MODERN METHODS
=overview of 3 anti-patogen strategy of cell and medicine
J. Skopalík
14.12.2021
Basic question from practiacal medicine:
• What does patogen (virus) need ????
a) aerosol b) extracelulár matrix c) cytosol ???
a) live cell b) death cell ???
Virus need
live cells.
Virus need cytosol,
where translation
of DNA is activated:
Where are the place of cell defence ?
1) Blocking of VIRUS landing by FUSION INHIBITORS analogy to
moon landing
2) Nucloside inhibitors
3) NON-nulceoside inhibitors
4) Protease inhibitors against HIV protease. (Because HIV protease is
responsible for processing of the gag and gag-pol polyproteins during
virion maturation. The activity of this enzyme is essential for capsid
finalisation and escape of virionnfrom cell)
• 5) KILLING OF THE INFECTED CELLS by lymphocytes or antoher
immune cells
TODAY 3 SHORT EXCURSION TO:
A – KILLING OF THE CELLS (nature immune raction against patogen)
B - FLOW-CYTOMETRY
(machine for T cell and another cell evaluation in patients)
C – IN SILICO design of ENZYME INHIBITORS (drug which stop not only the
HIV virus) IN SILICO versu IN VIVO realitě)
A – KILLING OF THE INFECTED CELLS
From last 3 lessons
you can know:
From last 3 lessons
you can know:
T cell
Cytotoxic T-CELL are the Most effective
„killing cells“. They can recognise the
infected cell, and they can start cell death
(by FAS ligand, or by perforins)
• Perforins casused
perforation of cell
mebranes and cell
death ane elimination
of virus replication
(dont forgot that
also some another T cells
exist: Th1 and Th2 )
Cytotoxic T Cells and T Helper Cells - Bing video
Very good overview of kinetic of CYTOTOXIC TCELL fight
against CANCER or INFECTED CELLS:
See the online movie:
Scheme of the T-lmyhpocyte perforin action
from textbook (Alberts 2003)
B cell
• B cells produce IgM antibodies.
IgM antibodies are pentamers, which have two main effect:
1) activation of another immune cells,
2) direct connectionm and neutralisation
of viruses and bacteria
Result: Result:
The IgM antibody is the largest antibody and the first to appear to fight off a new infection. The IgM is
basically the first line of immune defense against fighting pathogens. In inflammatory diseases, the
IgM can have both pathogenic and protective roles depending on the type of infection and tissue
affected. IgM is present in jawed vertebrates (gnathostomes) that existed already during the Devon
period over 400 million years ago. An IgM antibody test is very important because it generally comes
up earlier on an infection, and it is detectable 4 to 7 days after an infection starts.
Read more: Difference Between Antibody Test IgG and IgM | Difference
Between http://www.differencebetween.net/science/difference-between-antibody-test-igg-and-
igm/#ixzz7FEV3j9wQ
IgG antibodies are formed later than IgM antibodies and the test for IgG antibodies help determine
the immunity status following a virus infection or active immunization. It can also help to diagnose
persistent infection. So, IgG starts spiking as IgM starts coming down.
Read more: Difference Between Antibody Test IgG and IgM | Difference
Between http://www.differencebetween.net/science/difference-between-antibody-test-igg-and-
igm/#ixzz7FEUm5IGV
Also IgG antibodies, have similar function like IgM:
T lymph. / dendritic cell
Some microscopic photo of immune cells contact to patigen or cell-cell contact:
B -FLOW CYTOMETRY
• Flow Cytometry is a technique used to detect and measure
physical and chemical characteristics of a population of
cells or particles.
• In this process, a sample containing cells or particles is
suspended in a fluid and injected into the flow cytometer
instrument. Detectors detect if each one cell have CD8 or
another surface molecule. And computer compute how many
CD8 positive cells are in the sample and for example how many
CD4 and CD90 and CD 73 positive ….
CD4
CD4 is a T helper cell marker, which is a single chain
transmembrane protein. The extracellular structure
belongs to IgSF, and there are four IgSF domains.
The first and second domains can bind to MHC class
II molecules. CD4 acts as a co-receptor for the TCRCD3
complex recognition antigen and participates in
signal transduction by binding to the MHC class II
molecule, p56lek kinase.
CD8
CD8 is a cytotoxic T cell marker, a heterodimer
formed by the linkage of α and β chains by disulfide
bonds, and the extracellular structure is an IgSF
member.
CD4 and CD8 molecules divide T cells into two
distinct subpopulations. CD4 and CD8 are receptors
of MHC class II or MHC class I molecules,
respectively, and the changes in the number and
ratio of CD4+ and CD8+ cells reflect the immune
function status of the body.
Set of surface molecules. Specific antibody Antibody + fluorescence molecule
CD90
CD73
CD8
Detectors detect if each one cell have CD8 molecules
(GREEN FLUORESCENT)
Or CD 90 for example (in this case:
red fluroescence).
Modern multiparametric cytometry
(result presented on typical PC software protocol)
size of cell statistic: CD8 positivity statistic:
C –
IN SILICO inhibitors desing
At first step: atom 3D map of protease (or
another enzyme) had to be build:
At second step: computer asisted docking of molecules into the
active site of enzyme („looking for efective pieces of puzzle,
which is shape compatible with suroundig structure“)
• Effective inhibitors can „click“ to enzyme and „stop“ the enzyme
activity (for example they stop the protease which produce viral
capsid protein = HIV virus is not able to replicate in the cell and HIV
reproduction is stop)
• (after founding of good molecules IN SILICO, the IN VITRO and IN
VIVO test had to be started before use in practical human medicine)
The last note to BIOLOGY lessons: