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Laboratory Safety Training
Institute of Immunology
2009
Declaratory statements and concepts
about lab safety according to the year
1993. 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
•
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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
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
ADAPTIVE IMMUNITY
- immediate reaction
- non-transmittable
- not antigen-specific
- no memory
- 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. 2002
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
leukocyte
red blood
cell
CELLS OF THE IMMUNE SYSTEM
The human body contains ca 1.000.000.000.000 (1012)
white blood cells
pollen
red blood cell
white blood cells
pollen
leukocyte
red blood
cell
yeast
Staphylococcus
E. coli
Ebola virus
Ebola virus
Rhynovirus
Rhynovirus
Mimivirus
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:
2033.
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
FUNCTIONALLY DIFFERENT CELLS OF THE IMMUNE
SYSTEM
Resting lymphocyte
NK cell
Plasma cell
Mast cell
Monocyte
Tissue dendritic cell
Macrophage
Lymph node dendritic cell
Normal bone marrow smear taken from the posterior iliac crest
(middle aged person, about 50% cellularity)
granulocytic
precursors
erythroid islands
megakaryocyte
eosinophilic basophilic
precursors
plasma cell
erythroid precursor
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
-1% of circulating leukocytes
- large granules in the cytoplasm
- nucleus with 2 lobes
- mast cells, histamin, allergic reactions
- high affinity IgE receptors
- against parasites
NEUTROPHIL GRANULOCYTES
- 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
EOSINOPHIL GRANULOCYTES
- 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 FceRI 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 lymphocyte
(Bursa fabricii)
T lymphocyte
(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
B LYMPHOCYTES
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
PLASMA CELLS
-function: - antibody production
- humoral immun response
T LYMPHOCYTES
- 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)
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
neutrophil
granulocyte
eosinophil
granulocyte
MONOCYTE
neutrophil
granulocyte
LYMPHOCYTE
basophil
granulocyte
LYMPHOCYTE
DISTRIBUTION OF BLOOD CELLS AND LYMPHOCYTE
SUBTYPES
Percentage
5 – 10 x 103
WHITE BLOOD
CELLS
leukocytes
neutrophil
granulocytes
Cell number/mm3
50 – 70
2.2 – 8.6 x 103
eosinophil
granulocytes
2– 4
0.04 – 0.5 x 103
basophil
granulocytes
0.5 – 1
0.01 – 0.12 x 103
lymphocytes
20 – 40
0.8 – 3.5 x 103
monocytes
3 –8
0.2 – 0.8 x 103
RED BLOOD
CELLS
erithrocytes
4.0 – 5.2 x 106
PLATELETS
thrombocytes
1.5 – 3 x 105
LYMPHOID ORGANS
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
CELLS OF THE IMMUNE SYSTEM
T LYMPHOCYTES
LYMPHOCYTES
helper T cells (Th)
cytotoxic T cells (Tc, CTL)
ACCESSORY CELLS
dendritic cell
macrophage
B LYMPHOCYTES
PROFESSIONAL ANTIGEN PRESENTING CELLS
Collaboration/communication of the leukocytes during an
immune response
APC
T
direct
„communication”
indirect
„communication”
cytotoxic T cell
helper T cell
dendritic cell
Antigen
Alarm
B cell
plasma cell
antibody
macrophage
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
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
lectin
domains
SRC
domaines
cysteines
Ig superfamily
members
selectins
integrins
mucins
Immunglobulin superfamily members
FAMILY
FUNCTION
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
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% H2O
10% dry substance:
organic substance:
90% organic substance
10% mineral 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
ROUGH CATEGORIES OF HORMONES
hormons
cytokines
interleukines
chemokines
monokines
interferons
lymphokines
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 MOST IMPORTANT FEATURES OF CYTOKINES
-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 EFFECTS OF CYTOKINES
makrofág
macrophage
MHC
cytotoxicity
citotoxicitás
­
monokinek
­
monokines
adhesion
molecules
adhéziós
molekulák ­
TCR TT-sejt
cell
autokrin IL-1 parakrin
prosztaglandinok ­
prostaglandins
láz
fever
aluszékonyság
somnolency ­
fájdalomküszöb
slimming
fogyás
end okrin
bacterial
bakteriális
endotoxin
endotoxin
aktiváció
­
activation
IL-2R
­
IL-2R
limfokinek ­
lymphokines
CYTOKINES
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
IFNγ
produced by Th1, CD8+ and NK cells
main role is the activation of macrophages
CHEMOTACTIC FACTORS
function together with the chemokines in the inflammatory responses
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,
METABOLITES
C4b, C5b)
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