticoagulant (at the rate of 25 U / ml of blood). Peripheral blood is mixed in a 1: 5 ratio with 10% gelatin solution and settled for 10 min at room temperature, and then 30 min at 37 ° C. Leukomass is collected. Cells are washed with 0.9% sodium chloride solution, centrifuging them for 10 min at 200g. Determine the total number of selected white blood cells, prepare a concentration of 20 · 10 ⁶ cells in 1.0 ml of colorless medium 199.

To stimulate leukocytes, mitogens (PHA or concanavalin A) are used in a dose of 10-15 μg / ml. After 3 hours, the stimulator is removed, and the cells are washed three times from the stimulator and cultured in a colorless culture medium 199 for 20-24 hours. At the end of the cultivation, the supernatant is collected, sterilized by filtration through filters (Millipore-QS, USA, 0.22 μm pore diameter). The concentration of neutrophilic peptides (HNP-1-3) was determined using enzyme-linked immunosorbent assay using a system of the company HBT (Netherlands). The average concentration of HNP-1-3 in the autologous preparation of healthy donors is 4800.0 ± 2800 pcg / ml. The direct antibacterial and antiviral activity of an autologous preparation obtained from a culture of white blood cells from healthy donors was determined.

To study the direct antimicrobial activity of leukocyte supernatant for various pathogenic microorganism strains: Staphylococcus aureus (S. aureus ATCC6538P), E. coli (ATCC25922 E. coli) and candida fungus (C. albicans 865-465), we used the disk method impregnated with various concentrations autologous drug (50, 5.0, 0.5 mcg). Microbial suspensions were plated on nutrient media and then paper disks with different concentrations (dilutions) of the preparation were laid out. After 18 hours of incubation in a thermostat at 37 ° C, the diameter of the zones of inhibition of bacterial growth in mm was measured.

The sensitivity of S. piogenes was studied by dilution in a liquid nutrient medium, followed by inoculation on blood agar and determination of bacteria growth after 48 hours of incubation at 37 ° C.

All studied concentrations of the autologous drug depressed the growth of S. piogenes and the pathogenic strain of S. aureus staphylococcus.

The clinical effect of the use of the treatment method was evaluated according to clinical parameters: reducing inflammation, cleansing the wound of microorganisms, stimulating repair, the absence of allergic reactions, reducing the dose and timing of antibiotic use.

Table 1 presents the comparative results of the treatment of complicated wounds by the proposed method (15 patients) compared with the closest analogue (12 patients) and the basic treatment method (using antibiotics in 9 patients).

The proposed method was included in the complex treatment of 24 patients with moderate periodontitis. Basic therapy included both local and systemic treatment. Local treatment consisted in the removal of dental plaque, surgical surgery, the use of antiseptics and antimicrobials. General therapy included vitamin therapy, desensitizing therapy, antibacterial drugs in the presence of abscesses and suppuration.

The application of the method of personalized immunotherapy consisted in the use of an autologous leukocyte supernatant of a patient obtained from 20 ml of blood. The drug was applied for 5-7 days in the form of applications (0.5-1 ml) and submucosal administration. At the same time, 0.1 ml of chipping was performed at 4-5 points around the pathological focus of the periodontium.

The clinical effectiveness of the proposed method is presented in table.2.

As clinical observations have shown, the use of an autologous preparation of immunopeptides (antimicrobial neutrophilic peptides) contributes to:

the rapid disappearance of signs of inflammation, in comparison with the basic method of treatment, limits the use of antibiotics or completely cancels their intake, extends the remission period to 1 year, normalizes the ratio of microorganisms in saliva, gingival fluid. In patients with periodontitis, there is a deficiency of bifidobacteria and lactobacilli, and conditionally pathogenic microorganisms (St. aureus St. salivarius, St. epidermidis, Neisseria, Klebsiella Psevdomonas, etc.) predominate. An autopreparation of immunopeptides containing antimicrobial neutrophilic peptides has a direct bactericidal effect on opportunistic microorganisms and increases the content of lactobacilli and bifidobacteria in saliva and gingival fluid to normal values.

Chronic tonsillitis and adenoid vegetation are widespread, especially among children. Long-term persistence of infection in the body of such patients leads to the development of an immunodeficiency state both at the local and systemic levels. Traditionally, the use of antibiotics further exacerbates immunodeficiency. In this regard, the use of an autologous leukocyte supernatant with activity of endogenous antimicrobial peptides and cytokines is pathogenetically substantiated. The use of an autologous supernatant in 12 children with chronic tonsillitis and hypertrophy of adenoid vegetation reduced the degree of contamination of the tonsils of streptococcal and staphylococcal flora, by 8-10 days the pain syndrome completely disappeared when swallowed, and nasal breathing improved. In the group of patients treated with the basic method, receiving a course of antibiotics and washing the tonsils with an antibacterial drug of the sulfonamide group for 10 procedures, an exacerbation of the chronic inflammatory process was observed 4-6 times a year. The use of an autologous leukocyte supernatant reduced the frequency of exacerbation to 1-2 times a year.

The most common causes contributing to the development of immune disorders in the tissues of the eye are viral, bacterial infections, injuries. In this regard, in the treatment of keratitis of various etiologies in recent years, immunomodulators have been used to stop the clinical manifestations of immune deficiency and thereby increase the therapy of the underlying disease. A scheme is proposed for the use of autologous leukocyte supernatant in the complex treatment of viral keratitis in children (n = 16). As a control, we used the basic method of treatment in 10 patients and the method according to the closest analogue (n = 8), including the use of an autologous supernatant of mononuclear cells in combination with basic therapy. The results of clinical efficacy are presented in table 3. The use of an autologous leukocyte supernatant in the treatment of herpetic keratitis reduced the duration of the inflammatory process. The relief of corneal edema occurred a day earlier than during treatment with the closest analogue, and 3 days earlier than in the control group. Relief of corneal syndrome also occurred earlier in the main group. Complete epithelialization of the cornea in the main group occurred on the 6th day, while in the control group on the 11-12th, and in patients treated by the prototype method, on the 8th day. The restoration of visual acuity to 0.9-1.0 was observed in 56% of patients of the main group, in 12% of patients in the control group and 40% of patients treated by the closest analogue. The hospital stay of patients of the main group was reduced by 5 days.

Thus, during clinical observations of patient treatment results, the high effect of autologous leukocyte supernatant in the complex treatment of herpetic keratitis was confirmed, which manifests itself in stimulation of repair, more rapid relief of the inflammatory process, faster epithelization of the cornea, increased visual acuity and shortened patients in hospital almost 2 times.

The method is as follows. The supernatant of stimulated autologous white blood cells is obtained with a concentration of antimicrobial peptides in the supernatant of at least 2000 pg / ml, the supernatant is administered subcutaneously, or submucous, or as applications or as instillations for 5-10 days. With subcutaneous or submucosal administration, the supernatant is administered in a volume of 0.4-0.5 ml 1 time per day by chipping 0.1 ml in 4-5 points around the pathological focus. For applications, a supernatant is used in a volume of 0.2-2.0 ml once a day. For instillations into the nasal cavity, use a supernatant of 2 drops in each nasal passage 3 times a day. For instillations into the conjunctival, a supernatant of 2 drops in each eye is used 4-5 times a day.

Example 1. Patient T.P. K-ova, 32, was admitted to the hospital for pelvic presentation of the fetus.

A cesarean section was performed in the lower uterine segment due to a 40-week pregnancy, pelvic presentation, prenatal discharge. On the 7th day after the operation, a subaponeurotic suppurative hematoma, divergence of sutures to aponeurosis, was revealed.

After 2 days, leukocytes were isolated from 40 ml of venous blood and an autologous leukocyte supernatant (18 ml) was obtained.

For 7 days daily after cleansing the wound with hydrogen peroxide, the wound was treated in the form of applications of 2 ml of autologous supernatant solution (the concentration of antimicrobial neutrophilic peptides was 3580 pg / ml). Already on the 2nd day of using the drug, the wound edges cleared, bright granulations appeared, on the 3rd day in the wound discharge saprophytic and conditionally pathogenic microflora prevailed, on the 7th day the wound healed.

Example 2. Patient Cev was admitted to the Department of General Surgery of the Pediatric Faculty of the Russian State Medical University with a diagnosis of trophic ulcer of the leg (4.8 cm ² ) in the presence of chronic venous insufficiency, diabetes. Before admission, the patient was treated on an outpatient basis for a month and a half without a positive result.

From 40 ml of blood, 20 ml of leukocyte supernatant with neutrophilic peptide activity of 2340 pc / ml was obtained. After standard treatment, 1 ml of autologous supernatant was applied to the wound surface and 0.1 ml was administered subcutaneously at 5 points along the edges of the ulcer. Then a sterile dressing was applied. The course of treatment lasted for 10 days. On the 5th day of treatment, the ulcer surface was cleansed, and edge epithelization appeared at the same time. On the 7th day, active granulations appeared. After 14 days, 50% of the surface of the ulcer was epithelized. On the 20th day, the patient was discharged from the hospital in satisfactory condition.

Example 3

Patient K-va O., 11 years old, was hospitalized in the ENT department of the Children's Clinical Hospital of Roszdrav in Moscow with a diagnosis of exacerbation of chronic tonsillitis in combination with hypertrophy of adenoid vegetation. At endoscopic examination, hypertrophy of the tonsils of the III degree was noted, tonsil lacunae were dilated, deformed with rough commissures. Lacunae are made with mucous plugs. The tissue of the adenoids is hyperplastic and located on the entire surface of the nasopharynx.

Staphylococcus aureus, a-hemolytic streptococcus, and neisserial flora predominated in the microflora of the tonsils. An increase in submandibular, behind the ear lymph nodes was noted. The patient did not receive antibacterial and antiviral therapy.

On the 2nd day received 10 ml of autologous leukocyte supernatant. The concentration of antimicrobial neutrophilic peptides was determined in the obtained supernatant. It amounted to 6600 pg / ml. To 10 ml of leukocyte supernatant was added 20 ml of 0.9% sodium chloride solution. This solution was used for washing tonsils and instillation into the nasal cavity.

The pattern of application of the autologous leukocyte supernatant was as follows. Every day, sequential washing of tonsil lacunae with 0.9% sodium chloride solution was carried out once in order to clear the pathological contents of the lacunae. After that, 10 drops of a 0.9% sodium chloride solution were instilled into each nostril, after 5 minutes an autologous supernatant was instilled, 2 drops into each nostril. Sequential instillation procedures were performed 3 times a day. The instillation course was 7 days.

On the 3rd day after the treatment, there was a significant decrease in the severity of pain, there was a feeling of soreness and dryness in the throat when swallowing, hyperemia of the mucous membrane of the tonsils, swelling and inflammatory infiltration of the tonsils decreased, by the 5th - the lumpiness and deformity of the tonsils, decreased the size and depth of the lacunae, by the 10th day there was a complete relief of pain during swallowing, a further reduction in the size of the lacunae and the absence of contents in them, the nose was significantly improved Contents. The degree of seeding has significantly decreased, mainly due to a decrease in streptococcal and staphylococcal flora. The patient was discharged in satisfactory condition. The state of remission lasted 14 months. In patients treated with the basic method, exacerbations were observed every 3-4 months.

Example 4. Patient C-th, 15 years old, was admitted to the Morozov Children's City Clinical Hospital with a diagnosis of treelike keratitis.

On admission: manifestations of corneal syndrome (lacrimation, photophobia, blepharospasm, neuralgic pain), decreased visual acuity OS = 0.5, OD = 0.7, decreased sensitivity of the cornea. Biomicroscopy determined mucous discharge, pronounced pericorneal injection, the presence of a twig in the cornea of infiltrate.

In a patient from 15 ml of blood, according to the known method, 7 ml of autologous leukocyte supernatant with an antimicrobial neutrophilic peptide concentration of 4680 pg / ml was obtained. The supernatant was instilled into the conjunctival cavity, 2 drops in each eye 4-5 times a day for 7 days in parallel with the antiviral therapy, including Zovirax, vascular therapy (ascorutin), desensitizing therapy (tavegil). On the 4th day of treatment, pericorneal injection disappeared, there was a relief of edema of the cornea, corneal syndrome. Complete corneal epithelization was observed on the 7th day. The restoration of the transparency of the cornea led to the restoration of visual acuity OS = 0.9, OD = 0.9. The patient spent 8 days in the hospital and was discharged.

Thus, the use of autologous preparations obtained from the patient’s leukocytes significantly reduces the time of wound healing, stimulates the appearance of granulations, reduces pain, changes the composition of microorganisms defined in the wound discharge, and, most importantly, avoids taking antibiotics that exacerbate the course of secondary immunodeficiency , developing with long-term ongoing wound processes.

This method can be used in the treatment of a wide range of diseases in which there are disorders in the immune system.

Claims (6)

1. The method of personalized immunotherapy, including the use of stimulated autologous blood cells in the area of the pathological focus of the supernatant, characterized in that leukocytes with a concentration of neutrophilic antimicrobial peptides in the supernatant of at least 2000 pg / ml are used as autologous cells, and the supernatant is administered in the form of applications and / or subcutaneously, or submucous, or in the form of instillations for 5-10 days. 2. The method according to claim 1, characterized in that with subcutaneous or submucosal administration, the supernatant is administered in a volume of 0.4-0.5 ml once a day. 3. The method according to claim 2, characterized in that the supernatant is administered by chipping 0.1 ml at 4-5 points around the pathological focus. 4. The method according to claim 1, characterized in that for applications using the supernatant in a volume of 0.2-2.0 ml 1 time per day. 5. The method according to claim 1, characterized in that for instillations into the nasal cavity, a supernatant of 2 drops is used in each nasal passage 3 times a day. 6. The method according to claim 1, characterized in that for instillations into the conjunctival cavity, a supernatant of 2 drops in each eye is used 4-5 times a day.