Depending on the obtained results, all of these tests are performed in defined order, divided into 2 levels.

1St level tests:

• determination of number and morphology of lymphocytes in peripheral blood;

• determination of T and B lymphocytes based on CD receptors or in the rosetting methods;

• determination of serum immunoglobulins;

• determination of phagocytic activity of leukocytes;

• skin tests;

• X-ray examination of lymphoid tissues.

These tests are performed in any clinical immunological laboratory and sufficient for primary identification of patients with immunopathology.

2Nd level tests:

• histochemical analysis of lymphoid organs;

• analysis of CD markers of different subpopulations of lymphocytes using monoclonal antibodies:

• blast transformation of T and B cells;

• determination of cytotoxicity of lymphocytes;

• determination of synthesis and secretion of cytokines;

• determination of thymus hormones;

• analysis of respiratory burst of phagocytes;

• determination of complement proteins;

• analysis of mixture of cell cultures of lymphocytes.

These tests are performed in specialized immunological laboratories only.

Immunodeficiency is a condition resulting from a defective immune mechanism; may be primary (due to a defect in the immune mechanism itself) or secondary (dependent upon another disease process), specific (due to a defect in either the B or the T lymphocyte system, or both), or nonspecific (due to a defect in one or another component of the nonspecific immune mechanism: the complement, properdin, or phagocytic system).

Vaccines are immunobiological preparations which are used mostly for immunoprophylaxis of infectious diseases. They can include:

• Live, attenuated microorganisms;

• Those using inactivated microbial cells;

• Subunits of microorganisms (protective antigens):

• Secondary, secreted microbial compounds (e.g. inactivated toxin — anatoxins);

• Chemically synthesized antigens of microorganisms;

• Genetically engineered antigens of microorganisms.

Live, attenuated vaccines contained strains with decreased virulence, but retained immunogenicity. They can be obtained under the influence of unfavorable for microorganisms factors, including chemical (growth inhibitors),

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physical (temperature, radiation), biological (passages, cell cultures) ones. Example of this is anthrax vaccine.

Live, divergent vaccines are made on the basis of wild strains of microorganisms which have common antigens with human pathogens. Example of such vaccine is vaccinia vaccine used for prophylaxis of smallpox.

Live, recombinant vaccines are made with non-pathogenic microorganisms (e.g. some yeasts) in which genes of pathogens, responsible for synthesis of protective antigens, are incorporated. Example of this is recombinant hepatitis B vaccine.

Killed or inactivated vaccines contain strains of microorganisms killed by different methods (e.g. UV light, temperature, etc. ). As a result, such strains loose viability, but retain immunogenic properties. They can further be divided into whole cell (e.g. pertussis, cholera vaccines) or subunit (e.g. influenza, meningococcal vaccines) vaccines.

Anatoxins are immunobiological preparations which are obtained by inactivation of exotoxins (e.g. diphtheria, tetanus). For that purpose microorganisms are cultivated in broth, followed filtration of exotoxins and addition of 0.4% of formaldehyde solution. Inactivation is performed by incubation at +37-+40°C for 4 weeks, which allows complete loss of toxicity, but maintenance of immunogenicity. Anatoxin doses are measured more often in flocculation units (Lf). Sometimes, adjuvants are added to purified anatoxins in order to enhance immunogenic properties.

Adjuvants are group on non-specific substances which upon administration simultaneously with antigen, enhance the consequent immune response, Adjuvants can enhance immunogenicity in two different ways. First, adjuvants convert soluble protein antigens into particulate material, which is more readily ingested by antigen- presenting cells (e.g. macrophages). The antigen can be absorbed on particles of the adjuvant (such as alum) or made paniculate by emulsification in mineral oils. This enhances immunogenicity somewhat, but such adjuvants are relatively weak unless they contain bacteria or bacterial products, the second means by which adjuvants enhance immunogenicity. Although the exact contribution of the microbial constituents is unknown, they are clearly the more important component of an adjuvant. Microbial products may signal macrophages or dendritic cells to become more effective antigen-presenting cells. The bacterial constituents in the most adjuvants induce the production of inflammatory cytokines and potent local inflammatory responses. Interestingly, purified constituents of Bordetella pertussis are used as both antigen and adjuvant in the triplex DPT (diphtheria, pertussis, tetanus) vaccine against these diseases.

Combined vaccines are those containing a complex of different antigens which are used for simultaneous immunization against several infections. Examples of those are DTP, MMR (measles, mumps, rubella) vaccines.

The vaccination is classified into primary and secondary (booster) immunization. After primary immunization, active immunity and immunolo-

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gical memory are developed, allowing more effective and rapid response on antigen.

In Russia, there is an officially adapted immunization schedule (Federal Law No. 157, 2002) as listed in Table 23.

Table 23. IMMUNIZATION SCHEDULE USED IN RUSSIAN FEDERATION

Age	Vaccine
Birth (up to first	Hepatitis B: 1st vaccination
12 hours of life)
Birth (3-7 days)	BCG: vaccination
1 month	Hepatitis B: 2nd vaccination
3 months	Diphtheria, tetanus, pertussis, poliomyelitis: 1st vaccination
4. 5 months	Diphtheria, tetanus, pertussis, poliomyelitis: 2nd vaccination
6 months	Diphtheria, tetanus, pertussis, poliomyelitis: 3rd vaccination Hepatitis B: 3rd vaccination
12 months	Mumps, measles, rubella: vaccination
18 months	Diphtheria, tetanus, pertussis, poliomyelitis: 1st revaccination
20 months	Poliomyelitis: 2nd revaccination
6 years	Mumps, measles, rubella: revaccination
7 years	BCG: revaccination Diphtheria, tetanus: 2nd revaccination
13 years	Rubella: vaccination (girls; only when not vaccinated before) Hepatitis B: vaccination (only when not vaccinated before)
14 years	Diphtheria, tetanus, poliomyelitis: 3rd revaccination BCG: revaccination (only when not revaccinated at 7 years)
Adults	Diphtheria, tetanus: revaccination every 10 years of life

Vaccines used for the treatment of infections. These vaccines are administered to patients with protracted forms of infections (dysentery, brucellosis, ornithosis, typhoid fever, etc. ) in order to activate immunity and desensitize host.

Autovaccines (i.e. vaccines made of causative microorganisms isolated from the given patient) have the best therapeutic potential.

Immune sera and immunoglobulins. They are used for post-exposure prophylaxis (passive immunization) of the infections in persons, who are in incubational period of infection, or ill persons. All sera can be divided into two groups depending on mechanism of action: antitoxic (anti-diphtheria, anti- tetanus, anti-botulinum) and antimicrobial (anti-meningococcal).

Sera are used to produce concentrated preparation — immunoglobulins.

Heterologous immunoglobulin is a derivative of sera collected from hyperimmune animals (rabies, anthrax).

Homologous gamma globulin is produced from placental and donor human blood obtained from persons who have high antibody titers to certain antigens

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(anti-influenza, anti-measles, anti-tetanus). In contrast to heterologous immunoglobulins, homologous immunoglobulins and antisera extremely rarely cause hypersensitivity reactions.

Phages and eubiotics are another types of immunobiological preparations.

Phages are the preparations containing bacteriophages, which are capable to kill the bacteria (e.g. typhoid fever, dysentery, cholera). These preparations are also administered to contact persons as prophylactic agents.

Phages possess moderate therapeutic and prophylactic effect, so they are used only in combination with other medications.

Diagnostic phages are used for diagnostic and typing purposes which are of epidemiological significance.

Eubiotics are the preparations prepared from live non-pathogenic bacteria grown on nutrient media which are parts of normal flora (e.g. Bifidumbacterium spp., Lactobacillus spp., etc. ). They are normally sold in pharmacies in lyophilized conditions.

Also sometimes in clinical practice, non-specific immunostimulators of different originas are used. Examples of those are timalin, T-activin, pyrogenal, etc. which are stimulating different parts of immune system.

Main objectives of the session

1. To get acquainted with criteria for need, principles and methods of investigation of the immune status in some patient populations.

2. To get acquainted with immunobiological preparations used for prophylaxis, therapy of infectious diseases in humans.

Educational tasks

To know: 1. Immunodeficiency syndromes: classification, etiology,

pathogenesis, principles and methods of diagnostics.

2. Principles and methods of enhancement of both non-specific and specific resistance to infections diseases.

3. Preparation used for creation of active and passive immunity and enhancement of efficacy of non-specific protection against infections. Their classification and principles of production.

To be 1. To substantiate the need for investigation of immune status of capable: patients and interpret the results of such investigation.

2. To substantiate the necessity of usage of vaccines, sera and immunostimulators.

Methodical guidelines

For evaluation of functioning of the immune system and diagnosis of immunological pathologies, special clinical and laboratory methods were developed which allow differential characterization of functional activity of T- and B-lymphocytes.

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Enumeration of T- lymphocytes:

1. Reaction of spontaneous rosetting with washed sheep erythrocytes (E- rosettes).

2. Identification on the basis of CD markers.

Enumeration of B-lymphocytes:

1. Reaction of rosetting with sheep erythrocytes sensibilized with immunoglobulins and complement (EAC-rosettes).

2. Identification on the basis of CD markers.

Functional evaluation of cell immunity:

1. Reaction of blast transformation of T cells as a result of non-specific stimulation by T-mitogens (e.g. concavalin A, phytohemagglutinin).

2. Reaction of lymphocytotoxicity in mixed lymphocytes culture (MLC).

3. Production of lymphokines by T cells.

Functional evaluation of humoral immunity:

1. Determination of concentrations of main immunoglobulin classes using radial immunodiffusion (Manchini reaction).

2. Non-specific stimulation of lymphocytes by B-mitogens (e.g. lipopolysaccharides, etc. ).

Determination of subpopulations of lymphocytes is based on the following characteristics:

• Presence of antigenic markers (CD antigens) on the surface of lymphocytes. Those are glycoproteins specific for particular stage of differentiation. They can be detected by fluorescent monoclonal antibodies.

• Presence of receptors for some antigens and other chemical structures on the surface of lymphocytes:

Receptor for sheep erythrocytes — specific marker for T cells, which can be detected by rosetting reaction (E-rosetting).

Fc-receptor for Fc-fragment of immunoglobulins which present in B cells. If sheep erythrocytes are sensitized with immunoglobulins, B cells are able to form rosettes with them (EA-rosetting).

C receptor for complement which is present in B cells. Due to the fact that antigen-antibody complex is able to fix complement, this principle is used for detection of B cells in reaction of EAC-rosettes formation (sheep erythrocytes sensitized with antibodies and complement).

Functional differences in T and B cells can also be detected in vitro. Most often for such purpose, reaction of blast transformation is used. This reaction is based on the ability of small lymphocytes turn into blast forms which possess higher miogenic activity under action of mitogens during in vitro cultivation. As mitogens, mixed lymphocyte culture or some other substances of bacterial origin can be used. Also there are mitogens for T and B cells (e.g. concanavalin A and bacterial lipopolysaccharide, respectively).

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Laboratory work

1. Microscopy of preparations with E-rosetting reaction for enumeration of T cells.

2. Microscopy of preparations with EAC-rosetting reaction for enumeration of B cells.

3. Microscopy of preparation with reaction of blast transformation.

4. Interpretation of radial immunodiffusion (Manchini reaction) for determination of serum concentrations of different classes of immunoglobulins.

5. Interpretation of immune status based on the results of investigation of patients from immunological laboratory.

6. Acquaintance with different vaccine preparations, immune sera, immunoglobulins, allergens, etc.

7. Acquaintance with different eubiotics, phage, cytokine preparations.

Demonstrations

1. Microscopic preparations with E- and EAC-rosettes.

2. Microscopic preparations of reaction of lymphocyte blast transformation.

3. Manchini reaction in agarose gel for determination of different classes of immunoglobulins.

4. Kits with vaccine, immune sera, immunoglobulins, and allergens preparations.

5. Kits with eubiotics, phages and cytokines.

Control questions

1. What is the immune status of humans and what indicators are used for its characterization?

2. When investigation of the immune status is needed?

3. Immunodeficiency syndromes: principles of classification, etiology and pathogenesis.

4. Autoimmune diseases: origin and diagnostic use of immunological methods.

5. What markers are used for differentiation of T and B cells?

6. What are clusters of differentiation (CD) antigens? What is their practical significance in diagnostic immunology and how can they be detected?

7. What are the membrane immunoglobulins? What cells are they characteristic for and how they can be detected?

8. What are the direct (E-rosettes) and non-direct (EA-, EAC-rosettes) and when are they detected?

9. How can the functional activity of T and B cells be detected in vivo and in vitro?

10. What is the reaction of blast transformation?

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11. What mitogens are used in clinical immunology and what for? Please, give classification and examples.

12. How can the functional activity of humoral immunity be evaluated?

13. What is the principle of radial immunodiffusion (Manchini reaction) and what is it used for?

14. What are the principles of immunological correction of primary and secondary immunodeficiency syndromes?

15. What are the vaccines? Their classification and production methods.

16. When vaccines for treatment of infections are used?

17. What is the chemical vaccine?

18. What are the principles of production of genetically engineered vaccines?

19. What is anatoxin? How is it produced and what is it used for?

20. What are the adjuvants? What are they used for and what is the mechanism of their action?

21. What is ‘adjuvant disease’ and when can it be developed?

22. What is the immunization schedule? What vaccines are included into it?

23. What is population immunity? How can it be created and what is its role for prevention of infectious diseases?

24. What are the disadvantages of modern vaccines and how they can be corrected?

25. How can immune sera be obtained and what are they used for?

26. What are the homologous and heterologous immunoglobulins? How can they be obtained and what are they used for?

27. What complications can be developed in patients after administration of heterologous serum preprations? How can they be prevented?

28. What preparations are used for non-specific immunological correction and what is the mechanism of their action?