close

Вход

Забыли?

вход по аккаунту

?

IMMUNOLOGICAL EFFECTS OF MICROWAVES, ULTRASOUND, AND HYPERTHERMIA: B-LYMPHOCYTE CAPPING

код для вставкиСкачать
INFORMATION TO USERS
This reproduction was made from a copy of a document sent to us for microfilming.
While the most advanced technology has been used to photograph and reproduce
this document, the quality of the reproduction is heavily dependent upon the
quality of the material submitted
The following explanation of techniques is provided to help clarify markings or
notations which may appear on this reproduction
l.The sign or "target" for pages apparently lacking from the document
photographed is "Missing Page(s)". If it was possible to obtain the missing
page(s) or section, they are spliced into the film along with adjacent pages. This
may have necessitated cutting through an image and duplicating adjacent pages
to assure complete continuity.
2 When an image on the film is obliterated with a round black mark, it is an
indication of either blurred copy because of movement during exposure,
duplicate copy, or copyrighted materials that should not have been filmed For
blurred pages, a good image of the page can be found in the adjacent frame. If
copyrighted materials were deleted, a target note will appear listing the pages in
the adjacent frame
3. When a map, drawing or chart, etc , is part of the material bemg photographed,
a definite method of "sectioning" the material has been followed. It is
customary to begin filming at the upper left hand corner of a large sheet and to
continue from left to right in equal sections with small overlaps. If necessary,
sectioning is continued again-beginning below the first row and continuing on
until complete
4 For illustrations that cannot be satisfactorily reproduced by xerographic
means, photographic prints can be purchased at additional cost and inserted
into your xerographic copy These prints are available upon request from the
Dissertations Customer Services Department
5. Some pages in any document may have indistinct print. In all cases the best
available copy has been filmed.
University
Microfilms
International
300 N ZeebRoad
Ann Arbor, Ml 48106
8302997
Sultan, Michel Farid
IMMUNOLOGICAL EFFECTS OF MICROWAVES, ULTRASOUND, AND
HYPERTHERMIA: B-LYMPHOCYTE CAPPING
University of Illinois at Urbana-Champaign
University
Microfilms
I n t 6 r n a t l 0 n a l 30ON ZeebRoad.AnnArbor,MI48106
PH.D. 1982
PLEASE NOTE:
In all cases this material has been filmed in the best possible way from the available copy.
Problems encountered with this document have been identified here with a check mark V
1.
Glossy photographs or pages
2.
Colored illustrations, paper or print
3.
Photographs with dark background
4.
Illustrations are poor copy
5.
Pages with black marks, not original copy
6.
Print shows through as there is text on both sides of page
7.
Indistinct, broken or small print on several pages
8.
Print exceeds margin requirements
9.
Tightly bound copy with print lost in spine
10.
Computer printout pages with indistinct print
11.
Page(s)
Is
lacking when material received, and not available from school or
author.
12.
Page(s)
seem to be missing in numbering only as text follows.
13.
Two pages numbered
14.
Curling and wrinkled pages
15.
Other.
. Text follows.
University
Microfilms
International
• IMMUNOLOGICAL EFFECTS OF
MICROWAVES, ULTRASOUND, AND HYPERTHERMIA:
B-LYMPHOCYTE CAPPING
BY
MICHEL FARID SULTAN
B.S., University of Illinois, 1977
M.S., University of Illinois, 1979
THESIS
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy in Electrical Engineering
in the Graduate College of the
University of Illinois at Urbana-Champaign, 1982
Urbana, Illinois
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
THE GRADUATE COLLEGE
June 1982
W E HEREBY RECOMMEND T H A T T H E T H E S I S BY
MICHEL FARID SULTAN
F.MTTTT/F.n
IMMUNOLOGICAL EFFECTS OF MICROWAVES, ULTRASOUND, AND
HYPERTHERMIA:
B-LYMPHOCYTE CAPPING
BE ACCEPTED IN PARTIAL F U L F I L L M E N T OF T H E REQUIREMENTS FOR
THE D E G R E E OF DOCTOR OF PHILOSOPHY
(^Ji^fi^Xiu^ LLLtt*^".
J
Committee on Final Examinationf
LJ^MXA)LC4.
LAW-COUI^
Chairman
f^njey^M- CvtM,'kto
. ^ g X ^ a t - ^ t f W i MZsf
^eS^r*-^.
—b~?—'
^
t Required for doctor's degree but not for master's
0517
-
Director pi Thesis Rese:
Head of Department
iii
A la memo ire de mon pere
iv
EXPRESSION OF APPRECIATION
I
wish
Charles
to express my deepest gratitude to my advisor, Dr.
A.
dedication
Wayne A.
Cain,
to
F.
this
for
his
study.
valued
I
and
continuous
especially grateful to Dr.
Tompkins for his helpful counsel and expert advice
throughout this investigation.
other
am
guidance
Many thanks are also due to
all
staff members of the Bioacoustics Research Laboratory and
Oncology
Laboratory
who
have
been
willing
to
share
their
experience and knowledge.
Finally, I express my deepest thanks and appreciation to my
family and friends.
Their continued encouragement, support, and
help during the past years will take a lifetime to repay.
This investigation was supported in part by the
Office
of
Naval Research Contract NOOO14-79-C-0336, by PHS Grant number CA
20930 awarded by the National Cancer Institute, DHHS, and by PHS
Grant
number
AI
16793
awarded
Allergy and Infectious Diseases.
by
the National Institute of
V
TABLE OF CONTENTS
CHAPTER
I.
II.
Page
INTRODUCTION
1
GENERAL REVIEW
3
A.
HYPERTHERMIA
3
B.
MICROWAVES AND ULTRASOUND
9
l
C.
III.
IV.
BIOLOGICAL BACKGROUND
18
MATERIALS AND METHODS
29
RESULTS
39
A.
EFFECTS OF HYPERTHERMIA ON CAPPING OF
ANTIGEN-ANTIBODY
(Ag-Ab)
COMPLEXES ON
THE SURFACE OF B-LYMPHOCYTES
B.
39
EFFECTS OF HYPERTHERMIA ON ANTIBODYCOMPLEMENT (Ab-C)
CYTOTOXICITY AGAINST
B-LYMPHOCYTES AND ITS RELATION TO
CAPPING
C.
46
EFFECTS OF MICROWAVES ON CAPPING OF
A g - A b COMPLEXES ON THE SURFACE OF
B-LYMPHOCYTES
D.
56
EFFECTS OF AMPLITUDE MODULATED RADIO
FREQUENCY (RF) ON CAPPING OF Ag-Ab
COMPLEXES ON THE SURFACE OF B-LYMPHOCYTES
E.
. . .
60
EFFECTS OF ULTRASOUND ON CAPPING OF
A g - A b COMPLEXES ON THE SURFACE OF
B-LYMPHOCYTES
62
vi
V.
DISCUSION
72
A.
HYPERTHERMIC INHIBITION OF CAPPING
72
B.
HYPERTHERMIC ENHANCEMENT OF ANTIBODYCOMPLEMENT CYTOTOXICITY
C.
EFFECTS OF MICROWAVES AND HYPERTHERMIA
ON CAPPING
D.
74
77
EFFECTS OF AMPLITUDE MODULATED
RADIOFREQUENCY AND HYPERTHERMIA
ON CAPPING
E.
F.
APPENDIX.
REFERENCES
VITA
78
EFFECTS OF ULTRASOUND AND HYPERTHERMIA
ON CAPPING
80
SUMMARY
81
FURTHER COMMENTS ON METHODOLOGY
86
89
105
1
I.
Microwave
number
of
and
ultrasound devices have found an increasing
industrial,
applications
INTRODUCTION
over
the
medical,
consumer,
past decades.
and
Microwave and ultrasound
hyperthermia are presently being used to treat
become
a
widely
used clinical procedure.
has been a growing concern in both
communities
regarding
the
ultrasound radiation on
immune
system.
and
may
Consequently, there
public
and
scientific
deleterious effects of microwave or
biological
systems,
particularly
the
immuno-effects
These will be reviewed in the nest chapter.
Originally, these phenomena
result
the
cancer
Various microwave or ultrasound
have been reported.
military
of
the
heating
radiation,
but
many
were
considered
properties
scientists
to
be
a
direct
of microwave or ultrasound
are
now
convinced
of
the
existence of non-thermal field specific effects.
The present study was undertaken to evaluate the effects of
microwaves and ultrasound on
(Ag-Ab)
complexes
on
mouse B-lymphocytes.
after
specific
the
the
antibodies
bind
distributed
on the cell surface.
to
membrane-bound
antibodies, the receptor-antibody
under
normal
radiation) into a polar cap on
membrane.
antigen-antibody
Capping is an active process which
these
regrouped
of
surface of freshly isolated splenic
Originally,
and
capping
cell
membrane receptors.
receptors
are
uniformly
Following binding of specific
complexes
conditions
a
occurs
small
are
redistributed
(37°C,
no
drugs, no
portion
of
the
cell
The remaining larger portion of the membrane is thus
devoid of any of that particular receptor.
Before assessing
the
possible
effects
of
microwave
or
2
ultrasound radiation, it was necessary to determine whether heat
alone
had
any
effect on capping.
A series of experiments was
done for that purpose in the temperature range
43 °C.
Possible
37
and
thermal and non-thermal field specific effects
of microwaves or ultrasound were later
experiments
between
where
decoupled
by
designing
both irradiated and control cell suspensions
were maintained at the same regulated temperature.
While the exact immunologic role of
been
capping
not
yet
fully determined, it has been suggested that, for the case
of cell surface immunoglobulin (Ig) molecules,
the
has
triggering
has been
receptors
signal for B-lymphocyte proliferation.
speculated
may
that
capping
and
shedding
of
may
be
Also it
membrane
help the antigen bearing cells escape the immune
surveillance system; such a phenomenon
cells.
capping
may
occur
with
cancer
Any thermal or non-thermal effect on capping would then
seem to have important consequences on immunity.
3
II.
II A.
AI.
GENERAL REVIEW
HYPERTHERMIA
General biological effects oj. hyperthermia
The last two decades have witnessed an
in
increased
interest
hyperthermia and its possible application as a therapeutical
agent against cancer (1-3).
reported
the
effects
In
1967,
Cavaliere
et
al.
(4)
of perfusion hyperthermia on 22 patients
who had malignancies of the extremities.
The temperature in the
tumors was raised to 41.5
several
hours
blood
free
of
any
disappearance
of
the
regional
perfusions
- 43.5°C
with
chemotherapeutical agents.
tumor
was
observed
for
prewarmed
A
complete
in
25
in 10 out of 22 patients and a significant
decrease in tumor volume was observed in 5 out of the
remaining
patients.
Pettigrew (1974) treated 82 cancer
repeated
exposures
Clinical
with
to hyperthermia at 42 °C (5).
was well tolerated as long as the
41.8°C.
patients
and
temperature
several
The treatment
was
kept
below
radiologic evidence of tumor regression
was often obtained, but no
long-term
tumor-free
survival
was
reported.
At present, the mechanisms of hyperthermic cell killing are
poorly understood.
Many experiments
have
been
conducted
and
many theories are suggested, but none of them can be regarded as
conclusive.
Some of the best documented results show that
selective
heat
sensitivity
cells are apparently more
of
heat
there
cancer cells (4, 6-8).
sensitive
than
normal
is
a
Cancer
cells.
4
Cavaliere
(4)
reported
that
the
oxygen
hepatoma and Ehrlich a s c i t e s carcinoma
less
at
42°C
uptake
cells
of
was
Novikoff
considerably
than at 38°C, whereas for normal c e l l s
there was
l i t t l e d i f f e r e n c e in r e s p i r a t i o n at these two temperatures.
Different phases in the
sensitivities
to
cell
hyperthermia.
reduction caused by a constant
cells,
CHO
Bhuyan
cells
et a l .
were
(mitosis)
or
similar
dose
sensitive
S-phase
were the l e a s t s e n s i t i v e .
showed
By
show
determining
of
heat
than
cells
different
the
on
survival
synchronized
in
(DNA S y n t h e s i s ) .
Other
studies
on
the
M-phase
G^ and G, c e l l s
HeLa
cells
(9)
r e s u l t s , with the S and M phases b e i n g the most
heat s e n s i t i v e r e l a t i v e to c e l l s in other
cycle.
also
( 7 ) showed that the mid and l a t e S-phase
more
early
cycle
phases
of
the
cell
In c o n t r a s t , the S phase i s the most radio r e s i s t a n t
in
X-ray treatment.
Harris (10) was the f i r s t
to q u a n t i t a t e hyperthermic
killing.
His h e a t - s u r v i v a l curves show an i n i t i a l shoulder followed by an
exponential
decline
of
the
surviving f r a c t i o n .
These curves
show that there i s an accumulation of heat damage at
that
results
in
cell
killing.
correspond
to
the
exponential
accumulated a l l the damage
damaging
event
would
then
they
be
doses
At these sublethal doses, the
damage i n f l i c t e d by heat can be repaired.
that
low
can
At the
decline,
tolerate,
irreversibly
higher
doses
the c e l l s have
and
a
lethal.
single
Similar
r e s u l t s were obtained by Parlzer and Heidelberger on HeLa
cells
(9).
In 1963, Crile (11) found t h a t the d e s t r u c t i v e
effects
of
heat on tumors implanted on the f e e t of mice began at 42.°C.
He
5
also
found
that
the
increase in temperature above 42°C which
halves the time required to elicit the
same
lethal
damage
is
1.0 °C.
Other related studies show that hypoxia (poor
increases
the
oxygenation)
thermal sensitivity of cells (12, 13).
Schulman
(12) found that under hypoxic conditions,
killing
of
cultured
mammalian cells by hyperthermia started at
41°C whereas 43°C was
necessary to produce substantial cell killing.
The environmental acidity is another determining factor
the
hyperthermic
effect
(13,14).
Gerweck
(14)
in
observed an
enhanced cell killing when the cells were exposed to reduced
pH
and elevated temperature simultaneously.
It is not yet known how
Several
targets
have
hyperthermia
been
causes
suggested:
cell
proteins,
death.
RNA,
cellular membranes, lysosomes or any combination of these
be
directly
damaged
by
heat.
Overgaard
(15)
mammary
carcinoma,
and
hypothesized
that
lysosomally conditioned selective destruction of
cells
occurs
and
that
damage
in
heat
Mondovi
treated
organized system such as lysosomal
support
(8)
cancer
or
suggested
cells
a
a primary,
malignant
surface
that
the
resides in some
membranes.
In
of his conclusion is the fact that tumor membranes have
a different composition than those of normal cells.
in
of
a
this reaction is intensified by a high
acidity in the tumor milieu.
first
the
might
observed
pronounced lysosomal activity after a few hours treatment
murine
DNA
general
higher
levels of cholesterol.
have suggested that hyperthermia
indirectly,
may
cause
They
have
Other investigators
tumor
cell
death
either by affecting the vascular system (16,17), or
6
by s e n s i t i z i n g the immune system ( 1 8 - 3 0 ) .
A2.
Immunological e f f e c t s
of hyperthermia
Hyperthermia may have an i n d i r e c t e f f e c t on tumors v i a
immune
system.
Regression
of
human
reported to occur spontaneously, a f t e r
infection.
In 1975, S t e h l i n et a l .
malignancies
episodes
of
the
has
been
fever
and
(18) observed disappearance
of d i s t a n t metastases in some p a t i e n t s t r e a t e d with hyperthermic
perfusion,
and
suggested
that such e f f e c t might be r e l a t e d to
the stimulation of antitumor
immunity
other
correlate
investigators
now
of
the
an
patient.
elevation
Many
of
body
temperature with a better function of host defense mechanisms.
Experiments on b a c t e r i a l l y i n f e c t e d rabbits show that there
may be
an
optimum
febrile
range
for
an
animal
to
resist
i n f e c t i o n , with moderate f e v e r s b e n e f i c i a l and abscence of
or
presence
of
high f e v e r detrimental ( 1 9 ) .
l i z a r d s and fish show that the absolute
not
as
fever
Other s t u d i e s on
temperature
itself
is
important as is t h e e l e v a t i o n of temperature above that
normal for the s p e c i e s .
The b e n e f i c i a l
effects
of hyperthermia on response to v i r a l
i n f e c t i o n have been shown i n d i f f e r e n t animal s p e c i e s .
piglets
infected
puppies
challenged
with
gastroenteritis
with
canine
herpes
virus
(20),
virus
inoculated with herpes v i r u s (22) show increased
New born
newborn
( 2 1 ) , and mice
resistance
to
i n f e c t i o n when t r e a t e d with hyperthermia.
Direct e f f e c t s of hyperthermia on i s o l a t e d immune functions
have been reported in is, v i t r o s t u d i e s .
human
lymphocytes
appear
to
be
Mitogenic responses
enhanced
of
by
hyperthermia.
Roberts and S t e i g b i g e l ( 2 3 ) found that lymphocyte
transformation
7
responses
antigen
to
the
mitogen
phytohemagglutinin
streptokinase-streptodornase
r e l a t i v e to 37°C.
The
accompanied
acceleration,
40°C.
who
by i t s
elevated
were
(PHA)
enhanced
mitogenic
and
at
response
the
38.5°C
was
not
and t h e e f f e c t was not s e e n a t
S i m i l a r r e s u l t s were o b t a i n e d by Ashman and Nahmias
found
a
i n c r e a s e i n ( 3 H) Thymidine u p t a k e
significant
human lymphocytes i n c u b a t e d w i t h PHA o r CON-A
results
Bhow
that
hyperthermia
caused an e a r l i e r o n s e t o f
(25)
also
reported
an
lymphocyte c u l t u r e s
mitogen
(PWM)
at
not
only
enhancement
PHA,
of
38.5°C
was c o n s i s t e n t l y
stimulated
LIF
to
LIF
et
al.
CON-A,
or
pokeweed
Cytotoxic
T-cell
that
hyperthermia
at
a s s o c i a t e d w i t h g r e a t e r s p o n t a n e o u s and
(Leukocyte
exposure
Bruce
also
enhanced.
reported
Migration
p r o d u c t i o n by human mononuclear
after
Their
enhanced, but
40%.
r e s p o n s e s t o a l l o g e n e i c c e l l s were a l s o
(26)
39°C.
in
of DNA s y n t h e s i s i n mixed
with
temperature
R o b e r t s and Sandberg
at
the m i t o g e n r e s p o n s e .
incubated
a
(24)
Inhibition
leukocytes.
M i g r a t i o n at
produced at 3 8 . 5 ° C was g r e a t l y
r e l a t i v e to m i g r a t i o n at 37°C i n r e s p o n s e
Factor)
to
LIF
38.5°C
reduced
produced
at
37°C.
R e c e n t l y , i t h a s b e e n r e p o r t e d t h a t h e a t - t r e a t m e n t of
cells
modifies
enhancement
colon
cellular
of
In
vivo
hyperthermia
membranes
antibody-complement
tumor c e l l s
PARA-7 c e l l s
plasma
( 2 7 ) , and a g a i n s t
resulting
cytotoxicity
in
against
virus-transformed
tumor
the
human
hamsters
(28).
experiments
at
43%
have
(tumor
demonstrated
that
local
temperature)
results
in
the
s t i m u l a t i o n of the macrophage and T-lymphocyte
systems
in
the
8
treatment
of
Guerin
epithelioma
(29) and Mc7 sarcoma (30) in
rats.
Although
beneficial
most
of
the
reports
cited
(31,32),
reduced
the
(CTL's),
and
MacDonald
cytolytic
and
(33)
activity
show
the
of
the
immune
found
response.
that hyperthermia
cytotoxic
T-lymphocytes
that the P 815 mastocytoma cells did not show any
increased susceptibility to
heat.
far
effects of hyperthermia, other studies indicate that
hyperthermia may have adverse effects on
Harris
so
immune
lysis,
when
treated
with
The CTL's were shown to recover dramatically after being
inactivated
by
a
brief
hyperthermic
exposure.
Neither
suppression
of
protein synthesis nor damage to membrane lipids
seemed to be the causative mechanism.
The view that heat lowers host immune response is supported
by Schechter et al.
spleen
cells
(34) who
reported
that
heating
cells .in. vitro reduces their capacity for in vitro tumor
cytotoxicity.
metastasizing
However,
local
heat
treatment
of
carcinoma in Wistar/Furth rats caused a decreased
growth rate of the primary tumor as well as distant
Also
isolated
these
authors
observed
no
metastases.
detrimental
effect
on
cell-mediated tumor immune response of heated rats as tested
by
an ia. vitro lymphocytotoxicity assay 1 day later.
Other reports show that local
beneficial,
effects on
whereas
the
observed
that
followed
by
cell-mediated
whole-body
immune
local
tumor
heating
of
hyperthermia
response.
Shah
and
tumors
may
be
may have adverse
Dickson
(35,36)
heating of VX2 tumor-bearing rabbits, was
regression
and
a
marked
increase
in
immunity, as measured by skin reactivity to tumor
9
extract
and
dinitrochlorobenzene.
Total
body
hyperthermia,
however, led to temporary restraint of tumor growth, followed by
a
return
to an exponential increase in tumor volume.
accompanied by abrogation of the enhanced immune
This was
responsiveness
that followed local heating.
Discrepancies in
different
neoplasms
the
response
of
various
species
to heat-induced hyperthermia indicate that
more work is needed in this area.
Whether local hyperthermia or
whole-body hyperthermia induce a better immune response
yet
be
known.
highly
with
is
not
What is known is that different components seem to
heat
experiments
the
sensitive.
This
suggests
temperature
rise
induced
tumors or tissues must be uniform,
characterized.
non-invasive
well
that
in
future
by hyperthermia in
controlled,
and
well
Microwaves and ultrasound are now being used as
modalities
to
induce
thermal
rise
by
heat
generation in specified tumor or tissue volumes.
II B.
Bl.
MICROWAVES AND ULTRASOUND
Use of microwaves and ultrasound
as
hyperthermia-inducing
agents
Microwaves and ultrasound are relatively new clinical tools
that are being used in medicine.
they
can
be
human body.
generation
applied
reach
nature,
non-invaBively to different parts of the
Absorption of the waves in tissues results in
which
can
intensity of the wave
could
Becaase of their wave
the
be
is
levels
therapeutically
selected
beneficial.
properly,
the
heat
If the
temperature
needed for hyperthermic applications.
For a complete understanding of the mode of interaction of waves
10
with biological media, one must start by defining the electrical
and
acoustical
properties
coefficients,
of
reflection
various
at
tissues.
interfaces
tissues, and scattering properties must be
approximate
temperature-rise
Absorption
between
known,
different
so
that
an
profile in the irradiated tissues
can be estimated.
Because standing waves are
interfaces,
to
exist
at
and
near
undesirable "hot spots" with temperatures exceeding
the required levels, might be
interrupt
known
the
found.
These
hot
spots
might
uniformity of the temperature profile, and might
induce unwanted micro-thermal damage.
Various microwave
reported.
These
and
effects
ultrasound
might
function as clinical devices.
can
be
thermal
The
bio-effects
interfere
have
with their primary
possible
radiation
or non-thermal in origin.
hazards
The thermal effects
occur whenever the intensity of the wave is high enough.
effects
seem
to
be
to
sub-thermal
occur
operate
at
levels.
controversial in
are
not
These
of the same nature as those obtained when
heat is directly applied to the tissue.
reported
been
different
intensity
However,
these
nature,
well
Non-thermal effects are
and
the
levels,
reports
mechanisms
by
even
at
are
still
which
they
understood, even though many mechanisms
have been suggested so far.
B2.
Possible mechanisms for microwave bio-effects•
The interaction of microwaves with tissues
according
classified
to the Radiation Protection Guide (RPG) into "strong"
and "weak" interactions.
those
±a
which
take
place
"Strong" interactions are
defined
as
at intensity levels beyond 10 mW/cm .
11
They are mainly of thermal origin, but
non-thermal
effects,
such
as
they
may
field-induced
also
include
force
effects,
dielectric dispersion, and denaturation of biomolecules.
Field-induced
electrical
forces
potential
arise
energy
of
from
a
the
fact
particle
that
or
a
particles depends on the geometric arrangement with
the
applied
field.
The
field-induced
forces
the
system of
respect
to
lead
to
the
reorientation of individual particles in the field, and
to
the
rearrangement of a set of particles, such as in the "pearl chain
formation"
phenomenon (37). This effect is frequency dependent
and ha8 thresholds much larger than 10 mW/cm .
Dielectric
occur
at
dispersion
field
and
strength
denaturation
levels
of
effective
biomolecules
in
completely
overcoming Brownian motion, resulting in complete orientation of
polar molecular subgroups of biomolecules
(38,39).
"Weak" interactions are defined as those taking place
the
microwave
intensity
non-thermal origin, and the
is
less than 10 mW/cm .
suggested
mechanisms
when
They are of
include
the
excitation of cellular membranes, and the local amplification of
electric fields.
Several theoretical models have been
the
proposed
to
explain
excitation of cellular membranes by microwaves (40-42), and
by low frequency
(43-45).
amplitude
modulated
frequency
fields
Experimentally, Seaman and Wachtel (46) reported that
low energy microwaves were able to alter
individual
radio
the
firing
pacemaker neurons, while Bawin et al.
enhanced release of calcium ions from brain
tissue
amplitude modulated radio frequency radiation.
rates
of
(47) found an
exposed
to
12
Even though the
effects
are
to hot spots
responsible
microwave
intensities
in
many
reported
in the non-thermal range, microthermal heating due
in
for
the
microwave
these
field
effects.
distribution
Nilsson
and
might
be
Petterson (48)
suggest that microscopic-wedge-shaped boundaries between regions
with different dielectric constants,
present
in
the
mammalian
field-induced
radiation
local
intensities
are
likely
to
be
body, may give rise to local fields
about 100 times larger than
The
which
the
macroscopic
damage
10^
could
times
electric
fields.
then occur at incident
smaller
in
magnitude
than
intensities needed for regional damage.
B3.
Possible mechanisms for ultrasound bioeffects.
The bioeffects of ultrasound are
and
non-thermal interactions.
classified
into
Thermal effects usually occur at
ultrasound intensity levels at and above those needed to
destruction
of
tissues.
Non-thermal
interactions
generally high intensities, but many effects
intensities lower than 100 mW/cmS
have
been
effects
suggested
include
thermal
were
induce
require
reported
at
Some of the mechanisms which
as responsible for non-thermal ultrasound
cavitation,
acoustic
streaming
and
microstreaming, and radiation force effects.
Cavitation occurs when microbubbles in
respond
liquid
suspensions
to an acoustic field by executing expansion-contraction
pulsations.
This causes gas
to
diffuse
radially
inward
and
outward during each cycle in an asymmetrical manner, so that net
inward
flow
results.
diffusion, and collapse
very
high
temperatures
The
bubble will then grow by rectified
after sufficient expansion, leading
and pressures.
to
Bioeffects can then be
13
produced by mechanical shocks or by chemical changes.
the
of
bioeffects which have been attributed to cavitation include
cell membrane damage in cultured mammalian cells (49)
of
Some
growth
reduction
rate of plant roots (50), pyknosis of cultured human
lymphocytes (51,52), and the stimulation of
collagen
synthesis
in human embryonic fibroblasts (53).
Acoustic microstreaming might occur when shear stresses are
set up near an oscillating bubble lacking spherical symmetry
resting
on
a
solid boundary.
Large velocity gradients arise,
which might act on a cell by elongating it and then
There
is
experimental
evidence
cells,
and
lysing
it.
that these microflows play an
important role in the degradation of macromolecules
of
or
destruction
disturbance in the physiochemical states of the
cell membrane (54).
Radiation force effects arise when there is a net change in
the momentum of the wave, either by reflection or by absorption.
Cells in suspension are subjected to
This
such
a
radiation
force.
may explain the stasis phenomenon reported by Dyson et al.
(55), who have shown that red cells in blood
embryo
collect
into
vessels
of
chick
parallel bands spaced one-half wavelength
apart in an ultrasonic standing wave field.
In
many
bioeffects
reports,
are
not
the
yet
mechanisms
fully
responsible
understood.
These
bioeffects occur sometimes at intensity levels much
100
mW/cm?.
reported to
exposure
for
In
be
the
reported
lower
than
cultured human cells, cellular attachment was
significantly
a
for
total
power
reduced
output
equivalent to an average intensity
of
after
of
0.62
0.50
1.76
mW,
mW/cm2
minute
of
which
is
(56).
In
14
another experiment, the ultrasonic intensity reaching the uterus
of a mouse was less than 40 mW/cm2, and the exposure resulted in
induced contractions of the uterine smooth muscle (57).
It is important to note that
the
experimentally
measured
rise of temperature in irradiated tissues is extracellular.
intracellular
rise
in
temperature
measured outside of the cell.
problem
theoretically
100 mW/cm2
and
properties
of
Love
may
be
and
higher
Kremkau
The
than that
studied
this
(58), and found that for an intensity of
frequency
cellular
of
1 MHz,
components
and
they
for
the
thermal
assumed, the maximal
differential rise was of the order of 10~ 5 °K only, and occurred
at the center of the spherical
cell
model
they
used.
Other
microthermal effectB, similar to those suggested for microwaves,
are not to be excluded.
B4.
Immunological effects of microwaves
There have been many studies on the effects
on
lymphocytes
of
the
most
formation
and the immune system.
consistent
is
microwaves
As in hyperthermia, one
increased
lymphocyte
and activity (59,60), suggesting that the effects are
mainly thermal.
mitotic
findings
of
Prince
response
of
et
al.
monkey's
(61)
reported
peripheral
an
blood
enhanced
lymphocytes
stimulated in. vitro with PHA, three days after the monkeys
were
exposed to 1.32 W/cm2 pulsed radiation.
Increased "spontaneous
reported
by
lymphocytes
cultivation.
Czerski
from
He
lymphoblastoid
(60)
irradiated
also
to
occur
rabbits,
reported
that
transformation"
in
pheripheral
following
irradiated
in.
mice
is
blood
vitro
had
15
significantly greater numbers of
higher
serum
antibody-producing
cells
and
antibody titers following immunization with sheep
red blood cells.
Wiktor-Jedrzejeczak et
rectangular
waveguide
to
al.
2.45
significant increase in the
positive
(CR+)
However,
no
microwaves
of
and
was
did
later
reported
and
Thy-1
observed.
not
a
found
a
negative
spleen
cells.
They
concluded
that
that
the
by
acute
induced by whole-body
These
microwave
stimulate lymphoid cell proliferation per
substantiated
radiation.
in
complement-receptor
B-cell
activator,
to early maturation of noncommitted B-cells.
were
mice
immunoglobulin positive (Ig+), Fc
se, but appeared to act as polyclonal
led
exposed
change in the total number of Ig+ cells in spleens
of these mice
irradiation
(FcR+),
GHz
proportion
lymphocytes,
receptor positive
(62-64)
Sulek
transient
exposure
mice
et
to
also
Their results
(65).
lymphopenia
a
Liburdy
in
thermogenic
showed
splenic T- and B-lymphocytes (66).
al.
which
mice was
radiofrequency
relative
increase in
In another study he reported
alterations in lymphocyte distribution and
function
which
are
characteristic of a state of immunosuppression (67).
It
is
now
believed
lymphocytes
can
be
microwave
changed
radiation.
immunological
that
It
functional
following
is
significance
the
of
inconsistencies that exist among various
studies
indicate
lymphocytes and
others
indicate
that
microwaves
potentiate
depressed
the
in, vitro
difficult
these
to
the
because of the
reports.
While
many
responsiveness to
response
responsiveness.
of
exposure to
interpret
changes*
increase
immune
integrity
to
For
antigen,
a
better
16
understanding of the
immune
and
effects
of
thermoregulatory
temperature
determined,
it
is
radiation
on
the
systems, the characterization and
the control over the
whether
microwave
a
profile
must
be
accurately
of
local
or
whole body
done
to
case
irradiation.
Many in. vitro experiments
possibility
of
a
direct
were
action
of
investigate
microwaves
responsiveness of lymphocytes to different mitogens.
temperature
of
the
cultures
was
left
was
When
the
However, when the temperature of the irradiated cells
effect
observed
the
reported
was controlled so that it did not differ greatly from
no
on
to reach hyperthermic
levels, lymphoproliferative response of the cells
(23-25).
the
on
the
proliferative
(68,69).
This
response
strengthens
the
controls,
of lymphocytes was
belief
that
the
system
has
microwave effects are mainly thermal in origin.
B5.
Immunological effects of ultrasound
A variety of ultrasound effects on the
been reported in the last few years.
repair
was
lymphocytes
However,
observed
(70,71)
HeLa
DNA damage followed by DNA
in. vitro
cell
sonication
in
the
Gl
of
human
phase
(72).
other reports failed to confirm any chromosomal damage
in human lymphocytes
sonicated
and
after
immune
medium
and
HeLa
cells,
was avoided (73,74).
heating
of
the
Hedges and Leeman (51),
and Graham et al.
(52) observed
nucleus
by the eventual disruption of the entire cell
followed
a
while
degeneration
in human lymphocytes irradiated with ultrasound.
of
the
cell
They suggested
that sonication may damage lysosomal membranes via a
cavitation
17
process.
Ultrasound effects taking place at the cell membrane
have
also
been
reported.
Martins et al.
sonicated mammalian cells were rounded
vesicles
around
them
which
surface of control cells.
did
and
not
level
(49) observed that
had
characteristic
appear
on the smoother
Repacholi and Kaplan (75) found
that
the number of concanavalin A (CON-A) receptors on the surface of
human
peripheral
lymphocytes
significantly less
untreated
than
controls.
that
treated
found
Anderson
on
with
the
ultrasound
surface
insonated
the spleen.
the
and Barrett observed a state of
immunosuppression (76), and depression of phagocytosis
mice
of
was
(77)
in
with a low energy wave applied over the area of
These investigators suggested that the effects they
observed were caused by cell membrane damage during exposure
to
ultrasound.
The
mechanisms
ultrasound
may
determined.
reported
by
affect
which
hyperthermia,
cellular
functions
However, it seems very
effects
are
initiated
likely
at
the
microwaves,
are
that
and
adverse
immunocompetence.
necessary
is
therefore
understanding of the cell membrane biology.
of the next section.
many
a
of
variety
membrane-membrane interactions.
effect on cellular membranes may have
It
not yet fully
the
cell membrane level.
Immune recognition and response are modulated by
membrane-antigen
or
of
Thus any
consequences
on
to have a good
This is the subject
18
II C
CI.
BIOLOGICAL BACKGROUND
Cell membrane structure and properties
All living cells are delimited from their environment by
cell
surface
membrane,
the
plasma
membrane, which has vital
biological functions due to its strategic
membrane
a
location.
The
cell
receives information from and transmits signals to the
environment, controls the permeation of nutrients, regulates the
action of certain drugs and hormones,
recognition,
modulates
cell
to
cell
interaction and communication, and influences cell
locomotion, growth and differentiation.
The
most
satisfactory
current model for natural membranes that accounts for the latest
experimental
observations
is
the fluid lipid-globular protein
mosaic model presented by Singer and
They
consider
Nicholson
in
1972
that the basic structure of biological membranes
is a two-dimensional arrangement of globular proteins
in
a
matrix
amphipathic
of
fluid
molecules,
(non-polar)
and
lipid
i.e.,
the
dispersed
bilayer (Figure 1 ) . Lipids are
they
hydrophilic
hydrophilic-polar head groups
whereas
(78).
contain
both
(polar)
prefer
an
hydrophobic
regions.
aqueous
The
environment,
hydrophobic hydrocarbon tails are only stable in a
non-aqueous environment.
In
the
Singer-Nicholson
model,
the
lipids are arranged in a bilayer with their ionic head groups in
direct
contact
with the aqueous salt solutions on each side of
the membrane, and with their hydrocarbon tails aligned
back
to
minimize
their
with water.
configuration
which
proteins
also amphipathic biomolecules.
are
is
contact
thermodynamically
back
to
This leads to a
stable.
Globular
Their hydrophobic
19
Figure
1:
Representation of three-dimensional organization of
plasma membranes.
A bimolecular film of phospho-
lipids forms the matrix of the membrane, and globular proteins are embedded in the lipid core.
Some
proteins span the membrane; others are embedded in
one of the lipid monolayers.
20
regions are embedded
bilayer,
and
in
the
hydrocarbon
core
of
the
lipid
the more polar regions are exposed to the aqueous
solutions at one (peripheral proteins) or both
sides
(integral
proteins) of the membrane.
To fulfill its vital functions, the membrane
must
operate
differentially on the two compartments it separates, and thus it
must
by asymmetric.
within the lipid
The asymmetrical placement of the proteins
matrix
has
been
studied
extensively.
The
i
topological
asymmetry
of
phospholipids
in membranes has been
studied more recently (79, 8 0 ) .
The biological
affected
by
functions
of
the
cell
membrane
may
be
environmental factors such as temperature, pH, and
concentration of different chemical or biochemical constituents.
Non-thermal microwave or ultrasound effects are not yet
clearly
established.
precise
Membrane
proteins
have
fairly
environmental requirements for optimum activity, some
a
fluid
environment,
surroundings.
fluid,
whereas
others
The transition of
liquid-crystalline
the
phase
to
requiring
require much more rigid
membrane
lipids
from
a
a solid, gel phase is not
sharp, but occurs over an appreciable range of temperatures (81,
82).
Associated with any continuous phase
there
are
a
variety
of
process,
of pre- and poBt-melting phenomena (83).
Since the lipids in a biological membrane
degrees
transition
exist
within
a
their phase transition temperatures, they may be at
the temperature at which pre- and post-transition phenomena
expected
to
be
biologically
mixed lipid phases could occur
If
patches
few
of
solid
important.
at
are
It is possible that
physiological
temperatures.
lipids are present in mammalian cells at
21
3 7 % , they could play
mobility
and
the
an
important
arrangement
of
receptors such as antigen proteins.
and
human
that
the
surface
mobility
temperature
was
role
a
in
wide
controlling
range
the
of membrane
In an experiment
on
mouse
antigen mixing, Petit and Edidin (84) found
of
the
lowered
antigens
from
was
42°C
decreased
as
the
to 21°C, but with further
cooling, the mobility was increased with a new maximum occurring
at 15 °C.
They suggested that the decreased mobility in the 4 2 %
to 21°C interval is due to increases in
lipids
with
decreasing
viscosity
of
membrane
temperatures, and that between 21
and
1 8 % the effects were due to phase separations.
Lipid phase
Trauble
7
to
transition
temperatures
are
pH-dependent.
and Eibl (85) found that when the pH was increased from
9,
the
Fluid-ordered
transition
temperature
was
lowered
transition temperature.
transition
20°C.
transition at constant temperature can be induced
by use of divalent cations (Mg + + and Ca"*"*") which
the
by
increase
the
Monovalent cations (Li + , Na + , K + ) lower
temperature
and
therefore
have
antagonistic
in
establishing
effects to the effects of divalent cations.
C2:
Lipid-protein 3JUiSLSAS£iS&SL'
Lipid-protein interactions are
important
the function of the proteins included in a membrane.
The lipids
which surround a protein experience hydrophobic forces which are
different
a protein.
than those found in portions of the membrane far from
Jost et al.
(86) demonstrated a
highly
restricted
or immobilized component of the lipid corresponding to about 0.2
mg
of
lipid
per
mg
of
protein.
They
restricted "boundary lipid" corresponds to
suggested that this
a
single
solvation
22
layer of lipid about the circumference of protein where it is in
contact
with
lipid bilayer.
The existence of a boundary lipid
has been confirmed by many other investigators
and
(87-91).
Barantes (88) estimated that the proportion of lipid in the
immobilized component is greater than calculated
boundary
layer.
dependant
at
temperatures
well
temperature of the pure lipid.
temperatures
for
above
This
strongly
the
single
implies
temperature
phase
transition
that
the
higher
which can be biologically important are differrent
than those at which phase transitions of the pure
These
a
Other investigators (87) found that the amount
of lipid in the restricted environment is
temperatures
would
lipid
occur.
correspond to the range in which the
boundary lipids start their phase transition, because
in
Marsh
a
change
the fluidity of the annular lipids might bring about changes
in protein conformation and so changes in protein activity.
Although lipids
somewhat
al.
in
immobilized,
immediate
Grant
contact
with
protein
and McConnell (89) and Overath et
(90) reported that the entire lipid-protein complex
tendency
to
occupy
fluid
are
regions
of
the bilayer.
has
a
Very few
reports found no evidence for the existence of "boundary lipids"
(92).
Many
theoretical
protein-protein
models
interactions
in
for
bilayer
protein-lipid
membranes
and
have been
presented, based on thermodynamical arguments (93-95).
Lipid-protein interactions are
environmental
to
depend
on
many
factors such as temperature, pH, and the presence
of different
chemical
mobility
membranes
in
found
or
biochemical
depends
also
constituents.
Protein
on similar environmental
factors (96). Aggregation of a class of proteins (antigens)
on
23
cell
surfaces
following
antibody
mobility of these proteins.
aggregate
binding
is
related to the
The antigen-antibody complexes
together to form a single aggregate.
may
This phenomenon
is called capping.
C3:
Redistribution of surface receptors:
gapping.
Antigens found on the surface of cells are
are
embedded
on
of
An
antigenic
determinant
is
that
the antigen to which an antibody bj.nds by molecular
complementarity.
The antigens are initially diffusely scattered
all over the surface of the cell.
initiates
which
the lipid bilayer and which carry one or more
"antigenic determinants".
portion
proteins
The
binding
of
antibodies
a redistribution of the antigen-antibody complexes on
the cell surface.
The redistribution depends
receptor
the
and
on
particular receptor.
type
of
the
cell
on
that
the
type
carries
this
On many types of cells, the redistribution
starts with a regrouping of the antigen-antibody complexes
patches.
Patch
become
progressively
capping of the complexes:
cap,
into
formation is a passive phenomenon arising from
the diffusion of small complexes which upon collision
another
of
larger.
all patches
with
one
Patching is followed by
coalesce
into
a
polar
and the remaining part of the membrane is devoid of any of
that particular antigen.
In contrast with patching, capping
is
an active process which requires metabolic energy.
Capping has
conditions
(37°C,
B-lymphocytea
receptor,
serving as
been
no
(97,98).
surface
antigen
Ig.
very
drugs,
well
no
B-cells
The
receptors
characterized
under
normal
radiation) on the surface of
carry
the
immunoglobulin
number of Ig molecules presumably
has
been
estimated
to
be 10
24
molecules
on
Ig is found
the average per B human lymphocyte cell.
diffusely
throughout
nonrandom distribution.
discontinuous
the
plasma
membrane
a
with very small clusters which are often
interconnected by strands of a few molecules.
This
short-range
does not necessarily negate the fluid mosaic model
of membrane structure.
protein-phospholipid
It may
be
interactions
due
on
to
protein-protein
structure.
When
anti-Ig
or
the membranes, or to the
attachment of the Ig molecules in variable numbers
anchoring
in
The molecules are distributed in a lacy
network
organization
Surface
to
a
given
antibodies bind to the Ig
receptors, patching followed by capping takes place in a
of minutes, if the environmental factors are favorable.
matter
Capping
is inhibited at 4 °C, in the presence of metabolic inhibitors, or
in
the
presence
of
high
microfilament inhibitor.
has
some
been
enhancing
found
of cytochalasin B which is a
Colchicine, a microtubular
Cis-dichlorodiammineplatinum
has
doses
effects
on
inhibitor,
capping
II (cis-DDP), an anticancer
(97).
agent,
to inhibit capping of Ig and of concanavalm A
(CON-A) receptors on mouse B-lymphocytes (99).
CON-A is a lectin to which many cell
receptors.
(97,98).
CON-A
are known to cap on B-lymphocytes
normal and SV40 transformed human fibroblasts.
The
changes in the transformed cells occur more quickly than in
the
normal
cells,
show
surface
the
of
also
carry
on
surface
They
receptors
types
remarkable
redistribution
suggesting that the membranes of the transformed
cells are more fluid (100).
Cancer cells also show capping:
Rosenthal
et
al.
(101)
have found that antibodies specific for carcinoembryonic antigen
25
(CEA)
the
induce
polar redistribution of CEA which is expressed on
surface
colchicine
of
human
had
no
intestinal
effect
carcinoma
on
cells.
capping,
While
inhibition
of
redistribution was observed following cytochalasin-B treatment.
The
exact
understood.
mechanisms
However,
of
capping
better
are
capping
has
not
yet
fully
been observed many
times to occur under conditions where the cell membrane exhibits
higher fluidity, whereas poor capping
increased
membrane
has
been
attributed
viscosity, such as in lymphoma cells.
to
Upon
binding of the antibody, increased microfluidity may be obtained
by phospholipid methylation, which would then facilitate capping
(102).
It also appears that
directly
or
the
cytoskeletal
system
may
be
indirectly involved in the redistribution process.
This suggestion is supported by observation of
cytochalasin-B
and
microfilaments and
colchicine
which
microtubules—the
are
the
known
building
effects
to
blocks
of
inhibit
of
the
cytoskeletal system.
C4.
The cytoskeletal system:
involvement in capping.
Microfilaments (MF)
MF are fine structures of various
The
5 - 8 nm
thick
MF
are
lengths
and
diameters.
thick
MF
are made of actin, while the 13 - 25 m
made
of
myosin.
Actin
has
very
conserved
composition and characteristics throughout all Eukaryotic cells.
At
least
two
classes
of
MF
systems
can be found:
some MF
constitute a loose network of short interconnected MF forming
a
lattice localized at the anterior part of the cell, just beneath
the
plasma
membrane (PM), and may be associated with it.
They
are disrupted by cytochalasin but they do not seem to bind heavy
26
meromysin (HMM).
organized
cell.
as
The other set of MF consists of
bundles
of
fibres
parallel
MF,
in the posterior part of the
They are not disrupted by cytochalasin, but they bind HMM
(HMM is the globular ATPase portion of
obtained
by
trypsin digestion).
support the idea that
fibroblasts,
both
MF
sets
may
of
skeletal
muscle
myosin
Several investigators seem to
be
anchored
to
membrane.
In
MF are linked to the PM; the whole
cell length is spanned by long fibres which
converge
to
focal
points (98,103).
Microtubules (MT.)
MT appear as separate fibres of
diameter • 24 nm)
uniform
thickness
which run through the cytoplasm.
15 nm hollow core, and a dense cortex which iB
adjacent
protofilamenta,
4 - 5 nm
wide.
are built of subunits of tubulin dimers,
denaturation
vinblastine
reversibly
one
at
molecule
separate
disrupt
MT.
of
made
which
can
dissolution
into
up
of
13
bind
upon
and one molecule of
Colchicine
and
vinblastine
Low temperature, hydrostatic pressure
and high calcium concentration (>10 M) can induce
MT
They have a
These protofllaments
colchicine
Bites.
(outer
subunits.
The
a
reversible
equilibrium
of
assembly-disassembly between MT and tubulin can be shifted to MT
formation in vitro by D«0,
hydrophobicity-driven
which
association
supports
of
the
the subunits.
many lateral cross-bridges (2 - 5 nm thick by
between
concept
of
a
There are
10 - 40 nm
long)
adjacent MT or between a MT and all kinds of membranous
components, including membrane vesicles and PM.
reversibly destroyed by disruption of MT.
Cell
shape
is
27
Involvement pj. &&£. cytoafteUtPP i& capping
Early B
cells
spontaneous
show
rates
of
capping
and
even
capping of the Ig receptorB, as compared to capping
on mature B cells.
activities
higher
quite
This might be due to a balance of MT and
different
from
that
MF
of mature cells (104).
Many models related to the involvement of
the
cytoskeleton
in
capping have been proposed (98,105-107).
Loor (98) proposes that a contractile MF
directly
or
system,
via intermediate units to some membrane components
and responsible for their polar migration, would be
by
an
and
anchored
antagonized
MT network also associated with some membrane components
conferring
"thixotropic"
to
the
gel-like
cortical
cytoplasm
properties
(A
of
system,
the
cell
like the MT-MF
system, is said to have thixotropic gel-like properties when
becomes
liquid
when
and
as
long
it
as a force is applied, and
solidifies immediately after).
Berlin et
microtubules
associated
al.
is
(105)
to
suggest
influence
microfilaments;
microfilaments
the
the
that
a
primary
distribution
membrane
role
of
of
membrane
subtended
by
has different properties from bulk membrane:
it
has an increased "microviscosity" with an increased affinity for
antigen-antibody
complexes.
By
this
model
direct
microtubule-receptor connections are not an absolute requirement
for microtubule regulation of surface topography.
Clark
(106)
Albertini and
reported that CON-A capping induced redistribution
of cytoplasmic MT and colchicine binding proteins.
found
preferentially
concentrated
in
the
cell.
They suggest that the capped membrane
Tubulin
was
capped area of the
domain
may
be
a
28
preferred site of MT polymerization.
In contrast with what Albertini and Clark reported,
et
al.
(107) found no evidence of a redistribution of tubulin
after CON-A capping.
always
Singer
accompanied
Instead,
by
a
found
redistribution
actin and myosin under the
capping
they
cap.
They
that
capping
was
and concentration of
proposed
a
model
for
in which clusters of receptors become linked across the
membrane to actin- and myosin-containing structures.
linkage,
these
clusters
of
After
the
receptors are still mobile in the
plane of the membrane, and are actively collected into a cap
by
an analogue of the muscle sliding filament mechanism.
Finally,
categories
Emerson
and
Cone
(108)
found
that
different
of membrane proteins on the same cell surface may be
associated either with MF or MT.
Scientists have not yet
agreed
on
a
unified
model
capping, which would explain all of the reported facts.
one
fact
may be agreed upon:
However
whether via MF, MT, or both, the
cytoskeleton seems to be either directly or indirectly
in the capping process.
for
involved
29
III.
ANIMALS:
Young
adult
Swiss mice were obtained
MATERIALS AND METHODS
(12 to 16 weeks old) outbred female ICR
from Harlan Laboratories, Indianapolis,
and were used as the source of B-lymphocytes.
MEDIA:
Minimal Essential Medium
study.
MEM
was
supplemented
(MEM) was used
throughout
with 10Z heat-inactivated
bovine serum (FBS), 100 U/ml penicillin, 0.1 gm/ml
2 mM L-glutamine, and buffered with
20
mM
the
fetal
streptomycin,
HEPES,
and
0.075%
NaHC0 3 .
CELL
SUSPENSIONS:
sacrificed
by
In
each
cervical
experiment,
dislocation.
cells was obtained by mincing the
Essential
Medium
fetal-bovine
(MEM)
in
5
mice
suspension of
organs
in
complete
10%
heat
were
spleen
Minimal
inactivated
the cells
through
.The cell suspension was
then
MEM + FBS and centrifuged at 400 g for 30 minutes on
from
(123).
The
mononuclear
the gradient and washed
For antibody-complement
lysed
to
serum (MEM + F B S ) , and dispersing
Ficoll-Hypaque
removed
A
containing
a fine wire stainless steel mesh.
washed
3
leukocyte
was
three times in MEM + FBS.
cytotoxicity experiments,
by treatment with monoclonal anti-Thy
England Nuclear, Boston, MA) and guinea
band
T-cells
were
1.2 antibodies
pig
complement
(New
(Grand
Island Biological Company, Grand Island, NY) for 1 hour at 3 7 % .
The
remaining
B-lymphocyte-enriched
washed 3 times and resuspended
was better than 90% as
cell
population was then
in MEM + FBS.
determined
by
These cells served as the standard cell
trypan
Viability of
blue
population.
cells
exclusion.
30
Approximately 1 x 10 6 spleen cells per 12 x
HEAT TREATMENT:
75
mm test tubes were suspended in 300 yl complete MEM or in 100 yl
diluted
antibody
depending
on the experiment.
The test tubes
were sealed with rubber stoppers and immersed in a
The
heater
coils
were
fed
from
a
proportional
controller YSI model 72 (Yellow Springs Instrument
Springs,
Ohio).
The
temperature
thermistor probe (model YSI 403)
input
of
the controller.
was
which
sensed
was
Saddle
bath.
temperature
Co.,
by
Yellow
means of a
connected
to
the
The temperature was monitored at all
times on a digital thermometer, model BAT-8 (Bailey
Inc.,
water
Brook, N J ) .
Instruments
The temperature variation inside the
test tubes was less than 0.1°C.
CYTOCHALASIN-B TREATMENT:
from
SIGMA
Lyophilized
Laboratories, St.
dimethylsulphoxide (DMS0),
aliquots at -20°C.
cytochalasin-B
obtained
Louis Missouri, was dissolved in
10
mg/ml,
and
stored
in
0.1
ml
Before each experiment, the drug was diluted
in complete MEM to the desired final concentrations (10, 50, and
100 (i g/ml).
test
tubes
Approximately 1 x 10
were
cytochalasin-B.
and
immersed
temperature
suspended
in
spleen cells per 12 x 75 mm
300
yl
complete
MEM
+
The test tubes were capped with rubber stoppers
in
was
a
water
controlled
bath
as
at
37°C for 40 minutes.
above.
After
40
The
minutes
treatment, the cells were either washed in cold PBS or left with
the
drug,
then
tested
for
capping
using
a
direct
of
spleen
immunofluorescence technique.
MICROWAVE IRRADIATION:
cells
were
placed
in
Three and a half milliliters
cellulose nitrate tubes and sealed with
31
Parafilm.
The test tubes were irradiated in an exposure
described previously (124).
vertically
Briefly, the test tubes were placed
in a microwave-absorber-lined exposure chamber 45 cm
below the aperture of a 15 x 20 cm horn antenna.
microwaves
was
at 2.45 GHz.
Both water-bath and microwave-exposed
agitated
cycles/min.
A thin plastic test tube holder
a
was
wood
The source
by
the
same
shaker
cells
mechanism
in
the
were
at
75
microwave
mechanically linked to the shaker bath platform by
dowel
passing
Temperature-controlled
through
the
chamber
With
agitation,
maintained to
within
wall.
water from the water bath was circulated
through a shallow acrylic tray which supports the
holder.
of
a Litton Industries L-3501 Microtron, operating
constantly
chamber
system
the
0.1°C
internal
of
the
test
water
moving
flask
temperature was
bath
temperature.
Amplitude of sinusoidal platform displacement was 3.8 cm peak to
peak.
MICROWAVE DOSIMETRY:
cell
The specific absorption rate (SAR) in
the
suspension was estimated from a series of temperature-rise
measurements made after irradiation intervals of 15, 30, and
s.
The
test tubes were insulated with a 2 mm thick waterproof
polyurethane foam sleeve.
the
shaker
moving
but
The test tubes were
with
irradiation
estimated
Temperature
periods
from
the
increases
with
initial
were
a
irradiated
the circulating pump off.
temperature measurements were made
period.
60
within
digital
slope
linear
of
3
s
the
through
heating
the
Final
following
thermometer.
with
SAR
the
was
curves.
60 s heating
32
RF
EXPOSURE
SYSTEM:
Samples
were
exposed
in
a
specially
designed Crawford cell (Figure 2 ) . The cell has an upper cutoff
frequency
power
of
300 MHz.
signal
establishing
will
a
Below the cutoff frequency, an incident
propagate
quasi
only
in
the
TEM
mode,
far-field plane wave throughout the test
area of the cell (125).
An HP 3311A function generator provided
a low frequency sine wave which was used to amplitude
(>90%)
a
type
transmitted
different
oscilloscope
585), then amplified by a 200-watt broadbanded
amplifier (Amplifier Research, model 200L).
and
modualate
147-MHz carrier from an RF sweep generator (Textronix
TM 504). The modulated signal was monitored on an
(Textronix
thus
powers
were
Forward, reflected,
simultaneously
sampled
through
directional couplers and displayed on a digital power
meter (HP 436A) through a Transco 14704 SP3T coax RF switch
and
an HP 8481A power sensor.
The cell was terminated with a 50ftload impedance.
The SWR
on the input of the cell was less than 1.10, as measured with
a
digital BIRD RF Power Analyst model 4381.
Inside the
Crawford
cell,
two
round
bottom
thin
wall
cellulose nitrate tubes sealed with Parafilm and containing 3 ml
of
cell suspension were placed 6.4 cm apart in sample positions
1 and 2 (Figure 2 ) . Non-irradiated controls were
placed
temperature-controlled water bath with 45-liters capacity.
irradiated
a
Both
and non-irradiated cells were constantly agitated by
the shaker bath platform at 1.25 cycles/sec.
The Styrofoam test
tube holder in the Crawford cell was mechanically linked to
water
in
the
bath platform by a thin Plexiglas rod passing through the
cell wall.
The platform moved sinusoidally with a 3.8
cm
peak
33
DUAL DIRECTIONAL
CRAWFORD CELL SHAKER BATH
ISIOEVICWI
Figure
2:
DIRECTIONAL
COUPLER
Diagram of the temperature
modulated
RF
exposure
control
system.
and
(Not
amplitude
to
scale).
34
to peak amplitude.
Water was
supported
the
circulated
moving
through
test
tube
the
holder.
surrounding the test tubes was 1.3 cm.
cycles/sec
movement
controlled
water,
and
the
an
8
Plexiglas
tray
which
The depth of water
With
a
platform
1.25
1/min flow rate of temperature
internal
test
tube
temperature
was
maintained within 0.1°C of the desired temperature.
A Styrofoam shelf placed in the center of the Crawford cell
supported the Plexiglas tray such that the cell suspensions were
12.5 cm above the septum.
In that
region,
the
variations
in
electric (E) field strength are minimal (125), thus reducing the
1.25
Hz
amplitude
modulation component due to movement of the
shaker bath platform.
RF DOSIMETRY:
Although the quantity of energy being absorbed by
the samples was too small to be detected by the
any
of
total
absorbed
power
incident, reflected, and
absorbed
by
the
and
samples
fluid
was
and
of
samples
powers.
estimated.
the
net
power
measurements,
deviation
in
the
less
of any of the two measurements.
samples,
estimated to be less
intensity.
with
the
either inside or outside the Crawford cell.
than
than
the
Taking the
largest standard deviation as an upper limit for the
absorbed
power
surrounding fluid was estimated
The difference between the two measurements was
standard
was
calculated after measuring the
transmitted
after performing two series
samples
at
the power densities used, an upper limit of the average
specific absorption rate (SAR) for the
The
instruments
net
power
the average SAR for the samples was
42
yW/gm
per
mW/cm2
of
incident
35
This upper limit for
method
for
estimating
measurements in
the
insulated
a
sleeve.
with
2
in
one
mm
of
SAR
SAR's
cell
A thermocouple
inserted
the
made
ditital
within
thick
sec
thermometer
temperature-rise
irradiation.
on
test tubes.
following
test
tubes
were
Model
IT-18)
was
Thermocouple wires were
temperature
measurements
irradiation with a Bailey
BAT-8).
observed
another
proof polyurethane foam
(Bailey
Final
by
temperature-rise
The
water
(model
was
based
microprobe
the
3
confirmed
suspension.
perpendicular to the E-field.
were
was
Less
following
The SAR was thus estimated
to
a
be
than
60
0.1°C
sec
less
1
KW
than
35
per mW/cm2 incident radiation, confirming the upper limit
yW/gm
for the SAR estimated earlier.
ULTRASOUND
diameter
IRRADIATION
unfocused
suspensions.
AND
TEMPERATURE
transducer
The
transducer
was
was
used
CONTROL:
A
1-inch
to insonate the cell
driven
at
its
resonant
frequency (0.99 MHz) by a frequency synthesizer and an amplifier
system.
The transducer was mounted in a rubber-lined Plexiglas
tank containing 36
liters
Ringer's solution.
The sound intensity at the level of the cell
suspensions
calibrated
was
thermoelectric
thermocouple
detector.
was
of
temperature-controlled
by
Primary
means
of
a
calibration
degassed
transient
of
the
carried out by a radiation force method using
displacement of a stainless steel ball in the sound field (126).
Cell suspensions were placed in 6 mm
2
membrane
tubes
the
unique
Spectra/Por
(Spectrum Medical Industries Inc., LA-90054)
which are usually used for
because
diameter
dialysis.
structure
These
tubes
were
used
and 0.002-inch thickness of the
36
membranes offered
suspensions
good
and
the
thermal
conduction
between
the
cell
temperature-controlled Ringer's solution.
Furthermore, the same characteristics allowed the
membranes
to
act as acoustic windows thus minimizing reflections and standing
waves.
Dialysis
effects
were
minimal
during
the 20 minute
immersion in the Ringer's solution.
Inside the tubes, the cell suspensions were layered on
of
a
6
column
top
cm column of Ficoll-Hypaque solution, and below a 6 cm
of
arrangement
heavy
mineral
was
to
oil.
avoid
The
reason
behind
such
an
any solid-fluid interfaces (such as
between container and cell suspensions) and fluid-air interfaces
(such as between cell
reflections
of
the
suspensions
and
air),
thus
minimizing
ultrasound beam at interfaces between cell
suspensions and surrounding media.
The tubes were immersed in the irradiation tank
5
minutes
before starting the sonication to allow for equilibration of the
temperature
in
the cell suspensions to that of the surrounding
Ringer's solution.
that
the
volume
The tubes were
displaced
by
positioned
the
cell
vertically
suspensions (0.6 cm
diameter, 0.7 cm tall) was centered in the focal volume
beam.
such
of
the
In that region, the beam width (0.9 cm) between the half
power points was greater than the largest dimension of the
cell
suspension (0.7 cm), thus insuring a uniform field distribution.
Control samples were prepared in the same way and
in
the
samples.
Ringer's
solution
immersed
simultaneously with the irradiation
These controls were positioned behind
the
transducer
in an ultrasound-free region.
The temperature in the Ringer's solution
was
adjusted
by
37
feeding
the
heater
coils
controller YSI model 72.
a
thermistor
probe
a
proportional
temperature
The actual temperature was
YSI
input of the controller.
from
sensed
by
model 403, which was connected to the
Temperature variations in the Ringer's
solution were less than 0.1°C during experiments.
DIRECT
IMMUNOFLUORESCENCE
Treated
and
PBS
AND
CRITERIA
FOR
CAPPING:
untreated spleen cells were washed in cold PBS and
centrifuged at 500 g.
4°C.
ASSAY
The test tubes were then
transferred
Then 100 pi of FITC—labeled goat anti-mouse Ig diluted in
were
added to the cell pellets.
for the antibody to bind to surface
transferred
After allowing 10 minutes
Ig,
the
test
tubes
from 4 % to the desired temperatures.
cold paraformaldehyde.
cells were resuspended iu
mounted
under
in
containing
Following three washes in PBS, the
two
drops
of
glycerol-PBS
(10:1),
a glass coverslip, and examined for capping with
a Carl Zeiss fluorescence
done
were
Samples were
taken at different time intervals and fixed with PBS
2.5%
to
triplicate.
For
microscope.
Most
experiments
were
each sample of the triplicates, two
hundred cells showing fluorescence
(lg +
cells)
were
randomly
selected and scored for capping.
ANTIBODY-COMPLEMENT (Ab-C) CYTOTOXICITY ASSAY:
untreated B-lymphocytes (2 X 10
were
washed
and
Biological Company,
incubation
at
4°C,
cells per 12 x75 mm test tubes)
resuspended
anti-mouse-Immunoglobulin
Grand
the
Heat-treated and
(anti-Ig)
Island,
in
80
y1
antibodies
NY).
After
of
goat
(Grand
Island
a
10
minute
cell preparations were transferred to
water baths preset at the desired temperature.
After 12 minutes
38
of incubation, 20 yl of
Biological
Company,
suspensions.
intervals
duplicate.
selected
The
by
guinea
Grand
Ab-C
pig
Island,
reactions
transferring
complement
to
were
4°C.
stopped
The
at
viability
different
assays were done in
For each replicate, two hundred cells were
and
Island
NY) were added to the cell
randomly
was measured by trypan blue exclusion.
Further details and comments on methodology
APPENDIX.
(Grand
are
given
in
the
39
IV.
A.
EFFECTS
OF
RESULTS
HYPERTHERMIA
ON
CAPPING
OF ANTIGEN-ANTIBODY
(Ag-Ab) COMPLEXES ON THJ. SURFACE OF. B-LYMPHOCYTES
Al.
KINETICS OF CAPPING DURING HYPERTHERMIC EXPOSURE
B-lymphocytes collected from normal mice spleens were first
incubated with 100 yl FITC-labeled
minutes
at
4°C.
This
anti-Ig
antibodies
for
10
allowed the antibody to bind to B-cell
surface Ig, while preventing capping of the Ig-antilg complexes.
Different cell suspensions
from
were
then
transferred
4°C to 3 7 % , 39°C, 40.5°C, or 42°C.
immediately
Because the volume of
the cell suspension was small (100 yl), the temperature in
cell
preparation
each
reached
its
designated value in less than a
incubation
for
different
minute.
After
various
cell preparations were fixed with 2 milliliters of cold
PBS
containing
Ig-positive
2.5%
paraformaldehyde.
The
than
4
surface
minutes
were
The
results
sufficient
Ig
(Figure
3).
3
also
shows
the
different temperatures above
capping
of
for
indicated
Ig-antilg
complexes
that
the completion of
the
ability
to
Endocytosis of the redistributed
Ig-antilg complexes was observed by 12
Figure
time,
percentage
capping on the surface of those cells that had
cap
of
cells with capped Ig-antilg complexes was estimated
under a fluorescence microscope.
less
intervals
kinetics
37°C.
minutes
of
The
of
Ig-antilg
percentage
incubation.
capping at
of
cells
was gradually reduced from 85% at
37°C to about 10% at 42°C.
The data in Figure 3 indicates that
cells
with
prebound
heat
antilg inhibited capping.
treatment
of
B
To examine the
40
IUU
[
1
1
1
1
—
j
J 37°C
80
UJ
o
— 1 39 °C
•5* 60
u_
O
2
0.
CL
<
-J
.
•
40.5 °C
40
—
5? 20
1
T
a9°r.
•
1
1
A
1
12
MINUTES
Figure
3:
Kinetics
of
cells were
capping of
Ig-anti-Ig
first incubated
complexes.
B
at 4°C with anti-Ig
antibodies, and transferred to 37°C (O), 39°c (A),
40.5°C (•), or 42°C (•) for the times indicated in
the horizontal axis.
The reaction was stopped by
fixation with 2.5% cold paraformaldehyde.
represent the mean ± SD of triplicate assays.
Points
41
effects of heat on Ig-antilg binding and capping in more detail,
three sets of conditions were set up:
with
antilg
for
1) cells
were
incubated
10 minutes at 4°C and then transferred to the
desired temperatures and observed for capping (c in Figure 4);
cells
were
2)
incubated with antilg for 10 minutes at 4°C, washed
to remove unbound antibody and then transferred to
temperatures
to
observe
capping
(a in Figure 4);
heated at the desired temperatures for 60
the
desired
3) cells were
minutes,
washed
and
incubated at 4 % for 10 minutes with antilg and then transferred
to
37 C for capping to occur (b in Figure 4)< The results shown in
Figure 4 demonstrate that in all three cases (4a,b,c) capping of
the Ig-antilg complexes was gradually reduced from about 90%
37°C,
to
less
than 10% at 42°C.
The percentage of capping on
cells that were transferred to the indicated temperatures
washing
out
the
unbound
antibody
(4a)
suggests
This
the
binding
the antibody to surface Ig at the hyperthermic temperatures.
obsevation
pretreatment
hour
after
that
inhibition of capping was not due to an inefficiency in
of
at
in
the
incubation
of
was
further
the
cells
absence
with
of
anti-Ig
supported
by
the
fact
that
at hyperthermic temperatures for 1
antibody,
at
followed
37°C,
by
resulted
10
minutes
a
similar
in
inhibition of capping (4b).
A2.
RECOVERY OF B-LYMPHOCYTES FROM HEAT TREATMENT
AS
MEASURED
BY THE ABILITY TO CAP Ig-ANTI Ig COMPLEXES.
To determine
irreversible
whether
inhibition
hyperthermia
of
caused
Ig-capping,
reversible
B-lymphocytes
preheated for 30 minutes at temperatures up to 43°C.
They
or
were
were
42
37
38
39
40
41
42
TEMPERATURE (°C)
Figure
4:
Inhibition
of
capping of Ig-anti-Ig
hyperthermic levels.
complexes at
B cells were incubated at 4°C
with anti-Ig antibodies, and transferred directly
(c) or after washing (a), to the temperatures shown
on the horizontal axis.
were pretreated
Other sets of B cells (b)
for 60 minutes at the indicated
temperatures, washed, incubated in the cold with
anti-Ig, and transferred to 37°C.
All reactions
were stopped after 10 minutes incubation by fixation with cold paraformaldehyde.
the mean ± SD of triplicate assays.
Points represent
43
then
transferred
to
37°C and allowed to recover for different
intervals of time, after which they were incubated at 37°C
FITC-labeled
capping.
anti-Ig
with
for 10 minutes, then fixed and scored for
The results showed
that
less
than
two
hours
were
sufficient
for the cells to recover the ability to cap Ig after
30 minutes
heat
However,
pretreatment
cells
at
41°C
or
42°C
(Figure
5).
that were preheated at 43°C did not recover the
ability to cap even after 3 1/2 hours incubation at 3 7 % (Figure
5).
After heat
determined
by
treatment,
the
viability
the
cells
was
trypan blue exclusion and was found to be better
than 90%, even for cells heated for
another
of
experiment
in
which
30
cells
minutes
at
43°C.
In
were preheated at 42°C, a
direct correlation was found between the length of the
time
of
pretreatment and the decrease in percentage of capped cells, and
the time required for recovery of capping (Figure 6 ) .
A3.
EFFECT AND RECOVERY OF
B-LYMPHOCYTES
FROM
CYTOCHALASIN-B
disrupts
microfilaments
TREATMENT AT 37°C
Cytochalasin-B is
a
drug
which
(111) and as a result has a variety of effects on cell in vitro.
One
particular effect is the consistent, although in most cases
only partial, inhibition of capping
(112).
of
surface
immunoglobulin
To examine the possibility that heat inhibits capping by
impairing
microfilament
inhibited
by
function
cytochalasin-B,
the
in
the same manner as it is
rates
of
recovery
from
cytochalasin-B treatment at 37°C were compared with the rates of
recovery
from
heat
treatment.
B cells were incubated for 40
minutes at 37 °C with different concentrations of cytochalasin-B.
44
100
Figure
5:
Recovery of B cells from heat treatment as measured
by their ability to cap Ig-anti-Ig complexes.
B
cells were pretreated for 30 minutes at 37 °C (O),
41 °C (it), 426C (•) and 43°C (•).
Cells were then
transferred to 37°C, and samples removed at intervals to determine
37°C.
assays.
the precentage of capping at
Points represent the mean ± SD of triplicate
45
IUU
HOURS
6:
Recovery of B cells from heat treatment as measured
by their ability to cap Ig-anti-Ig complexes.
B
cells were incubated at 37°C for 40 minutes (a), or
at 42 °C for 10 minutes (b), 20 minutes (c) , and 40
minutes (d). Cells were then transferred to 37°C,
and samples removed at intervals to determine the
percentage of capping at 37°C.
Points represent
the mean ± SD of triplicate assays.
46
Part of the cell suspensions was then tested for capping at 37°C
in the presence of the drug, another part was washed once at 4°C
with PBS to remove the
capping,
drug
and
was
immediately
for
and a third part was washed with cold PBS, resuspended
in 300 yl MEM + FBS, and tested for capping
minutes
tested
for
recovery.
after
allowing
30
Cells that were prevented from capping
(<10% capping) recovered the ability to cap surface Ig within 30
minuteB (Figure 7 ) . In
fact,
the
large
variability
in
the
percentage of capping of pretreated cells which were immediately
tested
for
capping
cytochalasin-B
recovering
(10
(Figure
minutes
7),
while
heat
to
treated
that
after washing out
the
cells
recover
can
from
need
cytochalasin-B
about 2 hours for
therefore
be
suggested
microfilament function is not affected by heat in the same
manner or magnitude it is affected by cytochalasin-B.
is
were
In any case, it takes less
cells
complete recovery (Figure 5 ) . It
that
37 C)
suggests
while they were capping.
than 30 minutes for the cells
treatment,
at
Also
it
possible that heat affects other cellular functions required
for recovery of an intact microfilament system.
B.
EFFECTS
OF
HYPERTHERMIA
ON
ANTIBODY-COMPLEMENT
(Ab-C)
CYTOTOXICITY AGAINST B-LYMPHOCYTES AND ITS RELATION TO CAPPING
Bl.
SENSITIVITY OF B-LYMPHOCYTES TO
HEAT TREATMENT:
Ab-C
CYTOTOXICITY
ANTIBODY AND COMPLEMENT TITRATIONS
A cytotoxic antibody titration was performed
quantitative
DURING
measure
of
to
obtain
a
the relative amounts of anti-mouse-Ig
required to lyse unheated or heated mouse B-lymphocytes, in
the
absence
The
or presence of a fixed complement dilution (1:40).
100
to
0
10
50
100
CYTOCHALASIN-B CONCENTRATION ( / i g / m l )
Figure
7:
Effect of cytochalasin-B on capping
of
Ig-anti-Ig
complexes at 37°C, in the presence of cytochalasin
B at the indicated concentrations (•).
Other cells
were incubated with cytochalasin-B for 30 minutes,
washed, and incubated for 10 minutes with anti-Ig
directly after washing
(•), or 30 minutes after
washing (H). All reactions were stopped with 2.5%
cold paraformaldehyde.
SD of triplicate assays.
Points represent the mean ±
48
assay
was
described
performed
in
according
antibody
At
the
standard
procedure
highest
concentration
used
was cytotoxic in the presence and even in the
absence of complement activity,
cells
the
MATERIALS AND METHODS. and is further detailed in
the caption of Figure 8.
(1:10),
to
for
both
heated
or
unheated
(Figure 8 ) . Complement alone had no observalbe effect in
the absence
of
antibody
(=»
CONTROLS).
In
the
absence
of
antibody and complement, viability of the cells dropped from 91%
at 37°C to 72% at 42°C, demonstrating that 42°C hyperthermia was
causing direct cytotoxic effects on cells.
For antibody diluted
between 20 and 80 timeB, Figure 8 clearly demonstrates that Ab-C
cytotoxicity
was
enhanced
at
42°C
relative
to
37°C.
intermediate antibody dilution of 1:30 was selected and used
the
standard
An
as
antibody dilution in subsequent Ab-C cytotoxicity
assays.
With the antibody dilution fixed, another Ab-C cytotoxicity
assay was performed at 37°C or 42°C
dilutions.
Once
again,
with
different
complement
the results shown in Figure 9 clearly
demonstrate that 42°C hyperthermia enhances the cytotoxicity
Ab-C
against
normal mouse B-lymphocytes.
Cytotoxicity at 42°C
was most significant at the lowest complement
but
at
that
intermediate
subsequent
dilution
some
complement
studies.
Ab-C
dilution
lysis
of
1:30
of
dilution
(1:10),
occured at 37°C.
was
used
in
An
all
Complement alone was not cytotoxic at all
dilutions which were used (p > .1; Student's t-test).
49
100
I '10
I'20
l'40
I'80
CONTROLS
ANTIBODY DILUTION
Figure
8: Sensitivity of B-lymphocytes to
during heat treatment:
Ab-C
cytotoxicity
Antibody titration.
Cells
were incubated for 10 minutes at 4°C with different
antibody dilutions (CONTROLS = no Ab), then transferred to 37°C or 428C.
Complement (final dilution
= 1:40) or MEM + FBS were added after 12 minutes.
The Ab-C reaction was stopped after 45 minutes by
transferring
to 4°C.
trypan blue exclusion.
SD.
Viability was measured by
Error bars represent one
50
100
80
>-
t 60
-I
f
s<
>
f,
f
40
O 37°C No Antibody
• 37*C Antibody
20 -
A 42*C No Antibody
• 42°C Antibody
1*10
X
J_
l«20
l>40
J_
l>80
I
CONTROLS
COMPLEMENT DILUTION
Figure
9:
S e n s i t i v i t y of B-lymphocytes to
during
heat
treatment:
Ab-C
Complement
cytotoxicity
titration.
C e l l s were incubated for 10 minutes a t 4°C with or
without antibody ( 1 : 3 0 ) , then
or
42°C.
Different
transferred
dilutions
of
t o 37°C
complement
(CONTROLS = no C) were added a f t e r 12 minutes.
Ab-C r e a c t i o n was stopped by t r a n s f e r r i n g
viability
was measured by trypan b l u e
Error b a r s r e p r e s e n t one SD.
The
t o 4°C.
exclusion.
51
B2.
SENSITIVITY OF
B-LYMPHOCYTES
TO
Ab-C
CYTOTOXICITY
WITH
DURATION OF INCUBATION WITH COMPLEMENT
Cells were incubated with antibody for 10 minutes
at
4°C,
then transferred to 37°C or 42°C.
Complement was added after 12
minutes incubation with antibody.
The Ab-C reaction was stopped
after
different
intervals of time by transferring the cells to
4°C.
Viability
measurements
demonstrated
that
hyperthermic
enhancement of Ab-C cytotoxicity was more significant the longer
the
cells
were
incubated
with
complement
(Figure
10).
A
statistical analysis (Student's t-test) showed that a 45
minute
incubation
with
for
significant
enhancement
Further
complement
experiments
of
at
Ab-C
42°C
was
sufficient
cytotoxicity
(p
<
a
0.05).
were performed with cells incubated for 45
minutes with complement.
B3.
SENSITIVITY OF B-LYMPHOCYTES
TO
Ab-C
CYTOTOXICITY
AFTER
HYPERTHERMIA
The Ab-C cytotoxicity assays described above were performed
during
hyperthermia
distinguish
treatment
between
cellular components or
determine
against
whether
rather
complement
minutes
at
than
molecules,
37°C
on
or
by
performed
at
affecting
then
The
some
the
B-lymphocytes
37 C.
heat
enhanced
Ab-C
results
not
particular
chemistry.
To
cytotoxicity
cellular
activity
were
did
on
Ab-C
function
of antibody or
preheated
transferred
different periods of time, a standard
was
of
antibody-complement
affecting
42°C,
therefore,
effects
hyperthermia
B-lymphocytes
directly
direct
and,
for
40
to 37°C.
After
cytotoxicity
assay
demonstrate
that Ab-C
100
1
T
i
r
80
t 60
-J
m
<
> 40
20
O 37eC
• 37°C
A42°C
A42°C
Controls
Ab+C
Controls
Ab+C
I
I
I
I
15
30
45
60
MINUTES WITH COMPLEMENT
Figure 10: Sensitivity of B-lymphocytes to
Ab-C
cytotoxicity
with duration of incubation with complement.
were
incubated
for
10
without antibody (1:30).
minutes
at
Cells
4°C with
or
Complement (final dilu-
tion = 1:30) or MEM + FBS were added after 12 minutes.
The Ab-C reaction was stopped after different
time intervals by transferring to 4°C.
Viability
was measured by trypan blue exclusion.
Error bars
represent one SD.
53
cytotoxicity was enhanced immediately
(Figure
since
11),
it
implying
could
not
after
42 °C
hyperthermia
that heat sensitized the cells directly
have
affected
Ab
or
C
molecules.
Furthermore, sensitivity to Ab-C cytotoxicity returned to normal
levels
by
2-3
hours
post
suggesting that the target
exposure
cells
had
to
the
42°C
(Figure
ability
to
11),
repair
whatever cellular mechanism was affected by heat.
B4.
RELATION
BETWEEN
HYPERTHERMIC
ENHANCEMENT
OF
Ab-C
CYTOTOXICITY AND HYPERTHERMIC INHIBITION OF CAPPING
Kinetics of recovery from 42°C hyperthermia (Figures 5,
and
11)
strongly
suggest
that
the
increased sensitivity of
B-lymphocytes to Ab-C lysis was due to inhibition of capping
heat
treatment.
To
investigate
6,
further
this
by
possibility,
B-lymphocytes were heated for 40 minutes at 37°C, 42°C, or 43°C.
A
fraction
of
the
cell
suspension
was
then
tested
for
sensitivity to Ab-C lysis at 37°C immediately or 2.5 hours after
hyperthermia.
Another
fraction was tested for capping at 37°C
during the same periods.
direct
correlation
Immediately after
was
found
between
heat
the
treatment,
a
hyperthermic
enhancement of Ab-C cytotoxicity and the hyperthermic inhibition
of capping (Figure 12).
By 2.5 hours
post
exposure
to
42°C,
both the sensitivity to Ab-C cytotoxicity and the ability to cap
Ab-Ag complexes returned to normal levels.
were
still
sensitive
Cells heated at 43°C
to Ab-C cytotoxicity and did not recover
the capping ability even 2.5 hours after heat treatment
12).
and
(Figure
The reversibility of these effects after 42°C hyperthermia
their
irreversibility
after
exposure
to
43°C provide a
54
100
80
t
60
-J
CO
<C
> 40
%9
O 37°C
<fc 37°C
A 42°C
A 42°C
20
±0
Controls
Ab+C
Controls
Ab+C
JL
-L
90
180
MINUTES AFTER HYPERTHERMIA
Figure 11: Sensitivity of B-lymphocytes to
after hyperthermia.
Ab-C
cytotoxicity
Cells were heated for 40 minu-
tes at 37°C or 42°C, then transferred to 37*C.
different
time
intervals
after
hyperthermia,
At
a
standard Ab-C assay was performed at 37 °C (12 min
with 1:30 Ab, then 45 min. with 1:30 C ) . At the
end of each reaction, viability was measured by
trypan blue exclusion.
SD.
Error bars represent one
55
37°C
42°C
43°C
37»C
42°C
43°C
Figure 12: Relation between hyperthermic enhancement
cytotoxicity
capping.
and
hyperthermic
of
inhibition
Ab-C
of
B cells were heated for 40 minutes at the
indicated
temperatures then transferred
Immediately
or
2.5 hours
after
to 37°C.
hyperthermia, a
standard Ab-C assay was performed at 37°C on part
of the cells, while other cells were simultaneously
tested for capping at 37°C.
one SD.
city
Error bars represent
Statistical significance of Ab-C cytotoxi-
was
examined
using
the
Student's
t-test
(differences considered significant if p < 0.05).
56
strong argument in support of the hypothesis
enhancement
that
hyperthermic
of Ab-C cytotoxicity is significantly correlated to
hyperthermic inhibition of capping.
C.
EFFECTS OF, MICROWAVES (JN CAPPING pJF_ 4g.-Ab, COMPLEXES
ON
THE
SURFACE OF. B-LYMPHOCYTES
CI.
KINETICS OF CAPPING FOLLOWING 30 MINUTES HEAT TREATMENT
Before assessing the effects of microwave radiation on
the
ability of B lymphocytes to cap plasma membrane antigen-antibody
complexes, it was necessary to determine how fast capping occurs
at
37°C
following
heat
treatment
Cells were heated for 30 minutes
at different temperatures.
at
37°C,
41°C,
and
42.5°C.
following incubation at these temperatures, the viability of the
cells
was
determined
than 90%.
washed
and
Immediately
by
trypan blue exclusion and was better
after
the
treatment,
the
cells
were
and incubated at 4°C for 10 minutes with 100 yl anti-Ig,
then
transferred
temperature
in
to
37 °C
to
allow
for
capping.
The
each cell preparation reached 37°C in less than
a minute because of the small volume of the cell suspension (100
Pi).
After incubation for different intervals of time,
cell
preparations
were
tixed, washed, and scored for capping.
The results indicated that less than 4 minutes
for
the
various
were
sufficient
completion of capping on the surface of the cells that
had the ability to cap
surface
Ig
(Figure
13).
Endocytosis
(internalization) of antigen-antibody complexes started after 12
minutes
of
incubation
at
Ig-positive cells treated at
37°C.
37°C
While
more than 90% of the
exhibited
capping,
it
was
found that cells pretreated at 41°C lost partially their ability
57
100
MINUTES
Figure 13: Kinetics of capping of Ig-anti-Ig complexes at 37°C
following heat treatment.
Cells were preheated for
30 minutes at 37 °C (•), 41 °C (•), or 42.5°C (A),
washed, incubated with anti-Ig at 4°C, and transferred to 37°C for the times indicated in the horizontal axis.
The reaction was stopped by fixation
with 2.5% cold paraformaldehyde.
the mean ± SD of triplicate assays.
Points represent
58
to
cap,
and that capping on the surface of cells pretreated at
42.5°C was almost totally inhibited (Figure 13).
C2.
EFFECTS OF MICROWAVES AND/OR HYPERTHERMIA ON CAPPING
Cell suspensions were irradiated for 30 minutes
GHz
with
2.45
CW microwaves at intensities up to 100 mW/cm2 at 37°C, 41°C
and 42.5°C.
The specific absorption rate (SAR) was obtained
by
measuring the initial rate of temperature rise in the irradiated
suspensions
when
no
heat
exchange
was
allowed
irradiated cell preparations and the surrounding.
0.45
±
0.04
temperature
W/kg
in
non-irradiated
mW/cm2
per
the
of
incident
microwave-exposed
controls
was
taken
immediately after the shut-off
of
between the
The
the
was
radiation.
suspensions
just
SAR
The
and
the
before the onset and
microwave
field.
The
temperature variation between the irradiated suspensions and the
non-irradiated
controls
was
on
the
Immediately after the microwave exposure,
were
various
controls,
the
Figure 14
results
are
shows
in
Figure 13, that is an inhibition
percentage
of
capping
that
for
agreement
at
the
the
0.1°C.
preparations
pretreated at 42.5 °C.
microwave-treated
levels.
of Ig-positive cells was gradually
than
5%
for
Figure 14 also show*) that there is
capping
between
cells and the non-irradiated controls as
long as both were maintained at the
0.1; Student's t-test).
nonirradiated
hyperthermic
no significant difference in the percentage of
the
+
with those shown in
reduced from 90% at 37°C, to 52% at 41°C, to less
cells
of
tested for capping after 9 minutes incubation at 37°C with
100 yl anti-Ig.
The
order
same
temperatures.
(P
>
59
100
37°C
o— -"
W
80
UJ
O
•o»
6 0
4I"C
O
g 40
E
Q.
<
"
Controls
MW
20
at-
•--^i
J.
10
^j
£fc
''ft
X
25
X
50
too
INTENSITY (mW/cm2)
Figure 14:
Percentage of capping of Ig-positive
exposed to water bath (
Suspensions
of
B
42.S°C
cells pre-
) and microwaves (
B-lymphocytes
were
exposed
).
to
2.45 GHz microwaves for 30 minutes at different
intensities and temperatures.
The non-irradiated
controls were maintained at the same temperatures
at all times.
Samples were then washed and tested
for capping at 37°C.
Points represent the mean ±
SD of triplicate assays.
60
In another experiment, the possibility of a
of
the
microwave
field
on
capping
was
direct
investigated, while
capping of the Ig-antilg complexes was taking place.
incubated with 100 yl FITC-labeled anti-Ig
4°C.
Ig,
action
for
Cells were
10
minutes
at
Incubation at 4°C allowed the antibody to bind to surface
while
capping.
preventing
After
the
addition
antibody-antigen
of
ml
of
cold
to
PBS, the cell
were
chamber
to the control water bath, both adjusted to operate
at 38.5°C.
transferred
from
suspensions
or
immediately
3.5
complexes
the
microwave
After 10 minutes exposure to microwaves at different
intensities,
the
paraformaldehyde,
cells
fixed
with
2.5%
cold
and scored for capping.
As shown in
Figure 15, the results demonstrate that there is no
significant
difference
in
washed,
were
the
percentage
of
capping
between
the
microwave-exposed cells and the controls, even at intensities as
high as 100 mW/cm2.
D.
EFFECTS 0F_
AMPLITUDE
MODULATED
RADIO
FREQUENCY
(RF)
ON
CAPPING pj£ AgrAJb, COMPLEXES ON. THE SURFACE OF. B-LYMPHOCYTES
Dl.
EFFECTS
OF
AMPLITUDE
MODULATED
RF
FIELDS
AND/OR
HYPERTHERMIA ON CAPPING
Cell suspensions were irradiated for 30
frequency
amplitude
modulated
147
minutes
MHz
RF
intensities ranged between 0.1 and 48 mW/cm2.
groups
were
temperature.
the
onset
treated
fields.
Sham and
low
The
exposed
simultaneously and maintained at the same
Temperature measurements were
and
with
taken
just
before
immediately after the shut-off of the RF field.
Temperature variation between
the
irradiated
suspensions
and
61
100
10
25
100
INTENSITY (mW/cm 2 )
Figure 15: Percentage of capping of Ig-positive B cells during
exposure to water bath and microwave at 38.5°C.
B
cells were incubated at 4°C with 100 yl anti-Ig for
10 minutes.
PBS were
After antibody binding, 3.5 ml of cold
added,
transferred
to
and
the
the
cell
water
syspensions were
bath
microwave exposure chamber
(Q) or
to
the
(H), both at 38.5°C.
All reactions were stopped after 10 minutes incubation
by
Points
assays.
fixation
represent
with
the
cold
mean
paraformaldehyde.
± SD
of
triplicate
controls
was
less than 0.1°C.
Immediately after the exposure,
both control and irradiated groups were tested
37°C
for
capping
at
following the procedure outlined in MATERIALS AND METHODS.
The results are shown in Figures 16, 17, and 18 for 9, 16, or 60
Hz
modulation
parentheses
frequency,
represent
the
respectively.
actual
The
numbers
intensity that was measured
during the RF exposure of that particular sample.
demonstrate
The
of
was
capping
no
between
significant
the
difference
RF-treated
in
temperatures
(p>0.4,
student's t-test).
the
cells and the
non-irradiated controls as long as both were maintained
same
results
that at any of the modulation frequencies and power
densities used, there
percentage
in
at
the
While more than
90% of Ig-positive cells preheated at 37°C capped membrane-bound
Ag-Ab complexes (upper set of curves in Figures 16, 17, and 18),
less than 10% capping occurred when cells were preheated at 42°C
(lower set of curves in Figures 16, 17, and 18).
E.
EFFECTS OF ULTRASOUND ON CAPPING OF AjfAb COMPLEXES
ON
THE
SURFACE OF B-LYMPHOCYTES
El.
EFFECTS OF ULTRASOUND AND/OR HYPERTHERMIA ON CAPPING
Samples were prepared as described in MATERIALS AND METHODS
and immersed in
the
temperature-controlled
water
bath.
The
temperature in the cell suspensions reached its designated value
in
less than 2 minutes (Figure 19).
initiated 5 minutes after immersion.
were
tested
for
Exposure to ultrasound was
Sonicated and
cells
capping at 37°C immediately after a 15 minute
irradiaion at different temperatures and intensities.
demonstrates that
sham
for
the
non-irradiated
shams,
Figure 20
capping
of
63
100
i
r
«
1
T
(a,1)
(0S7
j 80
_J
bJ
U
'
f
1
,M„
r -
\
'
( 3 61
' '
»60
u.
O
- i 37°C
(45.1,
•Controls (no RF)
- R F O H z Modulation)
O
Z 40
E
a
<
u
^
20h
(0.55)
(48.4)
(2,67)
(14.1)
• -42°C
1
X
0.4
0.1
*"
1.6
J
J_
I2.B
51.2
INTENSITY (mW/cm 2 )
Figure 16:
Percentage of capping of
exposed
(
for 30 minutes
) and
to
(
cells pre-
37 °C or 42 °C water
9 Hz amplitude modulated
(RF) radiation
linear.
Ig-positive B
).
radio
bath
frequency
Horizontal scale not
Error bars represent one SD.
64
IUU
OT80
(O.ll)
(0.21)
(0.44)
(7.08)
(0.87)
(13.8)
(27.9)
(450)
(3,43)
•? 60
Controls (no RF)
R F ( I 6 Hz Modulation)
b.
O
(9
S 40
a
a,
<
u
sS 20 ',„„,
\\
nlfT*
0.1
0.2
J
W'*7'
(0.22)
I
•
|i
I
0.4
(0.87)
(1.72)
null
I
0.8
•
I
1.6
(3 44)
l II i
1
I
3.2
(6,89)
(13.9)
II I n
I
6.4
lift •
I
12,8
(276) (46.9)
•!
25.6
-~-TJ/\2'C
51.2
2
INTENSITY (mW/cm )
Figure 17:
Percentage of capping of Ig-positive
B cells pre-
exposed for 30 minutes to 37"C or 42°C water bath
(
(
) and 16 Hz amplitude modulated RF radiation
) . Horizontal scale not linear.
represent one SD,
Error bars
65
100
V)
- I 80
-J
UJ
<J
**
T
1
(0.M)
(0.22)
1
(043)
1
,0^0)
1
(,, 8 0 )
r
(3.72)
i
(7.04)
1
(14.1)
r
(28.1)
(44,9)
_
60
•Controls ( no R F )
R F ( 6 0 H z Modulotion)
O
Z
a.
a.
o<
^
40
20
"J"
(3.44)
{7 23
' '
(14.5)
J
0.8
1.6
3.2
6.4
I
(26.0) (46.0)
I
12.8
25.6
1—
51.2
INTENSITY (mW/cm2)
Figure 18:
Percentage of capping of Ig-positive
B
cells pre-
exposed for 30 minutes to 37°C or 42"C water bath
(
(
) and 60 Hz amplitude modulated RF radiation
).
Horizontal scale not linear.
represent one SD.
Error bars
66
43
-!_-/
,
,
,
t
j.rrr-\-:.-ir:--"-:r:--..:r--"r.---.-^. v .
42
/ "
41
^
U
£-40
UJ
K
H 39
No Ultrasound
5W/cm*
IOW/cm*
20W/cm*
<
K
UJ
| 38
UJ
r37
^r/-:--:A".tVfl:rZL~7~rr.:?.k
r
36 Immtrtion
Ultrasound
Ott
Ultrasound
On
I
5
1
10
15
20
TIME (Minutes)
Figure 19: Temperature variation in sonicated and control cell
suspensions.
A rapid 0.1°C temperature rise (fall)
component in the sonicated cell suspensions was
observed at the onset (shut-off) of the ultrasound
beam.
This artifact was due to visco-elasticity at
the boundary of the fine wire thermocouple which
was used.
67
100
"-*f
CO
Hii- • —•
f"
fti
80
Id
U
«? 6 0
At- fr-
O
a
z
a.
a.
<
20
-O—37°C
- A — 4 1 »C
-a—42eC
•Ultrasound
Controls
40
¥—-*2.5
5
INTENSITY (W/cm2)
Figure 2 0 :
10
Percentage of capping of I g - p o s i t i v e
B
c e l l s pre-
exposed for 20 min t o 37°C, 41°C, or 42*C hyperthermia
(-
- - ) and 15 min t o ultrasound
Horizontal s c a l e not l i n e a r .
one SD.
(
).
Error bars, r e p r e s e n t
68
membrane-bound
Ag-Ab
complexes was reduced from 90% at 37 °C to
60Z at 41°C, to 20Z for cells pretreated at 42°C.
significant
difference
in
the
percentage
There was
of capping between
sonicated and sham cells when the temperature of the
was
37°C
(p>0.6
Student's
t-test).
However,
significant reduction of capping was observed in
to
water-bath
a further and
cells
exposed
W/cm 2 when the temperature of the water bath was 41°C or
20
42 °C (p<0.05 Student's t-test).
have
no
been
observed
caused
in
by
cell
the
This reduction of capping could
0.5°C
suspensions
temperature-rise
exposed
which
to 20 W/cm
was
ultrasound
(Figure 19). To test this possibility, control cell suspensions
were heated for 20 minutes
37°C
and
at
different
temperatures
43°C, while other suspensions were heated at the same
temperatures during the first 5 minutes, and for
15
the
remaining
minutes the temperature was raised by 0.5°C, thus simulating
the ultrasound-induced temperature-rise in
to 20 W/cm 2.
control
suspensions
exposed
Immediately after heat treatment, all samples were
tested for capping at 37°C.
Figure 21 demonstrates that for the
cells (solid curve), capping was gradually inhibited at
temperatures above 37°C.
observed
on
cells
dashed curve).
one
between
obtained
A further
which
reduction
underwent
a
in
capping
0 . 5 % rise (Figure 21,
This reduction in capping was comparable to
in
cells
exposed
to
was
W/cm 2 ultrasound.
observed
No
reduction
in
temperature
was
(p > 0.8), Student's t-test), because at
that temperature capping was not very sensitive
increments
to
when
the
significant
37°C
capping
20
was
the
temperature
of the order of 0.5°C, as can be seen from the slope
of the solid curve in Figure 21.
Reduction of capping was
most
69
100
37
38
39
40
41
42
43
INITIAL TEMPERATURE (°C)
Figure 21:
Percentage of capping of Ig-positive
B
cells pre-
heated for 20 min at temperatures indicated on the
horizontal axis (
) , and cells pre-heated for 5
rain at the indicated temperatures followed by 15
min
incubation
at
0.5°C
above
(initial) temperatures (- - - ) .
sent one SD.
the
indicated
Error bars repre-
70
significant
at 41°C and 42°C (p < 0.05, Student's t-test) where
sensitivity to small temperature increments was greatest (Figure
21).
These observations strongly
support
the
idea
reduction of capping on cells exposed to 20 W/cm
that
the
ultrasound was
thermally induced.
E2.
RECOVERY FROM ULTRASOUND AND HEAT TREATMENT
To determine whether ultrasound and heat
reversible
or
irreversible
reduction
of
treatment
capping, cells were
exposed for 15 minutes to 20 W/cm2 ultrasound,
and
42°C.
at
37°C,
After different
intervals of time, samples were tested for capping.
demonstrated that for the non-irradiated controls,
heated
two
and
22).
which
an
hour
(85%
capping),
cells
were
heated
at
The shift between the CONTROL curves
(Figure
dashed curves), and the ULTRASOUND curves (Figure 22, solid
curved) which was observed at 41°C and 42°C, was believed to
due
42°C
Partial recovery of capping was also observed on
irradiated cells.
22,
cells
a half hours were required for partial recovery
(67% capping) when non-irradiated
(Figure
The results
at 41°C recovered almost completely the ability to
cap antigen-antibody complexes within
while
41°C,
Immediately after irradiation, the cell suspensions
were transferred to 37°C to allow for recovery.
were
caused
to
the
suspensions.
previous
0.5°C
These
studies
temperature-rise
observations
on
are
in
the
sonicated
in
agreement
with
be
cell
the
the inhibition and recovery of capping on
heat treated B-lymphocytes (Figures 5 and 6 ) .
71
100
O — 3 7 °C
A — 4 1 °C
a—42 °C
0.5
1.0
1.5
2.0
2.5
HOURS POST"IRRADIATION
Figure 22:
Recovery of I g - p o s i t i v e
B c e l l s from
h e a t (- - - ) and 20 W/cm
exposure t o
ultrasound (-
•)# as
measured by the ability to cap Ag-Ab complexes,
Error bars represent one SD.
72
V.
A.
DISCUSSION
HYPERTHERMIC INHIBITIONflj_CAPPING
There is increasing evidence that hyperthermia can
beneficial
effect
on host defense mechanisms.
to bacterial or viral infections
(19-22),
and
in
can have
different
animal
adverse
other
effects
activity
of
beneficial,
whereas
on
such
killer
that
local
whole-body
followed
by
local
tumor
cell-mediated
functions
heating
of
and
as
(31-33).
tumors
hyperthermia
Shah
heating
may
the
Some
may
be
result
in
Dickson
(35,36)
of VX2 tumor bearing rabbits was
regression
immunity,
immune
T-lymphocytes
immunosuppression.
observed
hyperthermic
studies indicate that hyperthermia
reports have indicated that
significant
species
enhanced mitogenic responses of human lymphocytes
However,
cytolytic
a
Better response
(23-25) have been reported to occur as a result of
treatment.
have
and
a
marked
increase
in
whereas total body hyperthermia led to
temporary restraint of tumor growth followed by a return
to
an
exponential increase in tumor volume.
The present
capping
of
study
Ag-Ab
indicates
complexes
on
that
the
hyperthermia
surface
inhibits
of normal mouse
B-lymphocytes (Figures 3,4). When the cells were heated in
presence
of
antibody,
the
percentage
of
capping
gradually from more than 90% at 37°C, to less than 10%
(Figure
4c).
Similar
results
the
dropped
at
42 °C
were obtained when the unbound
antibody was washed out before heating the cells, ruling out the
possibility
inefficiency
that
in
inhibition
binding
of
of
capping
antibodies
was
to
Ig
due
to
receptors
an
at
73
hyperthermic
was
temperatures
(Figure 4a) .
the fact that heat pretreatment
the
two hours.
were
ability
at
37°C,
for 30 minutes at
to cap antigen-antibody
However, no such recovery was
preheated
at
resulted
43 °C
in
a
(figure 4 b ) .
Cells that were preheated
recovered
idea
in the absence of antibody,
followed by incubation with antibody
similar inhibition of capping
In support of this
(Figure
41°C
42°C
complexes within
observed
5).
or
This
when
cells
threshold
for
irreversible inhibition of capping occurs at a temperature where
others have noted
mammalian
a
cells
transition
as
measured
survival curves (109).
time
at
43.5%,
in
by
the
a
thermal
change
A similar transition
in
response
slope of heat
temperature,
stem
cell
Moreover, the same researchers noted that
above
43.5°C the survival curve no longer exhibited a shoulder
implying
a
loss
damage ( 1 1 0 ) .
sane
in
ability
to repair sublethal
It is interesting to speculate that
region
heat-induced
perhaps
the
metabolic, cytoskeletal, and/or membrane function which is
necessary
for
capping
in
B-lymphocytes,
and
which
irreversibly inactivated above 43°C, may also be involved
control
of
sublethal
damage.
cell
proliferation
and
repair
of
completely
determined.
Capping
the presence of metabolic inhibitors
evidence
that
the
the capping process.
is
in the
heat-induced
The mechanisms which are responsible for capping
been
this
has been noted in experiments to determine the
thermal inactivation energy for granulocyte-monocyte
proliferation.
of
have
not
is inhibited at 4°C, or in
(97).
There is
increasing
cytoskeletal system is actively
involved in
Colchicine, a microtubular
inhibitor,
can
74
enhance
capping
microfilament
(97,112).
tumor
(97).
function
(111),
may
enhance
reactions by disrupting
and
inhibits
capping
that
capping
of
reversibly
cytotoxicity
and
preventing
Ag-Ab complexes
heat
by
impairs
that heat treatment of
antibody-complement
shedding
possible
Ig-antilg
drug which
the cytoskeletal system
subsequent
Similarily, it is
a
inhibits
Recently it has been suggested
cells
capping
Cytochalasin-B,
treatment
disrupting
the
(27,28).
of
B
cells
cytoskeletal
microfilament system directly, or inhibiting a process necessary
for its function.
the
The results presented herein demonstate
processes
involved
in
inhibition
of
capping
cytochalasin-B were fully reversible and much
more
recovery
Thus
from
heat pretreatment
(Figure 7 ) .
rapid
that
by
than
hyperthermia
may inhibit a slowly recovering metabolic process necessary
for
microfilament function, whereas recovery from cytochalasin-B may
be simply microfilament repolymerization or reorganization which
has
faster
kinetics
doses used in this
B.
HYPERTHERMIC
and
is fully reversible even at the high
study.
ENHANCEMENT OF. ANTIBODY-COMPLEMENT
Recent studies have indicated
may
that
in. vitro
hyperthermia
enhance tumor cell immunogenicity .in vivo (113), as well as
sensitivity
to
cytolysis
(27,28,35,114,115).
Jasiewicz
heating of synchronized
cultures
by
immunological
been
lymphoma
reported
cells
in
(115),
effectors
and Dickson (114) reported
of
rat
adenocarcinoma
markedly enhanced sensitivity to lysis by Ab-C.
have
CYTOTOXICITY
Similar
cells
results
other cell lines such as Moloney
human
colon
tumor
cells
that
(27),
virus
and
75
virus-transformed
hamster PARA-7 cells ( 2 8 ) .
In the present
B-lymphocytes
study,
were
it
was
found
heat
treated
more sensitive to the cytotoxic activity of
Ab-C than cells maintained at 37°C.
Ab-C lysis was observed during
treatment
that
(Figures 8 - 1 1 ) .
The enhanced
or
immediately
sensitivity
following
Ab-C cytotoxicity returned
to
heat
to normal
levels by 2.5 hours post exposure at 42 °C hyperthermia,
but
no
recovery was observed when cells were preheated at 43 °C (Figures
11-12).
Several
mechanisms
sensitization
suggested
Ehrlich
of
cells
may
to
explain
the
Ab-C lysis.
Mondovi et al.
that their observation of increased
ascites
cells
in
Swiss
and Dickson
(114) proposed that exposure
some
surface
antibody
with
modification
cell
immunogenicity
in. vivo
reactions
vitro
i&
antigens.
(113)
immunogenicity
Similarly,
to
heat
facilitating
Thus
the
(113) and increased
(27,28,35,114,115)
Jasiewicz
may
interaction
apparent
heated
cell
surface.
not suggest
to
immune
cells
may
cells
provide
more
antibody
binding
sites because both heated and non-heated human colon tumor
equivalent
antibodies
(27).
amounts
of
specific
anti-tumor
cells
cell
Other studies have indicated that the relative
efficiency of Ab-C cytotoxicity may reflect
to
on
However, studies on antibody adsorption do
that heated
adsorbed
of
increased
sensitivity
of
produce
represent an increase or more favorable exposure of antigens
the
of
mice was due to an antigenic
change in the cell membrane after heating.
cell
heat-induced
target
cell
repair
(116).
Thus hyperthermic
complement
enhancement
the ability
of
the
damage to the cell membrane
of Ab-C
cytotoxicity
may
76
be
the
result of inactivation by heat of these repair
so that fewer lytic events would be required
and cell
death;
complement
hence
lower
of
between
cytotoxicity
and
B-lymphocytes.
heat-induced
inhibition
It
was
found
of
capping
on
a
direct
of
the
Ab-C
surface
of
that both events (enhancement of
Cells
heated
at
even
43°C
not
2.5
were
occur
recover
hours
still
the
reversibly
affected
both
affected
by
42°C
at
immediately
42°C
post-exposure
to
to
cytotoxicity.
of
heat.
and
Ag-Ab
These
and
irreversibly
enhancement
responsible
of
Ab-C
However, a more plausible explanation may be that
heat affected
cellular components are primarily
for inhibition of capping and subsequent
endocytosis
complexes,
sites
acitivity.
Ab-C
components which are
hyperthermia,
capping
to
cap
by heat treatment at 43°C, may be directly
inhibition
(Figure
sensitive
ability
observations suggest that the same cellular
thus
leaving
Cell
concentrations
death
of
more
could
complement.
found that at least 30 minutes
were
or
42°C or 43°C hyperthermia, and that they return to normal
complexes
the
of
enhancement
cytotoxicity and inhibition of capping)
cytotoxicity and did
for
antibody
evidence
levels with comparable kinetics after heating
12).
damage
could mediate cell death.
correlation
after
for membrane
concentrations
The data presented herein provides
Ab-C
enzymes,
necessary
binding
then
Ag-Ab complexes, since
mediated
In support
of
of
Ag-Ab
complement
by
lower
of this idea, it was
incubation
for significant Ab-C
By that time, non-heated
be
for
responsible
with
complement
lysis at 42°C (Figure 1 0 ) .
cells would have capped and removed
these
processes
are
completed
all
within
77
10 - 12
minutes
at normal temperatures.
With no binding
for complement
activity, no
complement-mediated
take
This
with the observations
place.
agrees
dilutions of anti-Ig used, no Ab-C
Ab-C
cytotoxicity
complement
C.
increases
cytolysis
can
that with the
lysis occurs at
with
sites
37°C,
while
duration of incubation with
at 42°C (Figure 1 0 ) .
EFFECTS 0F_ MICROWAVES
AND HYPERTHERMIA ON CAPPING
Many of the microwave effects on immune reactions have been
attributed
to
different
components
non-thermal
a
thermal
action
of
effects
the
is
of
the
microwave
immune system.
still
field
on
The existence of
controversial.
To
determine
whether or not exposure to microwaves had non-thermal
effects on
capping,
control in
it was necessary
the irradiated
demonstrated
process
immediately
suspensions, since the earlier part
that capping
above
to have a good temperature
37°C.
after
is an extremely temperature
sensitive
The capping assay was performed
exposure
intensities and temperatures.
the
in this study
to
microwaves
during or
at
different
The results demonstrated
that for
contol cells, the percentage of capping dropped from 90% at
37°C, to less than 5% for cells that were
(Figure
14).
There
was
no
significant
preheated
at
difference
42.5°C
in
the
percentage of capping between control and
irradiated
any
used, as long as both
of
the
intensities and temperatures
control
and
microwave
preparations
were
(Figures
14,15).
synergism
temperature
specific and hyperthermia
No
kept
at
cells
the
between
effects on capping was observed.
at
same
field
78
D.
EFFECTS
fl£
AMPLITUDE
MODULATED
RADIOFREQUENCY
AND
HYPERTHERMIA OJ. CAPPING
Cellular
membranes
have
been
suggested
as
a
i n t e r a c t i o n between r a d i o f r e q u e n c y (RF) r a d i a t i o n and
systems.
RF
One of t h e most commonly r e p o r t e d e f f e c t s
radiation
has
been
the r e l e a s e
t i s s u e during i r r a d i a t i o n
reported
to
occur
at
narrow
of
biological
of
low-level
of c a l c i u m i o n s from b r a i n
(47,117-120).
a
site
The
power
effect
density
has
been
window
(0.83
mW/cm 2 ), and at a m o d u l a t i o n f r e q u e n c y window (16 Hz) w i t h i n
r a n g e of f r e q u e n c i e s a s s o c i a t e d with
Bawin
and
her
associates
the
cooperative
p l a c e between a d j a c e n t c a t i o n b i n d i n g
of
the
plasma
cooperative
proposed
to
biological
membranes.
phenomena
electroencephalogram.
( 1 1 7 ) have a t t r i b u t e d
c a l c i u m i o n s to a f i e l d - i n d u c e d
sites
the e f f l u x
taking
a t the o u t e r
surface
S e v e r a l t h e o r e t i c a l models b a s e d on
and
long-range
coherence
e x p l a i n the i n t e r a c t i o n of o s c i l l a t i n g
have
been
fields
with
membranes ( 4 3 - 4 5 ) .
a
change
such an e f f e c t
change
intracellular
might
(121).
of
in
does e x i s t ,
concentrations
functions
of
interaction
I t i s not y e t known whether a m p l i t u d e - m o d u l a t e d
induce
the
a
in
various
One p a r t i c u l a r
intracellular
fields
calcium c o n c e n t r a t i o n s .
change
alter
RF
intracellular
metabolic
event which
calcium
is
and
calcium
cellular
altered
concentration
If
by
is
a
the
r e d i s t r i b u t i o n and capping of Ag-Ab c o m p l e x e s on t h e s u r f a c e
of
B-lymphocytes,
to
following
surface immunoglobulins.
that
the
introduction
the
b i n d i n g of a n t i b o d y m o l e c u l e s
Schreiner
of
and
Unanue
(97)
reported
a calcium ionophore i n t o the
membrane o f B - l y m p h o c y t e s c o m p l e t e l y s u p p r e s s e s c a p p i n g .
plasma
If
the
79
ionophore-mediated calcium influx occurs
the
cap
is
completely
process which involves
These
observations
disrupted
the
and
a
lymphocyte's
cap
formation,
metabolically active
cytoBkeletal
system.
other considerations led Schreiner and
Unanue (97) to suggest a model for
dependent
by
after
capping
whereby
a
calcium
bond between antigen receptors and calcium responsive
cytoplasmic
receptors
microfilaments
through
the
effects
the
transport
of
the
plane of the membrane without affecting
other components of the membrane.
If intracellular calcium levels are shifted by
of
application
an amplitude modulated RF field, the change might affect the
capping
of
membrane
hypothesis,
a
there are any
capping.
bound
Ag-Ab
complexes.
series
of
experiments
effects
of
amplitude
To
test
this
was conducted to see if
modulated
RF
fields
on
The 16 Hz modulation frequency was chosen because the
reported effects on calcium efflux from brain tissue occurred at
16 Hz (47,117-120).
tested
because
Two other modulation frequencies were
there
is
no
membranes should react at
the
neurons.
of
A
wide
range
a priori reason why B-lymphocyte
same
frequencies
as
in
order
power density window.
to
those
of
power densities was investigated,
including the range over which the calcium efflux
reported,
also
reduce
effects
were
the possibility of missing any
the irradiations were
done
during
heat
treatment at 37 °C or 42 °C.
The results did not demonstrate
modulated
RF
radiation
on
capping
any
effect
(Figures
of
16-18).
modulation frequencies and power densities used, no
difference
in
the
amplitude
At the
significant
percentage of capping was found between the
80
controls and cells that were pre-expoBed to amplitude
modulated
RF radiation, as long as both preparations were kept at the same
temperatures.
The percentage,of capping was reduced from about
90% at 37°C to about 10% at
42°C,
in
agreement
with
results
obtained previously in this study.
Although no evidence of amplitude modulated field
effects
on
specific
capping was found, these results cannot be regarded
as definitive.
Much additional work is necessary
the
or
existence
nonexistence
to
determine
of frequency and power density
window effects on capping.
E.
EFFECTS OF ULTRASOUND AND HYPERTHERMIA ON CAPPING
Several field specific mechanisms
explain
some
(49-55).
of
have
been
proposed
to
the reported biological effects of ultrasound
These mechanisms include cavitation (49-53),
acoustic
microstreaming (54), and radiation force effects (55). However,
ultrasound
bioeffects
may
also
be
caused
induced heating of the sonicated tissues.
by ultrasonically
Marmor et al.
(122)
observed tumor regression after localized ultrasound heating and
suggested
of
that
patient
suggestions
such effect might be related to the stimulation
antitumor
immunity.
Similar
observations
and
were made earlier when hyperthermia was not induced
by ultrasound (18).
In the present study, the combined
and
effects
of
ultrasound
hyperthermia were analysed immediately after treatment.
before,
a
suspensions
good
temperature
control
was
necessary.
The
in
the
temperature
sonicated
was
within acceptable limits (0.1°C) at intensities up to
As
cell
controlled
5
W/cm 2 .
81
However,
at
the
highest
intensity
temperature-rise was sustained in
This
temperature-rise
(20 W/cm 2 ), a 0.5°C
used
the
irradiated
suspensions.
was sufficient for significant reduction
of capping on the surface of sonicated ceils with respect to the
controls, when the controls were
kept
significant
percentage
difference
in
the
at
41°C
or
42°C.
No
of capping between
sonicated and control cells was observed at lower intensities at
41°C or 42°C, or at any intensity when the temperature was
(Figure
20).
The
reason
that
no
significant
capping was observed on cells exposed to 20 W/cm2
that
37°C
reduction of
at
37°C
was
capping was less sensitive to small temperature increments
at 37 °C than at hyperthermic temperatures, as
21.
Partial
to
shown
in
Figure
total recovery from inhibition of capping was
observed within 2.5 hours post-exposure to 20
and/or hyperthermia.
W/cm 2
ultrasound
The difference in the kinetics of recovery
curves
between
sonicated and control cells (Figure 22) was due
to the
further
temperature-rise
sonicated
preparations.
These
which
results
was
observed
in
demonstrate
the
that the
observed ultrasound effects on capping are thermally induced.
F.
SUMMARY
It has been demonstrated that capping
was
significantly
B-lymphocytes.
above
42 °C.
of
Ag-Ab
complexes
reduced on the surface of heat-treated mouse
Total inhibition of capping was observed at
and
The ability to cap was recovered within two hours
post hyperthermia, as long as the
temperature
did
not
exceed
42°C.
A
strong
correlation
was
found
between
hyperthermic
82
enhancement
of Ab-C cytotoxicity and hyperthermic
capping, suggesting that the increased
sensitivity
resulted
more
from
complement
the
availability
activity.
Further
of
inhibition of
to Ab-C
binding
lysis
sites
for
studies are needed to test more
fully this hypothesis and, in particular,
to
determine
if
it
applies to cancer cells.
Figure
pretreatment
23
shows
at
the
in
a
significant
observation, it appears
essential
of
capping
during
1°C
increase
reduction
that
a
irradiation
in
of
good
temperature
experiments,
and properly performing
significant
non-thermal
following
exposure
effects
to
on
radiofrequency, or ultrasound.
may
From
this
control
is
especially
if
carefully
control system, no
capping
microwavea,
At any of
With the
temperature
heat
temperature
capping.
non-thermal effects are to be investigated.
designed
to
temperatures between 37°C and 43°C.
these temperatures, even
result
sensitivity
were
observed
amplitude
modulated
Further reduction of capping was
observed on cells exposed to 20 W/cm 2 ultrasound at 41° C or 42°C
bath temperatures, where capping was
temperature
variations.
most
sensitive
to
However, this significant reduction of
capping was explainable on the basis of thermal effectB
The mechanisms responsible for the hyperthermic
of
capping
are
not
known.
There
is
is
possible
capping by
that
disrupting
only.
inhibition
evidence
cytoskeletal system is involved in the capping
it
small
that
process.
heat treatment of B-lymphocytes
the
cytoskeletal
microfilament
directly, or by inhibiting a process necessary
This hypothesis was tested by comparing
the
Thus,
inhibits
system
for its function.
the rates of recovery of
83
100
CO
UJ
o
u.
o
e> 4 0 Q.
Q.
<
38
39
40
41
TEMPERATURE °C
Figure 23:
Inhibition of capping of
Ig-anti-Ig
temperatures associated with fever.
sent mean
study. .
complexes
at
Points repre-
± SD of all assays done during
this
84
B-cells
from
heat
cytochalasin-B,
a
treatment
to
the
microfilament
rates
of recovery from
inhibitor.
Recovery
from
cytochalasin-B was fully reversible and more rapid than recovery
from
heat
pretreatment,
which
suggests that hyperthermia may
i
inhibit a slowly recovering process necessary for
function,
whereas
microfilament
recovery
from
xepolymerizatiou
microfilament
cytochalasin-B may be simply
or
reorganization
which
has
faster kinetics and is fully reversible.
In
vivo
significant
hyperthermic
immunological
determined.
complexes
(97).
inhibition
It
may
has
of
consequences
been
suggested
capping
which
that
B-cell proliferation.
yet
to be
of
Ag-Ab
capping
the
with
immune
components
of
of
capping
might
On the other hand, inhibition of
antigen-Ig capping may facilitate
other
have
be involved in triggering B-cell differentiation
Consequently, hyperthermic inhibition
depress
are
may
the
cooperation
system,
of
B-cells
particularly
macrophages and helper T-cells, by slowing the removal of B-cell
surface Ig by endocytosis of capped Ag-Ig complexes.
Figure 23 shows that significant inhibition of
in
vitro
hyperthermia
associated with fever.
role fever (or
responses.
rational
An
basis
occurs
the
temperature
by
range
Only further research will determine the
hyperthermia)
understanding
for
over
capping
the
plays
in
regulation
of
immune
of this role will help provide a
design
of
therapeutic
hyperthermia
protocols.
Finally, the relevance of the
observations
made
in
this
study, and their applicability to cancer cells, are particularly
important.
Further
research
in
this direction is definitely
85
essential to determine fully whether hyperthermia
immunogenicity
of
cancer
cells
by
inhibiting
enhances
the
capping
and
subsequent shedding of membrane bound Ag-Ab complexes.
86
APPENDIX
FURTHER COMMENTS ON METHODOLOGY
During
the
initial experimental phase of this study, many
problems were encountered which resulted in poor reproducibility
and large variability
resolved
by
in
carrying
each type of
assay
experiments
was
the
results.
These
problems
out a set of preliminary experiments for
which
was
used.
The
purpose
critically
results
and variability.
reviewed
were
and
obtained.
usually necessary before
satisfactory
these •
for
improved
whenever
As
as three modifications were
many
obtaining
variability.
analysed
The experimental protocol was
non-satisfactory
reproducible
results
with
The final protocol for a particular
type of assay served as the standard protocol.
this
of
to develop an optimal protocol for each assay.
After each of these experiments, the results were
reproducibility
were
The
purpose
of
APPENDIX is to list the major factors which were generally
found crucial for success of the experiments.
i) Each experiment was
possible.
terminated
in
the
shortest
time
Before the animals were sacrificed, water baths were
set at the desired temperatures, all equipment and reagents were
prepared, and all test tubes to be used were
thus
possible
labeled.
It
was
to process the freshly isolated cells as quickly
as possible after sacrificing the animals.
ii) Cells were never kept at 4°C for more than ten minutes.
It was found that preincubation for thirty minutes
or
more
at
4°C or at 0°C (on ice) sometimes affected the capping efficiency
at
37°C.
This may have been caused by the depolymerization of
87
microtubules at low temperatures.
iii) Temperature control was the most decisive
obtaining
reproducible
results
with
factor
small
variability.
Temperature had to be measured in, the cell suspensions
in
the
surrounding medium.
because
the
and
The results were
temperature
varied
from
day
to
day.
poorly
in the cell suspensions
reached 37°C slowly (more than 5 minutes), with a time
which
not
Preliminary experiments on capping
at 37°C were done in an air incubator.
reproducible
for
constant
The variability in the time
constant depended on the volume of the sample, the
place
where
the samples were placed in the incubator, the circulation of air
in
was
the
incubator, and how frequently the door of the incubator
opened.
incubated
In
in
later
experiments,
a water bath.
cell
were
The temperature in the suspensions
reached its final value more rapidly than in
(about
suspensions
an
air
incubator
one minute), with less variability in the time constant.
Incubation
in
a
water
bath
thus
improved
greatly
the
reproducibility of the results.
iv)
Precise
experiment
was
timing
of
each
step
performed
during
necessary, especially when the cells were being
treated or when they were recovering from treatment.
important
because
This
was
if one has to determine the effect of change
of one particular parameter (e.g.
treatment)
an
temperature
or
duration
on one particular biological event (e.g.
of
capping or
Ab-C cytotoxicity) all other parameters must be held constant.
v) Control preparations had to
and
exactly
Temperature
in
the
same
differences
way
as
between
be
the
handled
simultaneously
treated preparations.
control
and
irradiated
88
suspensions were on the order of + 0.1°C.
vi) At the beginning of each complete
where
the
effects
of
and
simultaneously
uniformity
reagents used.
milliliter
experiments
in
sufficient
stored in small aliquots so that one aliquot would
be sufficient for one experiment.
the
of
changes in certain parameters had to be
compared, reagents were prepared
amounts
set
in
composition
Complete
test
This was necessary to
media
tubes
and
and
activity
(MEM+FBS)
of
were
stored at 4°C.
the various
stored
aliquots.
They
desired concentrations at
Monoclonal
anti-Thy
were
the
1.2
diluted
beginning
antibodies
in
50
FITC-labeled goat
anti-mouse Ig antibodies were stored in the dark at 4°C
milliliter
insure
in
of
MEM+FBS
each
diluted
in
0.3
to the
experiment.
1:100
in
PBS
containing 2.5% Bovine Serum Albumin, and non diluted guinea pig
complement were stored at -20°C in 0.1-0.5 ml aliquots.
Guinea
pig complement was restored to the desired concentrations at the
beginning of each experiment.
vii) Each complete set of experiments was conducted
short
a
time
period
as
possible,
weeks, in order to make sure that ail
same
condition,
takes place.
and
that
no
in
as
usually not more than two
reagents
remain
in
the
significant aging of the animal
89
REFERENCES
1.
H.
D.
Suit and M.
Schwayder.
as an anti-tumor agent.
2.
D.
E.
C.
Thrall, L.
Dewey.
Hyperthermia:
potential
Cancer 34, 122-129 (1974).
E.
Gerweck, E. L.
Gillette,
and
W.
Response of cells in vitro and tissues in vivo
to hyperthermia and X-irradiation.
Advances
in
Radiation
Biology 6, Academic Press (1976).
3.
R.
C.
Miller, W.
L.
M.
Boone.
therapy.
4.
R.
Connor, R.
S. Heusinkveld, and M.
Prospects for hyperthermia in human
cancer
Radiology 123, 489-495 (1977).
Cavaliere, E.
C.
Heidelberger, R.
G.
G.
Morrica,
Ciocatto,
0.
and
Johnson, M.
A.
M.
C.
Giovanella,
C.
Margottini, B. Mondovi,
Rossi-Fanelli.
Selective
heat
sensitivity of cancer cells. Cancer 20, 1351-1381 (1967).
5.
R.
T.
Pettigrew, G.
Circulatory
and
hyperthermia.
6.
D.
S.
Br.
and
Gait, C.
biochemical
Joahi, B.
Barendsen
M.
J.
F.
J.
Surg.
Deys,
M.
Ludgate,
effects
of
J.
B.
Kralendonk.
Biol.
7.
B.
K.
x-rays
and
U
al.
whole
body
61, 727-730 (1974).
A.
Kipp, G.
Comparison
mammalian cell-lines with respect to their
hyperthermia,
et
V radiation.
of
W.
three
sensitivity
Int.
J.
to
Rad.
31, 5, 485-493 (1977).
Bhuyan, J.
Ogunbasi.
K.
Sensitivity
Day,
of
C.
E.
different
Edgerton,
cell
and
0.
lines
and of
different phases in the cell cycle to hyperthermia.
Cancer
Research 37, 3780-3784 (1977).
8.
B.
Mondovi, R.
Cavaliere,
Strom, G.
and A.
R.
Pitilio, A.
Fanelli.
Finazzi
Agro,
R.
The biochemical mechanism
90
of selective heat sensitivity of cancer cells.
on cellular respiration.
Europ.
J.
Cancer
I.
5,
Studies
129-136,
Pergamon Press, (1969).
9.
R.
and
10.
J.
synchrony
on
Heidelberger.
Influence
33, 422-427 (1973).
M.
Criteria of viability in
Harris.
G.
Cell.
Crile.
implanted
Res.
the
drugs
heat-treated
cells.
44, 658-660 (1966).
The effects of heat and
on
of
the hyperthermic killing of HeLa cells.
Cancer Res.
Exptl.
11.
Palzer and C.
feet of mice.
radiation
on
Cancer Res.
cancers
23, 372-380
(1963).
12.
N.
Schulman and E.
J.
on
proliferative
and
Hall.
Hyperthermia:
plateau
phase
its
cell
effect
cultures.
Radiology 113, 207-209 (1974).
13.
J.
Overgaard and P.
Bichel.
The influence of Hypoxia and
acidity on the hyperthermic response of malignant cells
vitro.
14.
L.
in
Radiology 123,511-514 (1977).
E.
Gerweck.
elevated
Modification
temperatures:
The
of
pH
cell
effect.
lethality
Rad.
Res.
at
70,
224-235 (1977).
15.
J.
Overgaard.
carcinoma
Ultrastructure
exposed
to
of
a
hyperthermia
murine
in vivo.
mammary
Cancer Res.
36, 983-995 (1976).
16.
C.
W.
Levitt.
Song, M.
S.
Effect
of
Rang,
J.
G.
hyperthermia
normal and neoplastic tissues.
Rhee,
and
S.
H.
on vascular function in
Annals
of
the
New
York
Academy of Sciences 335, 35-47 (1980).
17.
P.
M.
Gullino.
Influence
of
blood
supply
on
thermal
91
properties and metabolism of mammary carcinomas.
Annals of
the New York Academy of Sciences 335, 1-21 (1980).
18.
J.
S.
M.
Stehlin, B.
Muenz,
perfusion
and R.
for
C.
Giovanella, P.
F.
Anderson.
melanoma
of
H.
de Ipolyi,
L.
Results of hyperthermic
the
extremities.
Surgery,
Gynecology, and Obstetrics 140, 3, 339-348 (1975).
19.
L.
R.
Vaughn and M.
J.
Kluger.
bacterially infected rabbits.
20.
21.
S.
Furuuchi
and
Y.
Fever and
Fed.
Shimizu.
Proc.
Survival
36, 511 (1977).
Effect
of
tenuated
transmissible
gastroenteritis
virus
born piglets.
Infect.
13, 990-992 (1976).
L.
E.
Carmichael, F.
Temperature
canine
Immun.
as
D.
Barnes,
in
in the bodies of new
and
D.
H.
Percy.
a factor in resistance of young puppies to
herpesvirus.
J.
Infect.
Dis.
120,
669-678
(1969).
22.
C.
Armstrong.
neurotropic
Some
recent
research
in
the
field
of
viruses with especial reference to lymphocytic
choriomenengitis and
herpes
simplex.
Mil.
Surg.
91,
129-145 (1942) .
23.
N.
Roberts J.
human
leukocyte
lymphocytes
capacity
24.
and R.
of
to
T*
Steigbigel.
functions:
mitogen
monocytes
and
and
on
and
neutrophils.
(1977).
R.
J.
Ashmans and A.
Effects
antigen
Immunity 673-679, Dec.
B.
Hyperthermia
Nahmias.
and
response
of
bactericidal
Infection
Enhancement of
and
human
lymphocyte response to phytomitogens in vitro by incubation
at
elevated
temperatures.
464-467 (1977).
Clin.
Exp.
Immunol.
29,
92
J.
B.
Smith, R.
Human
lymphocyte
40°C.
J.
N.
J.
D.
responses
Immunol.
leukocyte
are
and K.
Immunol.
W.
A.
F.
enhanced
Agarwal.
by cultures at
Hyperthermia
and
Inhibition
Factor
(LIF).
122, 5, 1990-1993 (1979).
Tompkins, G.
V.
R.
Rao, P.
Hyperthermia
antibody-complement
JNCI 66, 3, (1981).
C.
Cain, G.
Enhancement
V.
of
for
human
Rama Rao, and W.
antibody-complement
A.
radiation.
colon
F.
and
of
tumor
Tompkina.
cytotoxicity
virus-transformed hamster PARA-7 cells
microwave
Pantasatos,
enhancement
cytotoxicity
cells.
and
S.
functions, II, Enhanced production of and
C. A. Cain.
A.
S.
Sandberg.
response to Leukocyte Migration
J.
and
121, 691-694 (1978).
Roberts Jr.
human
Rnowlton,
treated
against
with
heat
Radiation Research 88, 96-107,
(1981).
St.
Szmigielski,
Jeljaszewics,
M.
and
Janiak,
G.
W.
Hryniewicz,
Pulverer.
Local
hyperthermia (43°C) and stimulation of the
T-lymphocyte
rats.
S.
Z.
A.
Res.
J.
microwave
macrophage
and
systems in treatment of Guerin Epithelioma in
Krebsforsch 91, 35-48 (1978).
Shah.
regression
J.
of
Participation
a
of
the
immune
system
rat Mc7 sarcoma by hyperthermia.
in
Cancer
41, 1742-1747 (1981).
W.
ionizing
Harris.
Effects of
radiation,
and
tumor cells by cytotoxic
36, 2733-2739 (1976).
tumor-like
hyperthermia
T-lymphocytes.
assay
conditions,
on immune lysis of
Cancer
Research
93
32.
J.
W.
Harris
hyperthermia
secondary
and
J.
J.
Meneses.
Effects
of
on the production and activity of primary and
cytolytic
T-lymphocytes
in
vitro.
Cancer
Research 38, 1120-1126 (1978).
33.
H.
R.
MacDonald.
functional
M.
of
hyperthermia
on
activity of cytotoxic T-lymphocytes.
Cancer Inst.
34.
Effects
J.
Natl.
59, 4 (1977).
Schechter, S.
hyperthermia
the
on
M.
Stowe, and H.
primary
and
host immune response in rats.
Moroson.
Effects
of
metastatic tumor growth and
Cancer Research 38,
498-502
(1978).
35.
S.
S.
Shah and J.
on
the
S.
S.
Dickson.
immunocompetence
Cancer Res.
36.
A.
of
VX2
Effects of
hyperthermia
tumor-bearing
rabbits.
38, 3523-3531 (1978).
Shah and J.
A.
Dickson.
Effect
on the immune response of normal rabbits.
of
hyperthermia
Cancer Res.
38,
3518-3522 (1978).
37.
A.
A.
Teixeira-Pinto,
Cutler,
and
J.
H.
L.
L.
Heller.
Nejelski,
38.
S.
J.
L.
The behavior of unicellular
organisms in an electromagnetic field.
Res.
Jr.,
Experimental
Cell.
20, 548-564 (1960) .
F.
Cleary.
biological
radiation.
Uncertainties in
effects
of
the
microwave
Health Physics,, Pergamon
evaluation
and
Press,
of
the
radiofrequency
25,
384-404
(1973).
39.
H.
Frohlich.
biological
(U.S.A.)
The extraordinary dielectric
materials
properties
and the action of enzymes.
72, 11, 4211-4215 (1975).
of
P.N.A.S.
94
C.
A.
Cain.
fields:
Biological effects of
Role
of
oscillating
voltage-sensitive
ion
electric
channels.
Bioelectromagnetics 2, 23-32 (1981).
S.
F.
effects
Barnes and C-L.
of
radio
membranes.
J.
and
IEEE
Hu.
Model for some nonthermal
microwave
Transaction
fields
on
on
biological
Microwave
Theory
and
Techniques, MTT-25, 9 (1977).
G.
C.
Berkowitz and
F.
S.
Barnes.
The
effects
of
nonlinear membrane capacity on the interaction of microwave
and
radio-frequencies
with
biological
materials.
IEEE
Transactions on Microwave Theory and Techniques, MTT-7,
#2
(1979).
J.
P.
Changeux, J.
Thiery, T.
Tung, and C.
the cooperativity of
biological
membranes.
Acad.
H.
Sci.
Proc.
On
Nat.
57, 335-341 (1967).
Frohlich.
oscillating
Rittel.
Collective behaviour of non-linearly coupled
fields.
Collective
Phenomena
1,
101-109
(1973).
I.
T.
Grodsky.
interaction
membranes.
R.
L.
to
C.
of
Ann.
Possible
electromagnetic
NY Acad.
Seaman and H.
W.
and
M.
Bawin, A.
mechanisms
tissue.
B.
0.
Sci.
Wachtel.
substrates
fields
with
for
the
biological
247, 117-124 (1975).
Slow and
rapid
responses
pulsed microwave radiation by individual
Aplysia Pacemakers.
S.
physical
J.
Microwave Power 13(1) (1978).
Sheppard, and W.
R.
Adey.
Possible
of weak electromagnetic field coupling to brain
Bioelectrochem.
Nilsson and C.
Bioenerget.
E.
Petterson.
5, 67-76 (1978).
A
mechanism
for
95
high
frequency
damage?
electromagnetic
IEEE
Transactions
field
on
induced
biological
Microwave
Theory
L.
and
and
Techniques, MTT-27, 6 (1979).
B.
I.
Martins, M.
Tobias.
Cell
R.
Raju, T.
E.
Radiat.
Res.
L.
Carstensen, S.
and
M.
Z.
W.
Child,
Miller.
W.
Acoust.
M,
G.
Soc.
Am.
Hedges
and
irradiation of human
35, 301-311
E.
62(3),
Graham,
M.
Leeman.
lymphocytes.
Hedges,
(1980).
D.
Webster, W.
S.
Harvey, M.
The role of ultrasound-induced
stimulation
of
collagen
Ultrasonics, 3, 1 8 ( 1 ) , 1-48
I.
Ye
El'Piner.
biological effect.
Dyson, B.
blood
572-573
on
R.
plant
roots.
(1979).
1.5
Int.
MHz
J.
Leeman,
bio-effects at 1.5 M H z .
224-228
F.
D.
Cavitation as a mechanism
66, 1285-1291
S.
Law,
ultrasound
Radiat.
Biol.
(1979).
Cavitational
E.
Abstracts
R.
for the biological effects of ultrasound
M.
A.
(1975).
Horowitz,
J.
C.
membrane damage in cultured mammalian cells
exposed to ultrasound.
June
Hayes,
cells
and
P.
Vaughn.
Ultrasonics 3 18(5),
Dyson, and J.
B.
Pond.
cavitation in the 'in vitro'
synthesis
in human fibroblasts.
(1980).
Non-cavitational
ultrasound
Biophysics 15 ( 2 ) , 354-365
Woodward, and J.
B.
Pond.
stopped by ultrasound.
Nature
and
its
(1970).
Flow
of
red
(London), 232,
(1971).
Siegel, J.
Goddard, E.
James,
Cellular attachment as a sensitive
and
E.
P.
Siezel.
indicator of the effects
96
of
diagnostic ultrasound exposure on cultured human cells.
Radiology 133, 175-179 (1979).
G.
Ter Haar, M.
induced
vivo.
L.
Dyson, and D.
contractions
in
Love and F.
W.
uterine smooth muscle in
Kremkau.
distribution produced by
S.
mouse
Ultrasonically
Ultrasonics 3, 16(6), 241-288 (1978).
A.
Am.
Talbert.
Intracellular temperature
ultrasound.
J.
Acoust.
Soc.
67(3) (1980).
Baranski.
white
Effect of microwaves on the reaction of
blood
cells
system.
Acta.
Physiol.
the
Polonica 23,
685-695 (1972).
P.
Czerski.
with
particular
Acad.
J.
Sci.
E.
monkey.
reference
to
H.
Cytology
Mori, J.
lymphocyte.
Ann.
NY
Aerospace Med.
response
of
W.
Frazer,
J.
C.
and
aspect of radiofrequency radiation in
43, 759-761 (1972).
Wiktor-Jedrzejczak, A.
Immune
the
system
247, 232-242 (1975).
Prince, L.
Mitchell.
W.
Microwave effects on the bloodforming
Ahmed,
P.
Czerski,
et
al.:
mice to 2450 MHz microwave radiation.
Overview of immunology and empirical
studies
splenic cells.
12, 209-219 (1977;.
W.
Radio Sci.
(Suppl.)
Wiktor-Jedrzejczak, A.
Microwaves
induce
and
Ahmed, K.
W.
increase
in
of
lymphoid
Sell,
the
et
frequency
complement receptor-bearing lymphoid spleen cells in
J.
Immunol.
W.
Wiktor-Jedrzejczak.
Increase
in
al.
of
mice.
118, 1499-1502 (1977).
the
A.
frequency
Ahmed,
of
P.
Czerski,
et
al.
FC receptor (FCR) bearing
ceils in the mouse spleen following a
single
exposure
of
97
mice
to
2450
MHz
microwaves.
Biomedicine
2 7 , 250-252
(1977).
K.
Sulek, C.
al.
J.
Biologic
Schlagel,
effects
W.
of
Wiktor-Jedrezejczak,
microwave exposure:
Threshold
conditions for the induction of the increase in
receptor
positive
exposure to 2450
(CR+)
MHz
mouse
spleen
microwaves.
complement
cells
Radiol.
et
following
83,
127-137
(1980).
R.
P.
Liburdy.
Radiofrequency
radiation
alters
the
immune system; Modulation of T- and B-lymphoeyte
levels and
cell-mediated
immunocompetence
radiation.
Radiat.
7 7 , 34-36
R.
P.
immune
Res.
Liburdy.
D.
A.
Holm
(1979).
Radiofrequency
system:
circulation.
by hyperthermic
Modulation
Radiat.
and
Res.
L.
radiation
of
in
80, 66-73
K.
alters
vivo
the
lymphocyte
(1980).
Schneider.
The
effects
of
nonthermal radiofrequency radiation on human lymphocytes
vitro.
Experientia 2 6 , 992-994
P.
Hamrick and S.
E.
culture
exposed
S.
D.
lymphocytes
Rat
H.
Bases, F.
Sister
after
Mendez, F.
chromatid
exposure
Repacholi,
synthesis observed
therapeutic
in
cell
and
in human
ultrasound.
J.
(1977).
to
J.
exchanges
G.
in
M.
human
ultrasound.
(1979).
Kaplan.
lymphocytes
Paper
Elequin, and
diagnostic
Science 205, 21, 1273-1275, Sept.
M.
lymphocytes
to 2450 MHz (cw) microwave radiation.
Liebeskind, R.
Roenigsberg.
(1970).
Fox.
Microwave Power 12, 125-132
in
exposed
DNA
repair
in vitro to
#1306, Proceedings of the
98
25th Annual Meeting of the Am.
Medicine, Sept.
D.
R.
ultrasound:
H.
D.
human
Bases, F.
Goldberg,
Elequin,
and
M.
Ultrasound
in
Neubort,
R.
S.
Koenigsberg.
Effects on the D.N.A.
animal cells.
of
(1980).
Liebeskind, R.
Leifer,
Inst.
Diagnostic
and growth patterns
of
Radiology 131, 177-184 (1979).
Rott and R.
Soldner.
chromosomes
in
The effect of ultrasound
vitro.
Humangenetik
on
20,
103-122
R.
Stern.
(1973).
J.
A.
Effects
Roseboro P.
of
Buchanan, A.
ultrasonic
chromosomes in vitro.
Norman, and
irradiation on mammalian ceils and
Phys.
Med.
Biol.
23(2),
324-331
(1978).
M.
H.
Repcholi and
alterations
Proc.
to
J.
the
G.
Kaplan.
lymphocyte cell surface.
of the 25th Annual Meeting of
Ultrasound in Medicine, Sept.
D.
W.
Ultrsound-induced
Anderson and J.
immunosuppressant.
T.
the
Am.
Paper #1307
Inst.
of
(1980).
Barrett.
Clinical
Ultrasound:
Immunol.
a
new
and Immunopathol.
14, 18-29 (1979).
D.
W.
Anderson
phagocytosis
and
J.
by ultrasound.
T.
Barrett.
Depression
Ultrasound in Med.
of
and Biol.
7, 267-273 (1981).
S.
J.
Singer and G.
L.
Nicolson.
The
model of the structure of cell membranes.
fluid
mosaic
Science 175, 720
(1972).
S.
E.
Gordesky.
Phospholipid
erythrocyte membrane.
asymmetry
TIBS, 208-211, Sept.
in
the
(1976).
human
99
L.
B.
Bergelson
asymmetry
and
L.
of p h o s p h o l i p i d s
I.
Barsukov.
i n membranes.
Topological
Science 197,
224
(1977).
A.
G.
Lee.
Lipid phase transitions and
Lipid phase transitions.
A.
G.
Lee.
R.
Lipid phase transitions and
Ubbelohde.
diagrams:
BBA 472, 237-291 (1977).
Mixtures involving lipidB.
A.
phase
phase
diagrams:
BBA 472, 285-344 (1977).
Melting
and
crystal
structure.
Clarendon Press, Oxford (1965).
V.
A.
Petit and M.
lipids
in
Edidin.
Lateral phase separation
plasma membranes:
Effect of temperature on the
mobility of membrane antigens.
H.
Trauble and H.
phase
transitions:
environment.
P.
C.
P.N.A.S.
W.
Bloom.
H.
Science 184, 1183-4 (1974).
Electrostatic effects
Membrane
P.N.A.S.
Jost, 0.
Vanderkooi.
F.
Eibl.
Evidence
D a h l q u i e t , D.
Deuterium
5435
D.
Marsh and F.
for
R.
A.
boundary
C.
and
ionic
Capaldi,
lipid
Muchmore, J .
magnetic
with
H.
resonnance
membrane
and
G.
in membranes.
David,
and
studies
proteins.
of
M.
the
P.N.A.S.
(1977).
J.
Barrantes.
acetylcholine
receptor-rich
torpedo-marmorata.
P.N.A.S.
M.
Grant and H.
lipid bilayers.
P.
lipid
70, 480-484 (1973).
74,
W.
structure
on
71 (1), 214-219 (1974).
Griffith
i n t e r a c t i o n of l i p i d s
C.
of
Overath, L.
P.N.A.S.
Thilo,
M.
Immobilized
lipid
membranes
in
from
75(9), 4329-4333 (1978).
McConnell.
Glycophorin
in
71(12), 4653-4657 (1974).
and
H.
Trauble.
Lipid
phase
100
transitions
and
membrane
function.
TIBS
186-189,
Aug.
(1976).
A.
G.
Lee.
Annular events: Lipid-protein
TIBS 231-233, Oct.
E.
Oldfield, R.
Co
Hshung,
and
D.
Rice.
investigation
(1977).
Gilmore, M.
J.
S.
Y.
Glaser, H.
Rang, T.
Deuterium
of
on hydrocarbon
chain
order
in
Owicki
J.
C.
and
and
Owicki, M.
membranes.
H.
A.
in
bilayer
Meadows,
resonance
M.
membrane
McConnell.
systems.
theory
of
76(10), 4750-4754 (1979).
W.
Springgate, and H.
M.
McConnell.
study of protein-lipid interactions in bilayer
P.N.A.S.
D.
King, M.
protein-protein interactions in bilayer
P.N.A.S.
Theoretical
Gutorvsky,
magnetic
model
J.
membrane.
S.
the effects of proteins and polypeptides
75(10) 4657-4660 (1978).
protein-lipid
E.
nuclear
P.N.A.S.
C.
interactions.
Pink and D.
75(4), 1616-1619 (1978).
Chapman.
membranes:
Protein-lipid
a lattice model.
interactions
P.N.A.S.
76(4),
1542-1546 (1979).
R.
J.
Cherry.
Protein mobility in
membranes.
F.E.B.S.
letters 55(1), 1-7 (1975).
G.
F.
Schreiner
cytoplasmic
and
changes
E.
R.
Unanue.
in
B-lymphocytes
ligand-surface immunoglobulin interaction.
Membrane
and
induced
by
Adv.
Immunol.
24, 38-165 (1976).
F.
Loor.
surface.
Structure
From:
Loor
immune recognition.
and
and
dynamics
of
Roelants:
the
B
Wiley, 154-184 (1977).
lymphocyte
and T cells in
101
99.
G.
C.
Tsokos and D.
immunoglobulin
D.
and
Choie.
Inhibition of capping of
concanavalm
cis-Dichlorodiammineplatinum
II
A
in
receptors
mouse
spleen
by
cells.
Cancer Letters 10, 261-267 (1980).
100.
M.
Yokoyamo,
Cytochemical
their
J.
P.
study
of
mobility
101.
K.
L.
and
W.
P.
C.
binding sites and
cystic
fibrosis,
fibroblasts
in
L.
Palmer, J.
A.
F.
A.
and
vitro.
Harris,
Tompkins.
J.
of CEA and isoantigen A.
W.
E.
Antibody-induced
redistribution of CEA on the cell surface:
separation
Moller.
28(6), 543-551 (1980).
Rosenthal, J.
Rawls,
normal,
human
Cytochem.
and
Concanavalm A.
in
SV40-transformed
Histochem.
Chang,
J.
Utilization
Immunol.
in
115(4),
1049-1053 (1975).
102.
F.
Hirata and J.
biological
Sept.
103.
E.
signal
Phospholipid
transmission.
Lazarides
and
cells.
P.N.A.S.
G.
Lyan,
B.
K.
visualization
Inhibition
R.
of
D.
R.
surface
Berlin,
surfaces.
and
From:
Active
actin
antibody:
filaments
The
in nonmuscle
71(6), 2268-2272 (1974).
E.
of
Microtubules
106.
and
Science 209, 1082-1090,
Weber.
Unanue,
J.
the
and
M.
J.
M.
Nature 250, 56-57 (1974).
Caron,
structure
and
and
J.
S.
Hyams.
D.
F.
Albertini and J.
M.
function
Microtubules, edited by K.
J.
Kamovsky.
capping of macromolecules by local
anaesthetics and tranquillisers.
105.
methylation
(1980).
specific
104.
Axelrod.
Oliver.
of
Roberts
cell
and
Academic Press, 443-486 (1979).
I.
Clark.
Membrane-microtubuie
102
interactions:
Concanavalm
redistribution
of
colchicine-binding
A
capping
cytoplasmic
proteins.
induced
microtubules
P.N.A.S.
72(12),
and
4976-4980
(1975).
107.
S.
J.
H.
H e g g e n e s s , and D.
and
Singer, J.
the
F.
Ash, L.
Louvard.
mechanisms
membranes.
J.
Y.
of
W.
Bourguignon,
Transmembrane
transport
Supramolecular
or
interactions
proteins
Structure
M.
9,
across
373-389,
(1978).
108.
S.
G.
Emerson and R.
colchicine
and
E.
Cone.
cytochalasins
B - c e l l membrane IgM and IgD.
Differential
on
the
effects
shedding o f
P.N.A.S.
72(12),
of
murine
6582-6586
(1979).
109.
W.
C.
E.
Dewey, L.
E.
Gerweck.
Hopwood, S.
Cellular
A.
Sapareto,
responses
to
Radiat.
Biol.
h y p e r t h e r m i a and r a d i a t i o n .
and
L.
combinations
of
123,
463-474
(1977).
110.
D.
Elkon and H.
E.
McGrath.
of g r a n u l o c y t e - m o n o c y t e
Thermal
stem c e l l .
inactivation
Rad.
Res.
87,
energy
368-372
(1981).
111.
N.
K.
Wessels B.
S.
Bradley,
M.
A.
and
M.
Yamada.
K.
Luduena,
developmental p r o c e s s e s .
112.
S.
DePetris.
cytochalasin
54-55
113.
B.
Spooner,
L.
Science
vinblastine
and
A.
Ash,
T a y l o r , T.
Microfilaments
Inhibition
B,
E.
J.
in
M.
T.
Wrenn,
cellular
171, 135,
(1971).
reversal
of
and c o l c h i c i n e .
D.
and
capping
by
Nature 2 5 0 ,
(1974).
Mondovi, A.
S.
S a n t o r o , R.
Strom, R.
Faiola,
and A.
103
R.
Fanelli.
Increased immunogenicity of
cells after heat treatment.
114.
M.
L.
Jasiewicz and J.
Dickson.
destructive effect of heat (42°C)
115.
in
Biol.
1, 221-225 (1976).
R.
Biochemical
Crifo A.
S.
S.
aspects
of
heat
117.
118.
Ohanian and T.
A.
S.
M.
Bawin,
Effects
of
system.
Ann.
F.
L.
K.
Killing of nucleated cells
In
Comprehensive
modulated
Kaczmarek,
VHF
NY Acad.
Blackman, J.
C.
A.
K.
Day and
Eichinger, and D.
E.
Effects
of
A.
G.
Lampe,
calcium-ion efflux
radiation:
W.
R.
Adey.
brain
M.
Weil,
House.
S.
G.
Induction of
tissue by radio-frequency
modulation
frequency
and
field
Radio Science 14, 65, 93-98, (1979).
Blackman, S.
J.
and
247, 74-81, (1975).
C.
radiation:
House,
Immunology,
on the central nervous
Elder,
from
strength.
fields
Sci.
efflux
F.
Mondovi.
sensitivity of tumor cells.
Borsos.
calcium-ion
C.
B.
Thermal
Good, Eds.), Plenum, New York, 115-135 (1977).
Benane, D.
119.
and
cancer
J.
2, Amplification Systems in Immunity (N.
R.
C.
synchronized
59, 7-22 (1977).
by antibody and complement.
Vol.
on
Rossi-Fanelli,
Recent Results Cancer Res.
116.
Potentiation of the
culture by cell specific antiserum.
Strom, C.
ascites
Cancer 30, 885-888 (1972).
A.
cells
Ehrlich
Effect
from
of
Benane, J.
A.
and
M.
Faulk.
tissue
by
brain
J.
sample
Elder,
number
frequency on the power-density window.
D.
E.
Induction of
radiofrequency
and
modulation
Bioelectromagnetics
1, 35-43, (1980).
120.
C.
F.
Blackman, S.
G.
Benane, W.
T.
Joines,
M.
A.
104
Hollis,
and
tissue:
Power-density versus field-intensity
at
50-MHz
D.
RF
E.
House.
radiation.
Calcium-ion efflux from brain
dependencies
Bioelectromagnetics 1, 277-283
(1980).
121.
R.
Eckert
and
R.
Kennedy editor, W.
Randall.
H.
Animal
Physiology.
D.
Freeman and Company, San Francisco,
Chapters 4 and 11, (1978).
122.
J.
B.
Marmor,
regression
C.
and
immune
ultrasound heating.
123.
A Boyum.
marrow.
Nager,
and
G.
recognition
Radiat.
Res.
J.
Clin.
Lab.
Hahn.
after
Tumor
localized
70(3), 633-634 (1977).
Separation of lymphocytes
Scand.
M.
from
blood
and
bone
97 (Suppl.
21),
Milner.
The
hyperthermia,
and
Invest.
77 (1968).
124.
G.
E.
Piontek, C.
effects
of
L-ascorbic
metabolism.
A.
Cain, and
microwave
acid
on
IEEE
J.
A.
radiation,
Ehrlich
ascites
Transactions
on
carcinoma
Microwave
cell
Theory and
Techniques, MTT-26, 535-540 (1978).
125.
M.
TEM
L.
Crawford.
Generation of standard EM
transmission ceils.
IEEE Trans.
fields
Electromag.
using
Compat.
EMC-16,4, 189-195 (1974).
126.
F.
Dunn, A.
J.
Averbuch, and W.
D.
O'Brien.
method for the determination of ultrasonic
the
elastic
(1977).
sphere
radiometer.
Acustica
A primary
intensity
38(1),
with
58-61
105
VITA
Michel
Lebanon.
Farid
He
in
was born on March 9, 1955 in Beirut,
attended
Urbana-Champaign
degrees
Sultan
the
where
He
Engineering
has
held
the
graduate teaching assistant from
research
assistant
of
Illinois
at
he received his Bachelor's and Master's
Electrical
respectively.
University
in
1977
positions
1978
to
and
1979,
of tutor in 1977,
1980,
and
graduate
in Electrical Engineering at the University
of Illinois since 1980.
He
has
authored
the
following
papers
with
Professors
Charles Cain and Wayne Tompkins*
Inhibition of
capping
surface
B-lymphocytes
of
of
antigen-antibody
by
complexes
hyperthermia.
on
the
Submitted
for
publication.
Hyperthermic
against
enhancement
of
antibody-complement
cytotoxicity
normal mouse B-lymphocytes and its relation to capping.
Submitted for publication.
Effects
of
microwaves
antigen-antibody
B-lymphocytes.
and
complexes
on
amplitude
B-lymphocyte capping.
Immunological
the
surface
on
of
capping
normal
of
mouse
Submitted for publication.
Immunological effects of
radiation:
hyperthermia
effects
of
modulated
radio
frequency
Submitted for publication.
ultrasound:
B-lymphocyte
capping.
Submitted for publication.
Mr.
Sultan has presented papers coauthored with Professors
Cain and Tompkins at
held
August
9-12,
the
1981,
Bioelectromagnetics
Washington,
DC,
Society
and
at
meeting
the North
American Hyperthermia Group/Radiation Research Society
meetings
held April 17-20, 1982, Salt Lake City, Utah.
Mr.
Honorary),
Sultan
Eta
is
a
member
of
Tau
Beta
Pi
(Engineering
Kappa Nu (Electrical Engineering Honorary), the
Institute of Electrical and Electronic Engineers, and
member in Sigma Xi (Scientific Reseach Society).
associate
Документ
Категория
Без категории
Просмотров
0
Размер файла
3 365 Кб
Теги
sdewsdweddes
1/--страниц
Пожаловаться на содержимое документа