Nonspecific immunity – inborne immunity

1. Nonspecific immunity – inborne immunity Immunity – multicomponent, highly regulated systém Tasks: immune survey of self and not self-autoimmunity, antitumor accurancy of reaction - alergia reaction agains infectious agents - anti infectious immunity Lines of antiinfectious immunity: - specific humoral - specific cellular - nonspecific

2. Immunity – lines of reaction • Specific – cellular – quantitative testing • - tests o functionality - i.d.tests (tuberculin) • - humoral –quantitative testing • - functional testing – antibodies against specific bacterial and virus antigens – SEROLOGICAL REACTIONS • Nonspecific - cellular- quantitative - no. of lymfocytes (rosettes), Blood count, - leu, lymfo, mono, eosino……… • - functional - phagocytosis, Activity, Index, test of activation of lymfocyts • - humoral - quantitative - IgG,IgA,IgM, C3,C4,CH50,CIK, proteins of acute phase of inflamation -CRP, • - functional – clinically, baktericidal activity of serum, activity of lysosym

3. Suspect deficiency of immunity • Screening tests: - general - blood count + diff., FW, CRP, ASLO, RF - immunological – humoral immunity: IgG, IgA, IgM, levels of antibodies after vaccination with protein antigens (diphteria, tetanus), poysaccharide ag (Hib), virus ag, ubiquitous ag (ASLO) • - cellular immunity – no of lymfocyts - rosettes, subpopulation – flow cytometry, intra dermal tests • - phagocytosis - diff. leukogram, FA,FI • - complement sy - CH50, C3, C4,

4. Demonstration • Lysosyme – function of macrophage sy • Baktericidal activity of serum - humoral immunity – functional test • CH50, C3, C4 - humoral immunity • CRP – protein of acute phase infalmation • T lymfocyts, B lymfocyts – quantitative testing of cellular immunity • IgG, IgA, IgM – quantitative testing of humoral immunity, functional testing of B lymfocyts • Phagocytosis – Activity, index,

5. Determination of lysosyme • Lysosyme – basic low molecular protein • Micrococcus lysodeikticus – bacterium soluble by lysosyme • Sample in which we are testing presence and level of lysosyme - saliva, blood, urine, CSF • Agar with suspension of Micrococcus l.on glass dish • Wells in agar with standards with known concentration of lysosym and unknown sera • We read the clarification next to the well with serum with lysosyme that cause the lysis of bacteria in suspension in agar • Diameter of the zone of clarification is directly influenced by the concentration of lysosym and we calculate it by comparison using a formula Standards of lysosyme Unknown samples Agar + bacterial suspension

6. Baktericidal activity of blood • Escherichia coli • Suspension of bacteria - nephelometry – measurement of shadow – with different concentration ex. 10, 100, 103 104 105 106 • + serum • On agar plates – cultivation • Reading the no of bacteria that grew in the presence of serum. Bactericidal activity is calculated using the formula in comparison of unknown sera result and result of known standard • Base – in the presence of healthy serum a certain no of bacteria are killed.

7. CRP - C reactive protein, protein reacting with C substance present in the wall of Streptococcus pneumoniae • Antibody agains CRP is poured to the capillary and carefully the serum is added to the top • If CRP is present in the serum, it will react with anti CRP present in the bottom of the capillary and a white precipitate will appear • Qualitative test in capillary • Quatitative test is done in the agar – difusion (compare with lysosyme, IgG, protein of acute phase of inflamation, C3, C4). Where zone of precipitation is compared with the zone of the standard • CRP positive in bacterial infections

8. Immune-diffusion in agar • Test for determination of concentration of proteins – ex.: IgG, IgA, IgM, C3, C4, proteins of acute phase of infalmation - ceruloplasmin, macroglobulin…………. • Base – the antibody against the substance we want to measure is added to the agar. In preformed wells the standards (4-5) with known concentrations and unknown samples are added. The diffusion occurs and a ring of precipitation is formed based on the reaction of AgAb. The diameter of the ring is directly dependent of the concentration of measured sample and is compared with standard.

9. CH50 • Complement system - cascade of protein components, each of which is activated by the previous. The systém is started when the reaction of Ag and Ab happened. The result is the formation of membrane complex – the structure that is incorporated to the bacterial cell wall and thist causes the lysis of the microbial cell. Complement is the principal condition for the comlex of AgAb cause the damage • Test – determination of the amount of serum that is able to lyse 50% of standard concentration of erytrocyts.

10. Number of lymfocyts • B lymfocyts has on the surface antibodies that are able to bind annimal erytrocytes sensibilised against them. • T lymfocyts has surface receptors binding annimal erytrocytes after 24 hour´s incubation in 4*C. • Lymfocytes are separeted from full blood using TELEBRIX. Sensibilised annimal erytrocytes are added and rosettes of B lymfocytes are read immediately. Annimal ery for T lymfo are added to the tube with lymfocytes for T cell testing and read next day in the same way

11. Microscopical picture of rosettes –we count the no of rosettes, pure lymfocytes till 100, no of rosettes represent % of B or T lymfo. Lymfocyt rozety Annimal.ery

12. Fagocytosis, activity and index. • Leukocytes (PMNL) are offered the foreign bodies - in a way that full blood is incubated with baker yeast. The foreign bodies are phagocytes by PMNL. The blood smear is done and stained by May Grunwald method. Under microscope with imersion we read the picture. • No of phagocyting PMNL and not phagocyting PMNL untill 100. No of phagocyting PMNL in % is determining the phagocytosis or phagocytic activity. In phagocying PMNL we register by addeing the no of foreign bodies – yeast that are phagocyted – localised intracellularly. The fagocytosis index is calculated by dividin the no of phagocyted foreign bodies by no of phagocyting PMNL. This is the value of functional properties of phagocytes. • Normal FI=2,5