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Laboratory Safety Training Institute of Immunology 2009

Declaratory statements and concepts about lab safety according to the year XCIII. act 87. § regulation Workplace: every place or faciltiy outdoors/indoors, where employees are working or residing in order to perform labour. Organized labour: labour initiated, controlled or approved by the employer under normal employment, civil services or civil service legal relation in schools under studentship relation, during the technical training (or qualification) and corresponding to the professional requirements.

Physiological effects of electricity Accidents may occur if a human body joins in an electric circuit and an outer electric source gets through it electric current. Though the destructive effects of electricity originate in several physical and physiological influences, even so the accidents can be classified into two groups: Electric shocks and injuries which may come off jointly. 1. Electric shock occurs when an electric current flowing through the body and causes muscular spasm, disorder or paralysis of ganglia. 2. Electric injuries: a typical lesion at the very places of the entrance and exit of the electric current. Another characteristic impairment is the metallization of the skin: it appears at the contacting surfaces of the metal conductor and the skin, if the metal ions gets into the skin during the electrolysis.

Chemical features The harmful effects of a chemical substance are depending on the circumstances and concentration in which the employee come into contact with. Alternatives of absorption: - surface of the skin - digestive organ - respiratory organ - conjunctiva of the eyes - ears The extraneous substance - after getting into the body – passes with the circulation and hinders the normal function of the organs. A single large dose-caused acute or a small but regular dose-caused chronic intoxication may lead to occupational disease.

Fire safety regulations storage and usage of flammable liquids and substance usage of fire extinguishers fire safety regulations of works with naked flame (e.g. bunsen burner) possibilities and ways of fire alarm and appropriate behaviour due to a fire alarm

General lab safety regulations reporting obligation of accidents and injuries knowledge of ergonomic, workplace lighting instructions hygiene handling of machines and equipments use of safety clothing, tools and accessories use of aerator and vent hood regulations of handling and collection of hazardous waste use of hazardous substances and products smoking at workplace

Lab instructions during practice food and beverages are prohibited in the lab electronic eqipments must be turn off after experiments to flush out chemicals to the duct or drain is prohibited do not place flammable substances near to the radiator smoking is prohibited in the whole building

General instructions of first aid On the site of accident the first thing is a rapid orientation about: –the number and status of injured people –what happened –a need for immediate intervention –the order of attendance goal of first aid: –to save life –to inhibit subsequent health lesion –to help healing

The role of aid donor to measure the site of accident quickly and correctly without risking his/her own safety to protect the site of accident without major alterations to attend the injured persons in order to seriousness (the most serious cases first)

THE CELLS AND MOLECULES THE CELLS AND MOLECULES OF THE IMMUNE SYSTEM

THE ROLE OF THE IMMUNE SYSTEM Differentiation between harmless and harmful impacts DETECTION OF STRESS AND DANGER SIGNALS INNATE IMMUNITY Differentiation between self and non-self structures Antigen-specific recognition ADAPTIVE IMMUNITY Neutralization and elimination of foreign and harmful structures EXECUTIVE FUNCTIONS COORDINATED AND REGULATED ACTIONS INNATE IMMUNITY - immediate reaction - non-transmittable - not antigen-specific - no memory ADAPTIVE IMMUNITY - developes in several days - transmittable - specific - has memory Humoral immunity Cellular immunity

Immunitas = exemption, protection Protection from / against what? Self or non-self substances? (What about the useful bacteria living together with us and what about tumors in this modell?) -„Danger model”: (Matzinger P., The danger model: a renewed sense of self. Science Apr 12;296(5566):301-5.) -harmful self / harmless self -harmful non-self / harmless non-self factors! DANGER SIGNAL / NO DANGER SIGNAL -obligate pathogen -facultative pathogen (Staphylococcus aureus)

About sizes

Human hair

Dust mite on the head of the needle

Dust mite, human hair

pollen red blood cell leukocyte

CELLS OF THE IMMUNE SYSTEM The human body contains ca (10 12 ) white blood cells pollen red blood cell white blood cells

yeast leukocyte pollen red blood cell

E. coli Staphylococcus

Ebola virus

Rhynovirus

Mimivirus is one of the largest and most complex viruses known. The virus was first isolated in 1992 from amoebae growing in a water tower in Bradford. La Scola, B. et al. (2003) A giant virus in amoebae. Science 299: 2033La Scola, B. et al. (2003) A giant virus in amoebae. Science 299: Mimivirus

THE SITES OF IMMUNE CELL PRODUCTION embryo: yolk sac, liver, spleen after birth:- epiphysis - flat bones – red bone marrow (sternum, ribs, vertebras, hip bone) CONSTANT REGENERATION FAST REGENERATION INTENSE ADAPTATION

Plasma cell NK cell Tissue dendritic cell Mast cell Monocyte Macrophage Lymph node dendritic cell Resting lymphocyte FUNCTIONALLY DIFFERENT CELLS OF THE IMMUNE SYSTEM

Normal bone marrow smear taken from the posterior iliac crest (middle aged person, about 50% cellularity) megakaryocyte erythroid islands granulocytic precursors erythroid precursor eosinophilic basophilic precursors plasma cell

MONOCYTES - origin: pluripotent cells of the bone marrow myeloid progenitors -size: 10-15um - nucleus: bean-shaped -localization: circulation out of circulation: macrophage TISSUE - VENTRICLE MACROPHAGES - phagocytic cells - antigen presenting cells (APC) -main types (based on tissue localization): a)microglia (brain) b)Kuppfer-cells (liver) c)histiocytes (connective tissue) d)osteoclasts (bone) e)alveolar macrophages (lung) - function: in cellular and humoral immun response

BASOPHIL GRANULOCYTES NEUTROPHIL GRANULOCYTES EOSINOPHIL GRANULOCYTES -1% of circulating leukocytes - large granules in the cytoplasm - nucleus with 2 lobes - mast cells, histamin, allergic reactions - high affinity IgE receptors - against parasites - highest number in blood (68% of circulationg leukocytes, 99% of circulating granulocytes) - phagocyting cells - does not present in healthy tissues - tissue damage, migration, elimination of pathogens (enzymes, reactive oxygen intermediers) - main participants in inflammatory processes - agains parasites - 2-3% of leukocytes - allergic reactions

MAST CELLS -origin: pluripotent cells of the bone marrow myeloid progenitors - localization: absent from circulation differentiate in tissues especially around small vessels - function: - upon activation they regulate the permeability of the vessels with their secreted molecules - native and adaptive immunity - allergic reactions (cell surface Fc  RI receptors) - main types: a) mucosal b) connective tissue

DENDRITIC CELLS -origin: myeloid or lymphoid progenitors -localization: the immatured dendritic cell migrates from the circulation into the tissues and upon pathogen uptake it differentiates to matures dendritic cell and migrates to the draining lymph nodes - antigen presenting cells (APC) -types : a) myeloid DCs: - Langerhans cells (mucosa, skin) - intersticial DCs (liver, spleen, etc.) b) lymphoid DCs: - thymic DCs - plasmacytoid DCs (pDC) Follicular DCs: stroma cells of the centrum germinativum of lymph nodes

COMMON LYMPHOID PROGENITOR CELLS B lymphocyteT lymphocyte (Bursa fabricii) (thymus) maturation: begins in bone marrow continues in bone marrow continues in thymus differentiation: peripheral tissues upon activation plasma cells effector T cells cytotoxic T cell helper T cell antigen recognition only via cell surface MHC molecules

origin: pluripotent cells of the bone marrow lymphoid progenitors maturation: bursa equivalent tissues (embrionic liver, later bone marrow) -localization: takes 5-10% of the circulating lymphocytes; migrate from the bone marrow to the secondary lymphatic organs thorugh the circulation - antigen presenting cells (APC) - activation: with antigens, via interaction with macrophages or T lymphocytes, lymphokines, cytokines - upon activation they differentiate to plasma cells or memory B cells B LYMPHOCYTES PLASMA CELLS -function: - antibody production - humoral immun response

- origin: pluripotent cells of the bone marrow lymphoid progenitors - maturation: thymus - localization: in the thymus the thymocytes mature into immunocompetent T cells and they enter to the peripheral (secunder) lymphoid organs as TCR expressing T lymphocytes - antigen recognition only in MHC molecules on the surface of APCs -types: - T helper (CD4+) - T cytotoxic (CD8+) - T regulator (suppressor) T LYMPHOCYTES

NK CELLS (natural killer) - origin: pluripotent cells of the bone marrow lymphoid progenitors - bigger than lymphocytes - several granules in their cytoplasm - has no antigen binding receptors („null cells”) - participants of native immunity

Professional phagocytic cells macrophages neutrophyl granulocytes dendrtitic cells the phagocytosed cells or molecules may modify the functions of the cell phagocytosis followed by enzymatic degradation Professional antigen presenting cells macrophages B lymphocytes dendrtitic cells they express MHC molecules the protein degradation products (peptides) can be presented to T lymphocytes by MHC molecules

WHITE BLOOD CELLS IN THE SMEAR OF HUMAN PERIPHERAL BLOOD LYMPHOCYTE LYMPHOCYTE MONOCYTE neutrophil granulocyte basophil granulocyte neutrophil granulocyte eosinophil granulocyte

DISTRIBUTION OF BLOOD CELLS AND LYMPHOCYTE SUBTYPES PercentageCell number/mm 3 WHITE BLOOD CELLS leukocytes 5 – 10 x 10 3 neutrophil granulocytes 50 – – 8.6 x 10 3 eosinophil granulocytes 2– – 0.5 x 10 3 basophil granulocytes 0.5 – – 0.12 x 10 3 lymphocytes 20 – – 3.5 x 10 3 monocytes 3 –80.2 – 0.8 x 10 3 RED BLOOD CELLS erithrocytes 4.0 – 5.2 x 10 6 PLATELETS thrombocytes 1.5 – 3 x 10 5

Primer lymphoid organs: - bone marrow - thymus Secunder lymphoid organs: - lymph node - spleen - tonsillas - MALT (Mucosal Associated Lymphoid Tissue) -GALT (Gut Associated Lymphoid Tissue) - BALT (Bronchus Associated Lymphoid Tissue LYMPHOID ORGANS

B LYMPHOCYTES PROFESSIONAL ANTIGEN PRESENTING CELLS T LYMPHOCYTES LYMPHOCYTES CELLS OF THE IMMUNE SYSTEM dendritic cell macrophage ACCESSORY CELLS helper T cells (Th) cytotoxic T cells (Tc, CTL)

Collaboration/communication of the leukocytes during an immune response helper T cell cytotoxic T cell B cell dendritic cell macrophage Antigen Alarm antibody plasma cell direct „communication” indirect „communication”

MOLECULES OF TEH IMMUNE SYSTEM Cell surface molecules: markers (CD) receptors (BCR, TCR, MHCI, MHCII, PRR, etc.) costimulatory molecules adhesion molecules (integrins, selectins, mucins, etc.) Soluble molecules: cytokines antibodies complements metabolites

DISTRIBUTION OF THE LYMPHOCYTE POPULATIONS IN THE BLOOD T cells B cells NK cells

THE MOST IMPORTANT CELL SURFACE STRUCTURES OF HUMAN LYMPHOCYTE POPULATIONS

The main types of cell surface molecules participating in the interaction between extracellular matrix and the lymphocytes Ig superfamily selectins integrins mucins members lectin domains SRC domaines cysteines

FAMILYFUNCTION integrins - cell-cell and cell-extracellular matrix interactions - cell migration - tissue organization - inflammation e.g. LFA-1 (Lymphocyte Function Associated Antigen) selectins - endothelial migration of lymphocytes and setle in tissues - mainly on leukocytes and endothels - binds the carbohydrates components of the cell membrane e.g. L-selectin – homing to the lymph node Immunglobulin superfamily members

mucins - highly glycosylated - lymphocyte migration though HEV e.g. CD34 binds L-selectin proteoglycans - interaction with the tissue environment Immunglobulin superfamily members

The main types of cell surface molecules participating in antigen recognition and the interaction between dendritic cells and T cells

SOLUBLE MOLECULES in the plasma and other body fluids plasma:90% H 2 O 10% dry substance:90% organic substance 10% mineral substance organic substance: polysaccharide (glucose) lipid (kolesterol, triglycerid, phospholipid, lecitin, fat) protein (globulin, albumin, fibrinogen) glycoprotein hormone (gonadothropin, erytropoetin, trombopoietin) amino acid vitamin mineral substance: ions in soluble form BIOLOGICALLY ACTIVE SOLUBLE MOLECULES AFFECT ON THE IMMUNE SYSTEM FUNCTIONS

hormons cytokines chemokines interleukines monokines lymphokines interferons ROUGH CATEGORIES OF HORMONES

CYTOKINES origin: - lymphokines - monokines - interleukines (IL) function: - role in acute phase reaction and inflammation - regulates maturation and development of immunocompetent cells - regulates the activation and differentiation of lymphocytes

-The cytokines are the most important mediators of indirect cell communication in the immune system („hormones” of the immune system). - Cytokines can be act in low concentrations. - The responsiveness of the given cell is based on the expression of cytokine-specific receptors. - Cytokines can affect to the productive cells by autocrine way, to the nearby cells by paracrine way, to the distant cells by endocrine way, so they can modulate lots of cell types (pleiotropic effect). - Cytokines can act by synergistic or antagonistic ways to each other, a given cell may affected by many cytokines resulted in the same effect (redundant effect). -Cytokines can be devided into sub-groups by origin and functional properties. - Cytokine receptors are categorized into molecule families. THE MOST IMPORTANT FEATURES OF CYTOKINES

THE EFFECTS OF CYTOKINES bacterial endotoxin cytotoxicity monokines adhesion molecules prostaglandins fever somnolency slimming activation IL-2R lymphokines T cell macrophage

CHEMOKINES enhance adhesion chemotaxis (IL-8) leukocyte activation produced by macrophages, endothels, keratinocytes, fibroblasts, smooth muscle cells, T lymphocytes TYPE I INTERFERONS native immunity protection agains viruses, glycoproteins IFNα: produced by virus infected macrophes and and other leukocytes IFNβ: produced by fibroblasts TYPE II INTERFERONS - immune interferons IFNγproduced by Th1, CD8+ and NK cells main role is the activation of macrophages CHEMOTACTIC FACTORS function together with the chemokines in the inflammatory responses CYTOKINES

COMPLEMENT Inactive enzyme cascade system in the serum activation of complement system- production of cell surface binding and soluble factors - opsonin - anaphilactic (C3a, C4a) - chemotactic (C5a) - phagocytosis (C3b, iC3b, C4b, C5b) METABOLITES Regulate the functions of the cells belonging to the immune system ANTIBODIES – multifunctional proteins Produced by plasma cells differentiated from activated B lymphocytes roles: antigen recognition and binding binding to cells connecting immune cells triggering effector functions elimination of antigens activation of complement system