1. health and fitness
2. therapy

QUANTITATION OF INTERFERON IN PLASMA OF
CANCER PATIENTS BY UTILIZING A
VERTICAL LIGHT PATH PHOTOMETER
A Senior Honors Thesis
Presented to
the Faculty of the Department of Biology
University of Hawaii at Manoa
In Partial Fulfillment
of the Requirements for the Degree
Bachelor of Arts with Honors
by
Leo K.L. Yotmg
November, 1988
.ABSTRACT
QUANTITATIOO OF IN'1'ERFEROO IN PLASMA OF CANCER PATIENTs BY
t1l'ILIZING A VERTICAL LIGHT PATH PHOTCMETER h Yotmq, University
Qf. Hawaii st. Manoa, Honolulu, HI. 96822.
Interferon (IFN) is :inqx)rtant in fighting virus and
microbial infections. IFN pretreated cells have the ability to
inh:ibit the growth of viruses and may also have i.Jrnu.maoodulatory
roles on natural killer cells and macrophages. Plasma in
selected patients (lung 1 breast 1 renal cancer 1 and non-hodgkin' s
disease) are analyzed for the ability to inhibit infections in
WISH (Human Amnionic) cells challenged with Vesicular Stanatitis
Virus (VSV). Patient sanples with high IFN activities exhibit
low VSV infections. The measurements are recorded in Optical
Density ( OD) readings using a vertical light path photaneter
{ELISA reader) and in Cytopathic Effects (CPE) readings. The OD
readings of Cell Controls (CC 1 WISH cells not treated with virus)
are ccmpared with Virus Controls (VC1 WISH cells treated with a
virus challenge with no IFN protection) . The mean OD for cc is
1 . 176 ± 0.214 (N=34) and VC is 0. 323 ± 0. 115 (N=32) . The
deviations in the CC and VC indicates reasonably reliable OD
reading in this assay. An inverse relationship is found between
the CPE and OD readings . The patient plasma IFN levels are
calculated by probit analysis using IFN standards made in each
assay. The mean standard IFN units found to provide 50% VSV
protection in the IFN titration was 0. 693 ± 0. 777 units/rnl1 (N=
3) . The log of the dilutions of patient plasma are found to have
a linear relationship with the log of the % protection fran virus
infections. Pre-stained CPE and Post-stained CPE correlated with
each other with a slope of 0.737 ± 0. 179.
The mean IFN units of cancer patients (4.86 ± 5. 1 units/rnl1
N= 4) is lower than the mean IFN units of the controls (29. 04 ±
37. 25 units/rnl1 N= 4) . SUpernatants of HSV-1 treated patient
effector cells haw a mean IFN leYel ef 3. 27 x 10"'7 units/ml as
canpared to 43. 6 units/rnl in the control. Because of the limited
sanple size in this study I no conclusive statements can be made.
Additional studies in rore patients are planned in the future to
provide rore data for staistical evaluation.
T.ABLE OF CONTENTS
List of Tables
i
List of Figures
ii
Abbreviations used in this Paper
I.
II.
iii
Introduction
A.
Topic Selection
1
B.
Patient Population
2
c.
Purpose and Scope
2
D.
History of Interferon Research
3
E.
Function of Interferon
4
F.
Types of Interferon and other BRM' s
5
Materials and Methods
A.
IFN Assay
7
B.
WISH Cells
7
c.
Cell CUlture and Media
8
D.
VSV Virus
8
E.
9
SUpemates of Herpes Simplex Virus-1
(HSV-1) treated. Patient Effector Cells
F.
Cell staining
9
G.
Data .Analysis
9
Probit Analysis M:xiel
11
IV.
TCID50 Measurements
13
v.
sample Calculations
14
Results
15
VII.
Discussion
17
VIII.
Conclusion
23
IX.
References
25
III.
VI.
I. Deficiencies of interferon (IFN) production and :their
possible origins
II.
Biological Response M:xiifiers
Transformation of percentages to probits
III .
OD of CC and VC Reproducibility
IV.
V.
CPE
VI.
vs
OD Readings
IFN units in Standards
VII.
Plasma IFN Levels of Hu-IFN-Alpha fran Normal individuals
and cancer patients
VIII.
IX.
Pre-stained CPE and Post-stained CPE Comparison
IFN Levels in Control Patient SUpemates
i
la.
Reciprocal of IFN Dilution
vs
Percentage ProteCtion
log of Reciprocal of IFN Dilution
1b.
Protection
vs
log of Percentage
2a. Reciprocal of Patient 307-6 Plasma Dilution
Protection
vs
Percentage
2b. log of Reciprocal of Patient 307-6 Plasma Dilution
Percentage Protection
ii
vs
log of
ABBREVIATIONS � lr! ll:lm, PAPER
Biological Response M:xtifiers
BRM,
cell control
cc,
COnA, Concanavalin A
Cytopathic Effect
CPE,
Dul.beco' s Minimum Essential MEdia
DMEM ,
DNA, Deoxyribonucleic Acid
Fetal calf Serum
FCS,
HBSS,
Hank's Basic Salt Solution
HSV-1,
Herpes si.nplex virus-type 1
Hu-Leu-IFN or Leu-IFN-alpha,
Human Leukocyte interferon
IFN, interferon, 3 major types:
IL2,
alpha, beta, and gamna
Interleukin 2
LAK, Lymphckine activated Killer Cells
NED , nonnal equivalent deviate
NK,
Natural Killer Cells
OD,
Optical Density
FBL,
per1pherai blOOd lymphOcytes
PHA,
phytohemagglutinin
TCID50, Tissue CUlture Infection Dose at 50% infection
VC,
VSV,
virus control
vesticular Stomatitis Virus
WBC, White blood cells
WISH,
Human Amnionic Cell Line
iii
INTRODUCTION
Topic Selection
This topic is chosen for the following reasons:
1)
IFN,
Interferon, aroong other Biological Response M::xiifiers ( BRM' s) is
making tremendous progress in cancer related research; 2)
an
IFN
study in patients could detennine in which disease does IFN play
an essential role for bodily defense; and 3)
Identification of
IFN deficiencies in patients may help clinical researchers to
continually develop new effect1ve therapies [3,38].
This project is significant to the field of clinical
immunology research because it relates the units of interferon in
a patient's body to possible correlation between any ailment to
interferon deficiencies [ 36] .
Furthenrore 1 low lymphokine
activated Killer Cell ( LAK) activities may have a direct
correlation to low IFN units since both involve similar
lymphocytes in their production.
Therefore I an IFN assay when
used in conjunction with a LAK assay may provide nruch 1nfonnat1on
about the patients' state of readiness on their immune system.
This assay may be used in similar assay protocol to test for TGF
(Tumor Growth Factor) or any
The procedure
was
other lymphokines measurements.
carefully developed to determine the best
concentrations of IFN standards and virus stock to be used for
the protocol.
I spent the early m::>nths establishing a w::>rkable
method �able to that of several published papers [ 7,14]
learning tissue culture techniques to take care of the cell line.
The current state of research throughout the world employs
1
similar methods in this paper [ 10,13, 14,29].
Patient Population
Native Hawaiians have the highest incidence of cancer
canpared to other ethnic groups .
This fact is �jnt�esting
because NK activities may be correlated with cancer cases [ 4]
•
If an inverse relationship exists between the two, we have a finn
reasoning to dete:rmine connections between Hawaiian's high rate
of cancer and low NK activities.
Dr. MacDonald of the Hawaii Department of Health
Epidemiology Division assisted my unsuccessful efforts in finding
anyone else actively involved with IFN research in Hawaii.
I
hope to establish a reliable IFN assay in Hawaii for IFN
-
research.
Purpose and Scope
Cells foreign to the body such as turror cells are recognized
by the body's lymphocytes:
and NK cells
namely macrophages, T cells, K cells,
These defense cells are activated by varjous
lymphokines such as interleukins (IL) and
IFN [ 5, 33,36] .
Therefore, an individual with turror is suspected to:
1) have
defective cells unable to interact with lymphokines, or 2)
IL deficiency or IFN deficiency, or both .
;
have
This paper examines
the possibility that cancer patients may have elevated IFN units
in their plasma samples due to the presence of turror in their
bodies, and exami.I?.es the IFN units in supernates of HSV-1 treated
patient cells to test for the cells' ability to produce IFN after
a HSV-1 infection.
Wheelock's report of "interferon-like"
2
substance in supernatants of human leukocyte culture treated with
PHA (phytohaemagglutinin) acting as an antigen [ 38] leads to the
speculation of using HSV-1 treated patient leukocytes to see
whether IFN is produced with such antigen.
This 'WOUld give us an
idea of how actively the leukocytes are participating in
producing IFN for the body defense system.
t��-lThis
experimental technique gives physicians a powerful tool
if\, . ,
til detennining possible IFN deficiencies in patients.
One could
correlate diseases with the imnune system breakdowns at the
cellular level where the lymphocytes could not be activated to
kill or to produce IFN upon stimulation.
History Qf. Interferon Research
A certain protein was noticed to protect chicken chorio­
allantoic membranes from influenza virus infections after the
cells were pretreated with heat-inactivated influenza virus
.
This
led to the discovery of interferon (IFN) by Isaacs and Lindermann
in 1957.
Many ncre discoveries on the potentials of IFN has been
made over the last 30 years such as its inmmoregulatory roles
and cell proliferation regulation roles on the lymphocytes [ 2, 3,
10-15] .
Before the technology of recanbinant DNA, research on IFN
was very limited.
Today, E. coli is genetically altered by
---
recanbinant DNA technology to produce IFN [ 34] .
IFN is a potent
substance, so only a small amount is produced thus only tiny
ancunts could be extracted.
To compound the extraction obstacle,
IFN is also labile and loses its reactivity when exposed to heat
for prolong period of time.
----- ·
Many separation methods have tried
3
to extract the maxinun anamt of IFN such ·as gel filtration,
precipitation, chranatography, electrophoresis, and highperformance liquid chranatography.
These methods are able to
extract a limited am:::>unts of purified natural leukocyte,
fibroblast interferons.
Purification of beta and alpha IFN in
the late 70's open many research opportunities in this growing
The advent of genetic recanbination helps to make IFN
field.
rrore accessible to the researchers.
Recanbinant DNA technology
makes human therapy possible using dosages carparable to dosages
possible only in animal studies before.
F\mction Qf. lm
Interferon was �irst characterized to inhibit virus growth
by Lindennann has been proven to be true over the years, but
-
there are diseases associated with IFN deficiency not necessarily
involving viruses (see Table I).
Methods of IFN application
include direct application on the skin to injection into the
blood stream,
IFN.
providing both prophylactic and therapeutic use of
Treatment in animal has given us sane ideas of IFN 1 s
ability to inhibit virus infections. IFN seems to be useful for
many facets of clinical usages, it is both a lymphokine and a
hotm:>ne.
Interferon function is so diverse that it is
interesting to note that inmmologists see IFN as biological
response rrodifiers (BRM 1 s), while virologists see it as antiviral
agents, and oncologists see it as· antiturror agents.
4
Types of
Interferon ,emi other BRM1s
The first type of IFN was discovered by Isaacs and his
coworkers in 1957 [ 24] .
At the time , they proposed ·that viral
and foreign nucleic acids sti.rm.ll.�m:>st body cells (fibroblasts
and epithelial cells).
In 1980, after many new discoveries of
different types of IFN, an international group of scientists met
tmder the sponsorship of the National Institute of Allergy and
Infectious Diseases and the WOrld Health Organization
-
US
National Center on Interferon and proposed a standardized
nanenclature of interferons.
Three major types of IFN were
named: alpha (also leukocyte interferon), beta (also fibroblast),
and ganma (also called irmume interferon, T interferon, type 2
interferon).
They all have differe."lt amino acid canposition, and
their origins are different.
Yet all of them have the same
underlying purpose in protecting cells from viral infections [23,
25].
Alpha and beta IFN are tanned classical interferon.
They
are differentiated by their origin. Alpha and beta can be
produced by
non-irmume system cells.
Beta IFN is produced upon
stimulation from foreign cells, virus infected cells, tumor cells
and bacterial cells which stimulate B lymphocytes, and
macrophages.
Ganma interferon 1 s sole source of production is
from the T lymphocytes. It has greater antitumor activity,
inm..tnosuppressive activity, and cell lytic effects than the other
IFN 1 s.
All IFN types protect cells against virus infections by
inhibiting nucleic acid or protein synthesis and not by direct
effect on virus.
IFN is also considered as a lymphok.ine.
5
Lymphokines are
non-antibody soluble mediators of cellular inm.mity produced by
B- and T-lYI'IPhocytes.
They can activate inflanmatory cells and
induce cellular proliferation.
.MJst :importantly, lYI'IPhokineS
exert regulatory control of the immme system.
IFN has a very general sensitivity.
Different stimuli can
induce the same IFN and that IFN can have nonspecific resistances
against a broad range of viruses.
IFN is only specific in the
sense that human interferon (HuiFN)
related families of organisms .
works only for closely
For example, HuiFN work for hl.mlail
and possibly m:>nkey cells but not roouse or chicken cells.
Biological Response M:xtifiers (BRM' s) are substances that
activate a certain part of the irrmme system for increased bodily
defense.
IFN is one such BRM, other BRM' s include interleukins,
TNF ('l'Urn.':>r Necrosis Factor) , TGF ( 'l'Urn.':>r Growth Factor), and many
other lYI'IPhokines.
However, BRH' s are not limited to only
proteins produced by the body though, they also consist of
rnitogens such as PHA and Concanavalin A (COllA) and
polysaccharri. des from plant extracts in its arsenal (Yn1mg, I·
-=--
Pal, B. 1987 tmpublished).
Table II shows the various BRM's.
6
.MATERIALS Mm METHOD
m,Assay
WISH cells (Hmnan amnionic cells) are grown as oonolayers in
flat-bottaned 96-well microtiter plates (Linbro).
The medilUll is
replaced with 0 .1 ml volumes of 2 fold dilution of plasma, with
an initial dilution of 1: 2.
In addition to control wells which
contains fresh DMEM with 2% FCS , a series of dilutions of the
human leukocyte reference interferon, Hu-Leu-IFN (X43620;
National Cancer Institute, Bethesda, MD) are included on each
microtiter plate.
After each incubation, the oonolayers are
drained on sterile gauze, and washed twice with Hank's Basic Salt
Solution (HBSS)
.
After 24 hr, the medilUll is renoved and replaced
with fresh medium containing vesicular stanatitis medilUll (VSV,
Indiana strain) in DMEM + 2% FCS .
The plate is then incubated
for about 24 hr or until approximately +3 to +4 cytopathic effect
( CPE) is de�TDnStrated by the unprotected cell (VC) oonolayer.
At
that time the cultures are observed under an inverted-scope for
CPE readings.
The plate is rinsed, fixed, and stained with a
crystal violet/formaldehyde solution.
The oonolayers are
carefully washed with tap water to rem:>ve excess stain.
A second
CPE reading is done after the staining procedure.
� Cells
The WISH cell line (American Type CUlture Collection CCL
225, Bethesda, MD) is derived fran a human amnionic cell line and
is stored in a -100 degree c freezer. The cell culture is
maintained in 25-crn""2 flasks (Corning) containing
7
Dulbeco' s
Mi.nim.mt Essential Heditun (DMEM) supplemented with penicillin (100
U/ml), streptomycin (100 ug/ml), and 10% fetal calf serum (FCS;
Giboo Laboratories).
rredi�
� CUlture and Media
The growth
made of Dulbeco ' s M:inimJm Essential
Media (DMEM) containing 10% heat-inactivated fetal calf serum
( DMEM-FCS 10%) with 100 units of penicillin per ml, 100 ug of
streptomyci."'l per ml, 2rnM glutamine, and 20mM HEPES.
Maintenance
Media is used after the first day of experimentation to sustain
the cells but do not prarote continual proliferation-of the--·-f.,& I'­
rronolayer.
It contains the same ingredients as the growth
medii
I
. \);> (I)>Jv: :. : � •
excep� that there is only 2% FCS instead of 10% Qf' the growth
1
medii I
Each ingredient has a specific purpose for its inclusion,
.a.
DMEM provides the essential salts for normal cell sustenance.
The Fetal calf Serum ( FCS) brings proteins for the manmalian cell
growth, L-Glutamine is needed for tryptophan synthesis, pyruvate
is an important component of Kreb cycle, and finally penicillinstreptarrfcin are added to Y�ll off any extraneous bacteria.
vsv Virus
Vesicular Stomat1tis Virus (VSV} Indiana Strain (Whitaker
pharmaceutical) , is found mainly in livestocks .
This virus
mfects WISH cells and is sensitive to IFN-alpha [27] .
The
viruses are proliferated in vero cells and stored in aliquots at
-80 c .
8
Supernates Qt Herpes Simplex Virus-1 (HSV-1) treated
Patient E£fector Qells
Patient effector cells are plated in 96-well microtiter
plates and treated with HSV-1 virus then incubated for 24 hours.
The plates are spun at 1200 rpm for 10 minutes in a centrifuge.
Then the supernates are collected and stored in 1 rn1 eppendorf
tubes.
�Staining
A Crystal violet-formaldehyde stain is used to stain the
cell monolayer.
540
nm
and
can
It has a light absorption wavelength optin'aJm at
be read on the ELISA reader.
of 25% Crystal Violet, 50%
EtOH, 5%
The stain consists
Formaldehyde, and 0.85%
NaCl.
Data AnalysJ.s
Optical density {OD) reading acquisition is carried out on
an IBM-PC compatible computer interfaced to an ELISA reader.
OD
re�dings from the ELISA reader are analj"Zed in a 2 step process:
flist, OD readings are processed through the Q and Student's T
test on data evalua�ion to eliminate any aberrant values.
The 3
cell control (CC) and virus control (VC) wells are each averaged
and a mean value of OD for each assay is used to calculate the
range of OD readings.
The range is used to define the OD at
I
�
which 50% protection by IFN is achieve.
Each assay dilution is
perfonned in triplicate to yield 3 sets of OD values.
The mean
OD values of ecch dJ.lution is used to calculate the % protection
relative to the cell and virus control derived range {see eq. 1) .
The % protect� on and the % standard IFN protectJ.ons
0
are conver-: �:.·
into probit m.unbers and processed in probit analysis.
The IFN
standard determines the IFN units/ml. at 50% protection, then the
patient's dilution is canpared with the IFN standard to measure
the IFN level of the patient.
In each IFN reference, the precise
conditioP.s of IFN assay is strictly defined by using an NIH
standard reference IFN.
10
PRQBIT ANALYSIS M)I)EL
PhYSical � can be exeRi>lified by
R
at the
measurement of an unknown 'Weight with a �t__gf s�ed
'Weight.
The reproducibility of the results is consistent and is
not questioned.
Biological Assays, however, need to take into
consideration the variability in the reaction of the test
subjects and consequent inp:>ssibility of reproducing at will the
results in successive trials.
A biological assay makes canparisons between the strengths
of alternative but similar biological stimuli .
In its widest
sense 1 the term means the measurement of the potency of any
sti.mulus, physical, chemical, biological, physiological or
psychological, by means of the reactions that it produces in
living matter.
The biological method of measuring the stilraJlus
is adopted whether for lack of any alternative, or because an
exact physical or chemical measurement of stinu.ll.us intensity may
need translation into biological 1mits before it
Call
be put to
practical use .
Estimation of the potency of a natural product I such as a
drug extracted from plant material, in producing a biological
effect of a certain type, is often impossible or impracticable by
chemical analysis.
·
Even if the chemical constitution of the
material has been determined, there may be little knowledge of
the magnitude of the effect which the constituents will produce.
The difficulty is not confined to natural products but occurs
also with many manufactured compounds, such as aspirin where the
11
same dose to different subjects cause varied dose response
intensity.
The ca1iX)UDd to be tested can be made to precise
chemical specifications but there is still tmeertainties due to
Therefore, the IFN in this
unknown biological activities.
experiment must in fact be tested and standardized by a method
appropriate to its future use.
1-bdern statistical techniques began with Gaddurn in 1933.
proposed to
He
measure the probability of response on a transfonned
scale, the normal equivalent deviate (NED).
Bliss later
suggested in 1934 a slightly different response metameter.
He
defined the probit of P ('probit' =probability unit) [8, 9, 16).
The Probit table is shown in Table III.
In practice,
results should finally be expressed by median effective doses,
relative potencies, tolerance variances, or other suitable
quantities.
Researchers routinely speak of the 50% effect in the
experiments.
The OD readings are first converted into % protection with
the following formula:
(sample OD)
cc
-
-
VC
=
%
protection
vc
eq. 1
Probit Analysis is a linear regression using the help of the
probit table with nunbers corresponding to certain percentages.
The % protection is matched up on this list and the probit number
is recorded
as
the y value while the x value are the log of the
--
reciprocal of the dilutions.
12
TCID50 MEASUREMENTS
Virus particles are too small to be
such as number of viruslml solution.
meas ured
in quantities
To measure the
am::lt
lm
of
virus present in a dilution, a scale is made with an arbitrary
determination that one ' virus unit' would cause an infection in a
nonolayer of cells.
SUch a unit is tenned. a tissue cu1 ture
infection dilution at 50% infection (TCID50).
titrations
The virus
are prepared in each assay plate.
A calculation of TCID50 in the set up of the experiment is
as dem::mstrated below:
Virus
ratio of culture
rumul.ative
Cl.UliUlative
dilution
wells/inoculated
infected
not infected
ratio
10"'-5
3 I 3
6
0
6/6
100
10"'-6
3 I 3
3
0
313
100
10"'-7
0 I 3
0
3
01 3
0
percent
To interpolate the dilntiOD where 50\ illfecti.oll:
infectivity above 50% - 50%
------ =
infectivity above 50% - infectivity below 50%
100 - so
=0. 5
100
eq. 2
Fran the above equation, the calculation of TCID50 is the
average of the log (10"'-6) and log (10"'-7) which gives a dilution
of 6. 5 per dilution of 0. 1 ml.
The TCIDSO is 5. 5 per m1 for the
vc.
13
SAMPLE CALCtJLATIC!iS
A semple calculation to show how the I:m units are measured:,
•
Assay 220B
cc
=
vc
=
1. 268
0. 312
cc - vc = 0. 956
LOO OF
IFN (UNIT/WELL)
12
6
3
1
0. 5
% PROl'ECTIC!i
76
71
79
76
59
RECIP DILU
0. 602
0. 903
1. 204
1. 669
2. 000
PROBIT
5. 71
5. 55
5. 81
5. 71
5. 23
A linear regression was done with the log of the reciprocal
of dilution
vs.
the probit and the dilution best fitted to a
probit of 5. 0 (50% protection) was detennined to be 1. 59
units/ml.
To calculate the IFN level of a patient, the patient semple
provided the WISH cells the following protection:
CONTROL 307-6P
RECIPROCAL OF
DILUTION
2
4
8
16
% PROTECTION
L03 OF
PROBIT
-·tt>
RECIP DILU
0. 301
0. 602
81
69
5 88
1. 204
31
4. 19
5. 50
The result of the linear regression performed on the patient
results where the dilution factor gave a 50% protection was
multiplied by the IFN standard level obtained fran the result
above and further standardized to give the I:m level of 13. 8
units/ml.
14
e ; �· :;>
- .s (..
;
f\
-:.
-
q1
RESULTS
AsSay Reproducibility
The mean OD reading at 540
nrn
for Cell Control (CC), (N=34)
is 1.176 ± 0. 214 and for Virus Control (VC),
(N= 32) is 0. 323 ±
0.115 {see Table IV).
CPE�m
CPE reading took approximately 20 minutes to read the \\bole
plate while OD reading took 2 minutes to read the plate.
The CPE
readings range fran zero {no infection) to 4+ (100% infection)
wi. th increments of 0. 5.
The OD readings and the CPE readings
were inversely related wi. th a slope of -0. 26 ± 8. 995 (see Table
V)
•
.mi actiy:i.ty in Standard gng Patients
Table VI shows the IFN units needed by the standards to
establish a 50% protection on the WISH cells.
Patient sarrples
providing 50% protection to VSV infection is analogous to IFN
level to the 50% protectioo �Qe<i by the IFN stand!lrd.
results of experiments are listed in Table VII.
'fhe
Patients wi. th
ltmg cancer and renal cancer have IFN units CClll'Pai'able and
possibly higher than that of the controls.
Patients diagnosed
with breast cancer and non-hodgkin's disease have IFN units
substantially lower than the control IFN level.
The OD readings representative of its antiviral effect is
converted to
% protection and plotted as a ftmction of the
reciprocal of the sarrple dilution.
Fig. 1a and 2a
shows the
percentage protection produced by each sample dilution after
15
incubatioo.
Fig. lb and 2b s� the data plotted as the log
I
percentage protection as a function of log reciprocal dilution of
patient sar�i>le, a linear relationship can be seen.
The 50%
protection point can be linearly extrapolated fran such a curve
[ 14] .
Five cancer patients (4. 86 ± 5. 1 units/ml) and five nor:mal
controls (29. 04 ± 37. 25 units/ml) lolei"e analyzed for their IFN
units by measuring the OD readings by percent protection against
virus infection.
cancer patients have plasma IFN units lower
than that of the controls.
Pre � fQa.t � Cgnparison
CPE measurements are made before and after staining prior to
the OD readings.
Since 4. 0 CPE means all of the WISH cells are
infected and thus are expected to be dead.
Wells with higher CPE
readings have more readings with tissue gone ( TG) .
The b«> CPE 1 s
carrparison result in a slope of 0. 737. The results are reported
in Table VIII.
IFN Levels in Patient SUpernates
The supernate taken from patient 1 s effector cells treated
with HSV-1 are tested in similar ways as the plasma.
Effector
cells of normal patients treated with HSV-1 virus retains their
ability to produce IFN.
The supernates of cancer patients are
tested to see whether they exhibit the same level of IFN units as
the nonnal controls.
The mean IFN level of the HSV-1 treated
supernate is 3. 72 x 10"'7 units/ml, substantially higher than the
measurements of plasma IFN {see Table IX).
16
DISCUSSIOO
The presence of IFN in patient samples WJU.ld provide
interferences to viral replications, and in tum nore WISH cells
w::ruld survive the virus challenge.
The IFN protection mechanism
does not act directly on the virus particles, it activates
antiviral activities on the cells [ 15] .
Protection fran virus
infections is a cellular property of cell surface receptors.
Briefly, it involves the production of IFN by an infected cell
and attaching onto the surface receptors of neighboring
lynphocytes.
This causes a confonnational change that interrupts
virus replication while also activating defense cells such as NK
cells or macrophages.
NK activity [ 29] .
IFN is known to have a regulatory role to
Once botmd to receptors, it acts in a
classical pathway of hornone action lrodulating intracellular
concentration of cGMP and cAMP [ 37] .
The infected cell is not
expected to survive the ordeal, but it serves to protect other
cells from its own death.
Transmitted light is described by the ratio of light
emerging to light entering a solution.
This may be described in
terms of percent transmission or by units of optical density
( OD) .
Percent transmission is the negative logarithm of the
ratio between light emerging and light entering the solution, OD
is 100% - (% transmission).
Note that the highest percent
transmission is found when no light absorption takes place.
The
ELISA r�ader reads (at its optimum absorption wavelength of 540
nm) the OD of the crystal violet fonnalin stained wells
vertically .
The OD readings of the cell control (CC) wells
17
serve as the basis of canparison.
When nore dye remains in the
wells of patient scurples relative to the control 1 there will be a
higher OD reading than the control.
When fewer dye remains in
the patient scurple wells relative to the control 1 a lower OD
reading is recorded.
� of � and VC Readings
All wells contained 2. 0 x 10"' 4 WISH cells at the beginning
of the experiment.
cc received no VSV challenge while all other
wells received a VSV challenge of 6. 5 TCIDSO/well.
When the cc
were stained I all the cells should grow nonnally and w:W.d take
up stain thus picking up the most stain.
Whereas the VC wells
would have the most virus infected cells and \olOuld have less
cells that are able to absorb the stain during fixing.
The
difference between the two controls seiVes as the divisor of the
% protection fonm.Ua seen il1 eg. 1.
Table IV illustrates the average OD readings of CC and vc
wells.
With the given definition of the cc and VC 1 the CC wells
provided the upper lirnits and the VC provided the lower lirnits of
the OD readings.
Although
�
assay �lated with the same
amounts of cells I the cells may have been in different growth
phases.
Some assays plated with WISH cells in the log phase may
have OD readings above 1. 0 while wells with OD readings below 1. 0
may have plated cells in the stationary phase.
of IFN :r:�- f' --: �-
\.'2 �
done on
an
The measurements
assay by assay process.
This
process preserves the reliability of the assay because its
percent of virus protection is based on OD readings within the
same assay.
A relatively higher cc reading plated with log
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phasic cells are offset by higher patient plasma treated wells
and higher VC wells as CCJJpared to assays using WISH cells in
stationary phase.
Nonetheless, both the CC and VC readings
showed consistent range of readings.
� � Ql2.
CPE readings have only 9 designations: 0, +0. 5, +1, +1. 5,
+2, +2. 5, +3, +3. 5, and +4.
With the data obtained, we could
make out a CC�iti>arative picture of the IFN-alpha characterization
in CPE and OD readings.
The CPE cotmting method took about 15-20
minutes per plate and involves a high degree of subjectivity. The
quantitation method requires just 5 minutes of adding the stain
solvent and 1 minute of reading the plate on the ELISA reader
with no eye strain of the experimenter.
Also the ELISA reader
gives objective data through the use of optical density reader .
m. Measurements
The first titers of IFN standards made included
concentrations of up to 5000 units of IF'N per ml.
The results of
the earlier experiments showed a bell type curve where cells with
IFN units over 1000 were dying just the same as cells receiving
little or no IFN protection.
the duel effect phenomenon.
It was first suspected to involve
The dual effect phenanenon is seen
when IFN of 25-2500 u, provide increased Antibody production
stimulation on spleen cells from mice but inhibits further
antibody production at 5000 to 10, 000 u of IFN [ 3] .
A
ncrphological examination showed the cells dying in the high IFN
titers were not dying because of VSV infections .
19
Their
norphology showed they were dying because of sanething else in
the well, too nuch IFN.
The cells showed signs of cytotoxicity
by the shriveling cell membrane and irregular cell growth.
Reduction in the IFN dose were made and IFN unit calculations
presented a lTDI"e definite probit curve .
However, one high titer,
1000 units/ml, is still being included to later assays to provide
a limit of IFN toxicity.
The plasma of cancer patients were tested for protection
......
against VSV infections.
This protection is analogous to IFN
activity because of the IFN ' s antiviral property against vsv .
The mean IFN units of the controls was ruch higher than the mean
of any cancer types tested (see Table VII).
Note there were
unusually high standard deviations to the mean control IFN units.
This exemplified the wide range of IFN units even in control
donors .
Forti (1986) reported IFN level results where the
standard deviation is greater than the mean and cancer patients
had canparable IFN units with the controls [ 14] .
Preliminary
results did not show a correlation between IFN units of cancer
patients with IFN deficiency.
But the limited assays prevented
any definitive conclusions on t;he correlation between the two .
�
Also, the stage of the cancer disease also need, to be considered
in each patient.
Pre-Post CPE Cgnparison
Coolparison between pre-stained and post-stained CPE values
were made.
In keeping the prestained cells as a constant, the
two wells can be canpared using the least square fit foiTm.ll.a.
In the CPE, a problem may arise if the experimenter did not
20
rinse the plate thorough enough and leaving residual dye to
darken a well or sprayed water on too strongly sloughing off sane
cells that WJUl.d have . otherwise stayed if not for the turbulence.
HSV-1 treated 9Ul, SUpernate of Patients
Inmune canpromised illnesses present a difficult situation
for the clinician.
In ordinary illnesses, the lYlTQ?hocytes in the
inmune system is activated to defend the body fran foreign
�
invaders utilizing BRM 1 s such as IL2 and IFN-ganma. The 2 major
mechanisms used for this defense are the llunoral and cellular
mechanism.
mechanisms.
The interferon system are involved in both
IFN is established as a regulatory factor for NK
activity [ 19] .
Inm..m e deficient patients lack this defense
either in part or in whole (such as in AIDS).
Opportunistic
organisms may infect the body that otherwise \loOuld have been
eliminated by the body 1 s natural defense.
Kaposi 1 s sarcana are
-- -
contracted by nostly young horrosexual males with AIDS and organ
transplant patients.
There is evidence that turror growth is
related to a breakdown in the i.mm.me system because in instances
where imm.mosuppressed transplant patients whose turrors can
regress spontaneously when inmunosuppression is lifted.
Because
IFN has antiviral and antitumor activities, it could serve as a
therapeutic agent in those patients who \loOuld not easily tolerate
conventional cytotoxic chenotherapy [ 36] .
In Table IX, the HSV-1 treated cell supernate of patients
are tested.
The supernate exhibited a ruch higher IFN level than
the plasma.
HSV-1 treatment would cause normal lyrrphocytes to
produce IFN while IFN deficient individuals may lack such
lyrrphocytes thereby not producing nn.tch IFN.
21
The results would
indicate whether an individual's lyrrphocytes can respond to
stimulation to produce IFN
.
The IFN units in supernate is higher than the IFN units in
plasma.
The HSV treatment nust have caused the cells to produce
IFN during incubation and thus m::.>re IFN was released in the
supernate.
22
cmcr,usxoo
IFN is i.oix>rtant in the body's defense system. · It is
i.oix>rtant to evaluate the immunological system as a whole which
serves as the body's front line defense in antiviral protection.
Along with other known factors that augment inmune responses such
as LAK activation with IL-21 IFN becanes a vital part of the
immunological team.
pa.§t
because past
Studying IFN was difficult in the -=--
;;;..----
protocols depended a lot on subjective readings and vulnerable to
This bioassay1 using a photaneter 1
possible observer bias.
eliminated many of the disadvantages of the past such as observer
bias in plaque reading I and a protocol requiring many researcher
hours.
The anount of assay time was about the same in this
assay I but the
,time
in this assay are nostly just waiting time
whereas the past assay required teclmical work on the experiment
·
during the same arrount of time.
The assay CC and VC readings showed same stability among the
cc and the VC counts.
This showed the reasonable dependability
on the assay to provide adequate upper and lower range OD
readings.
M::>re cc and VC readings will be collected in the
future experiments to detenn:i.ne a 'true' reading.
'----The comparison between OD readings and CPE readings showed
little or
no
correlation.
The OD readings are quite consistent
at above 0. 900 1 s while CPE has increase.
IFN units was examined in 4 types of cancer patients I nonhodgkin 1 s disease I lung I breast I and renal cancer I
and controls.
All four groups reported IFN units within the standard deviation
23
of the other groups .
No correlation is made bet.\een IFN
activities and cancer patients because only a limited number of
assay were done.
----- �-
The HSV-1 treated patient effector cell
supernates of two control individuals were tested for IFN
activity.
The results showed
a
significant anotmt of IFN
presence in the HSV-1 treated supernates.
This suggests that
ability of nonnal individuals have lymphocytes that are capable
of producing IFN after HSV-1 sti.nnla
l tion.
It is hoped that nore
testing could be done to correlate cancer patient plasma and HSV1 treated supernate with IFN presence.
In this study, I found that an assay is very challenging to
reproduce even with the most descriptive instructions.
However,
I was able to produce sane results of significance in that the
basis of the IFN measurements can be established and further
studies can be done with relative confidence.
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