December. 1996. 729. Cytotoxicity of peritoneal murine macrophages against encapsulated pancreatic rat islets: in vivo and in vitro studies. Laurence. Kessler$.
Cytotoxicity of peritoneal murine macrophages against encapsulated pancreatic rat islets: in vivo and in vitro studies Laurence Kessler$ Cathy Jesser Yves Michel Pinget7 and Philippe Poindront *Jeune
Equipe
M#{233}dicale Universitaire,
and
Lombard,t
V#{233}ronique Karsten
tLaboratoire
d’Jmmunopharmacologie,
in
vivo
of macrophages necrosis of encapsulated
and
Several macrophages
Alain
BeIcourt
Facult#{233}de Pharrnacie,
Strasbourg,
France
Abstract:
The
and in vitro inter1eukin..13
aim
of this
islets during xenograft protective efficiency
6 days
work
the involvement (ff.1) in the
was
to study
and to evaluate the of the AN69 membrane.
immunoIn vivo,
face
after implantation, 65% ofthe membrane surof the devices containing the islets was colonized
with
macrophages
compared with only 5% ofthe surcontrol devices. The morphological islets was altered and their insulin
face of the empty aspect ofimplanted release isolated
decreased significantly ones (265 ± 50 vs. the
vitro,
for in
2 the
insulin
ofencapsulated
with
ofanti-IL-113 and islets
islets
membrane. damaging
probably failure.
In
cultured
respectively, The
pancreatic for islet 60: 729.-736;
encapsulated
partly
responsible Riot.
J. Leukoc.
the
are
xenografts [5-71- Like after xenotransplantation, devices whereas
ad-
Words: cytokine tran.splantation
brane
bioar4frcial
.
pancreas
islets devices
through Kasuga
caused
the
et al.
ulins.
but
The
the
aim
of this
involvement
islets
rejection
probably
the
in the
treatment
protect to
the
low
that both
islets
from them
must glucose
antibodies
ported
that
allo-
sulated
islets
several
months
stricting
together the
in
the
with long-term
system,
insulin
diffusion
xenografts
Although reaction
a necrosis efficiency
while cells. of
cavity
were
encapsulated
of the
graft.
cultured
3
islets
the
of immunoglob.
passage
conditions, and
was
cytokines
damage
to study
the
in vivo
and
during
AND
Cytokines
been
al-
IL-13
xenograft
and
in vitro
in the
and
of the
might islets.
necrosis
to evaluate
AN69
the
membrane.
METHODS
and other
reagents
Recombinant
IL-i
human
polyclonal
were on their islets
13
were
(sp.
act.
purchased
=
from
lU/mg)
108
Genzyme
and Corp.
anti(Cam-
reAbbreviations:
functional
interleukin
antibody
the
macro/microencap-
the membranes was observed of the
and has
(ILFurtherof IL-i
mem-
preventing It
from
me-
To
proposed
artificial
nondegradable,
immune
was
Iinterleukin-113
a molecular mass not only a satisfac-
efficiency
MATERIALS
mellitus. it was
cytotoxicity
are
transplantation
diabetes
immune
peritoneal
[1-4J. a cellular
islet
a semipermeable
and and
auto-immunity
in
biocompatible,
and
of
face
of
factors
the in
be
passage
compatible,
recurrence
limiting
of insulin-dependent
encapsulate
brane
and
major
the
nitric oxide 9J. the cytotoxicity
of macrophages
of encapsulated
by
exhibiting prohibited
such
work
IJ#{149}
cellular or cytoislets
cells
release
membranes
immunoprotective
Immune
and that
a at
response.
that
cytokines
also
under
through
the
like
membranes 50,000 Da
However,
pass
I NTRODUCTION
de-
recognized
against
of insulin
diffusion
be
cellular
demonstrated
factorl proven
inhibition
insulin
and the
directed
factors
Selected of about
islets triggered the as secreted proteins from encapsulated
initiating
macrophages
by soluble
I1OJ. cutoff mem-
of and
[7, we observed that, membrane surfaces of
membrane
thus
1), tumor necrosis more, it has been
.
et al. AN69
involvement in both allo-
were colonized with macrophages, remained free from cellular
the
cells,
murine
diated
are
artificial
.
pass
Recently of
involved
islets and transplantation 1996.
Weber the
These results suggest that the reaction. Indeed, antigens such plasmic components released might
AN69
predominant reaction
posit 181. Using barium alginate, Horcher et al. showed similar discordance in the cellular reaction occurring the surface of both empty and islet-containing capsules
tory
Key
containing empty
specialized
antibody to the co-culture of macrodid not modify this loss of functional
Furthermore, IL-13 passed through In conclusion, macrophages
activity.
freshly
.tU/mL).
days decreased to 32 and 28%, presence of IL-i 3 and macrophages.
dition phages
in
release
compared 507 ± 81
groups reported the in the pericapsular
for
biosurre-
interleukin-1
ELISA.
Correspondence:
Dr.
sitaire,
H#{244}pitaux
ologie.
67091
Received tember
Journal
enzyme-linked
immunosorbent
assay;
IL-13,
.
12,
L.
Kessler,
Universitaires,
Strasbourg
June
10,
Jeune
Pavillon cedex,
1996;
revised
Equipe
M#{233}dicale Univer- Service
Leriche
d’Endocrin-
France.
September
9,
1996;
accepted
Sep.
December
1996
729
1996.
of Leukocyte
Biology
Volume
60,
UK). RPMI-1640
bridge, calfserum,
were
culture
1% gentamicin,
obtained
from
medium
and
GIBCO
lOx
supplemented
Eagle’s
(Cergy
with
minimal
Pontoise,
10%
essential
fetal
medium
The uated
with
France).
of rat pancreatic
islets
of macrophages
on
means
of a phagocytosis
test
hematoxylin-eosmn
empty
Encapsulation
adhesion by and
1640
in
the
Pancreatic ing
islets
were
220-250
digestion
were
the
in
in our
membrane
laboratory
of three
a collagen
using
f8J.
the
Briefly,
CO2
gel
islet
in air,
and
this
device
50
was
an
rat
the
at
with
the
performed
with
developed
were
buffer
solution
(pH
cohols,
stained
with
The
fixed
light
percentage face
as
was
from
the
solution
were
sential
medium
mM) the
mixed
islet
rapidly
and
a sterile
in
2
flask
with
sealing
the
ice
40
.tL
of this
and
Eagle’s
bicarbonate
suspension for
10
the
minimal
es-
was
poured
mm
at 37#{176}Cbefore
the
outbred
rendered oem
male
mice
diabetic
(Zanosar
diabetic
1G,
state
ating in
islets
their
500
by
blood
glycemia
1131. Three
Paris)
in tail
weighing of 200
injection a glycemia
samples
among
weeks
the
above
by means
20-30 g were mg/kg streptozot-
after
injection.
3 gIL
measured
the on
of a glucometer
experimental
animals
(Life-
was
3.85
p.tL
for
(CEA,
Diabetic
mice
anesthesia.
were
toneum
by
use
cultured
of
only di-
culture
the
were
medium.
adhesion
bated
for
by washing
filter
dishes
heart
with
under
by washing
Cell
dish
the
culture
containing
4 days above
and
with
IL-13
(1000
in
macrophages
RPMI-1640
islets
more
with
anti-IL-13
culture
37#{176}C in 5%
at
incubated
(500,000
cells/mL)
CO2
macrophages
polyclonal
for
or
2 days.
IL-13
antibody
of non-encapsulated
and
Fifty
served
(25
islets
or
About
of culture,
incubated
iL/mL)
was
added for
with
co-
were
Two
and
and
the
the
device
of the macroencapsulation six days
after
implantation.
macroencapsulation
tion.
The
and
the
was
examined
device
islets
dylate
buffer
dried,
mounted
was
were solution on
six
devices to evaluate
then
fixed (pH
aluminium
in each The
to analyze The
stubs,
group
the
were
implantation
necrosis
and/or artificial
0.2
glutaraldehyde
2%
7.4).
mice
tissue
dismantled
in a
device
removed.
killed area
rings
and
membranes
coated
with
gold,
and
cacowere
by
2 days
December
aspect
compared
with
and
those
of Alamar 36
h. The
spectroscopy
func-
of freshly
rat islets.
2 days.
was
in 500 density 570
=
Blue
of the the
cul-
islets
of RPMI of the
activity islets
maintained
by use
were
medium
culture
at
medium
nm).
functional isolated
tL
incu-
tested
1151. Alamar
CA)
Blue
and
freshly
islets
for
optical (X
aspect with
IL-13
Briefly.
of encapsulated
is-
for the
in vivo
study
for
2 days
for
in culture
and the
in
islets
in
of encapsulated ILl
enzyme-linked
was
immunosorbent
permeability
of the
37#{176}Cin
a silicone-coated
MO).
The
volumes
chamber
of 3 and
113.10 3 was
AN69
measured
assay
in the
method
The
to compartment
1 and
sorption
of
lL-13
pg/mL
concentration was
the degradation
chamber 24
at the
was filled
super-
(Intertest-13
lL-13
3 mL
of IL-I 3 were
incubated
and/on
under in the
B, with
At time
concentrations
were
To evaluate medium
The
membrane
3 onto
meathe
membrane
conditions.
of the
0, 700 ad-
supplemented
with the AN69
of IL-I
nespec-
being
same
in St
(surface
concentration
of RPMI the
studied
(Sigmacote, membrane
method.
absence
the adsorption
was
RPMI-1640.
final
ELISA
surface,
measured
AN69
with
A, the by the
performed
IL-ill A and
by the
h of diffusion,
compartment
to
chamber
two compartments,
separated
mm2). After
membrane diffusion
had 8 mL
added
in each 700
or non-encapsulated
3 release
Genzyme).
kit.
at
IL-ill
and diftusion
and
same
ex-
to evaluate
the
walls
s
were
ofthe
diffu-
chamber.
Statistical
analysis
significance
60,
glu-
same
by radioimmunoas-
activity.
Students
Volume
g/L
of the
air in a
devices.
Biology
measured
to a metabolic
implanted
Leukocyte
0.8
modification
studies,
of
by evalu-
solution
a color
After in vitro and in vivo The comparisons between
Journal
with
2 h of culture
containing
Sacramento,
dry atmosphere before examination under a Philips 501B scanning electron microscope. Their structure and the cellular adhesion were analyzed and compared with that of implanted empty devices and non-
730
studied
After
inducing
of macnophages.
sured
sion
(Alamar,
of co-culture
by the
peniment
membrane
stored
.tL for
compared
ELISA The
with of
inflamma-
M sodium
25 CO2
presence
pg/mL.
Analysis
test
or
indicator
release
the
=
and
stained
study.
IL-i
2 days.
was
or non-encapsulated
macrophages
morphological
IL-i
tive
Further-
to the
of encapsulated
measured
Louis,
non-encapsulated
were
morphological
were
light
above
microscope.
glucose
was
islets
in response
37#{176}Cin 5%
vitro
pg/mL)
.tm)
islets
The
removed and
as described
stimulation.
release
in the preswere
microscopy
solution
France).
of
islets
(0.5
in a 4-gIL
insulin
Blue
medium
natant
50
incubated
as a control.
macrophages
sun-
by cells.
of incubation
a light
bicarbonate
incubated
murine
Alamar
After
cells
medium.
500,000 macrophages adhered to the filter. After rat pancreatic islets were encapsulated as described murine
was (106/mL)
Nonadherent
of the
membrane
activity
fixed
with
to glucose
of encapsulated
viability
of the
vitro
pen-
enrichment
suspensions
C02).
micros-
evaluation
covered
sections
response
to non-encapsulated
ether
the
in a plastic
2 h (37#{176}C, 5%
the
the
the to the
were
of encapsulated
1 h. The
with
lets
Macrophage
method.
Millipore
medium
from harvested
permitted
of al-
light
islets.
The
tune
islets
examined
Gif-sur-Yvette.
isolated
was
cultures
aspiration
cells
on a 0.45-.tm
harvested
islets
by blood
exudate
with RPMI-1640
2 mL of culture were
pancreatic
killed
Peritoneal
obtained were
and
by
encapsulated
activity
were
activity
The
Macrophages
cacodylate
sequence
adhering
by phase-contrast
the
Krebs-Ringer
islets
the
Semi-thin
is an oxido-reduction ±
0.25 gIL. Fifty encapsulated islets or an empty device, containing collagen gel, were implanted in the right ilea fossa of anesthesized abetic mice (n = 24) by means of a small laparotomy.
latter, then
insulin
the
say
Caw, Iffa Credo)
confirmed
days
Mean
(lOPS
intraperitoneal
Upjohn,
was
2 successive scan).
by
the
functional
tional
OF1
on the membrane
examined
or 2 days
IL-1I.
examined
in araldite.
The
cose,
membrane.
of encapsulated
or and
For
embedded
For into
device
medium
Implantation
sus-
phagocy-
M sodium
functional
of implantation
hematoxylin-eosin.
(0.14
gelation.
6 days
microscopy.
collagen
solution
immediate
to gel
to
of cold
lOx
of
to prevent
allowed the
according
7 volumes
sodium
on
with
reconstituted
1 volume of
kept
a pipette
chamber
was
with
volumes
encapsulation,
chamber
gel
1121. In brief,
et al.
yeast
3 h of incubation,
a graded
of membrane
aspect and islet cells
2 and
4#{176}C.Collagen
of cells
percentage
Morphological encapsulated
forming
of macrophages
at
0.2
examination
of RPMI-
After
After
and
of both
1 mL
cerevisae
adhering
through
kinds
membranes in
eval.
identified
made
membrane
After
of Montesano
dehydrated
was
were
blue.
cells.
the cells
hematoxylin-eosin,
the
ence
stored
7.4),
different
the walls of the encapsulation chamber, and a collagen gel to immobilize the islets. Type I collagen was prepared with adult Wistar rat tail tendons that were dissolved and stirred in a sterile acetic acid solution (1/1000, v/v). After centrifugation at 16,000 g for 1 h at 4#{176}C, the supernatant method
Coomassie
glutaraldehyde
microscopy
of the
as well
Saccharomyces
and
in a 2%
the
of
as blue
washed
cells
incubated
with
jiL
appeared
were
surface
copy.
100
surface
other
Briefly
were
stained
macrophages
the membranes
a dis-
of a support
artificial
tosis,
pancreatic
device
is composed
rings,
under
encapsulated
encapsulation
weigh-
of RPMI-1640
macroencapsulation
poly-tetrafluoroethylene
rats
collagenase
hand-picked in 5 mL
of 5%
Pancreatic
Wistar
by standard
of culture
4 days
immobilized
AN69
outbred
After
atmosphere
membrane.
adult France)
ref. 11j and
37#{176}Cin a humidified
AN69
male
L’Arbresle.
Kostianovsky,
microscope.
islets
from
Credo,
and
Lacy
secting
isolated
g (Iffa
of
cells/mL)
membrane
while
1141.
devices
presence
(108
pension
staining
islet-containing
the
1996
t-test.
Statistical
each
mean
values
parameter was
were assumed
±
calculated.
performed
by use
for
P < 0.05.
of a
RESU LTS Analysis Two
of the
and
six
containing They
were
area
remained
tion
of
and
the
days
after were
retrieved
covered
only
with
intact. vaseularization
Scanning colonization
The
preservation
that
the
tween after
implantation,
brane
surface
the
of implantation,
adhering
to the
volved
being
sulated
islets
of
containing
pared thermore, with
the
the
tively,
the
full
Morphological Two
days
most
of the
outline
Fig.
1.
of membrane empty
implantation, a few
Scanning
electron
after
the islets
islets
was
micrograph
preserved,
broken.
ofthe
surface
tral
part
(Fig.
enee
of
intact
3D).
macroislets
of
outline in
the
round
4A).
(Fig.
their
was
the after
(Fig.
in the
culture
observed
only
In islets
outlines
necrosis
of
islets
(Fig.
encapsulated
showed
necrosis
ofencapsulated
center
of encapsulated
gaps
regardless
a cellular
part
cen-
conditions.
in the culture
periph-
in the
pres-
IL-iD.
Insulin
studies release
days
in the
from
that
606
±
2).
aspect
of the
after
6
membrane
Kessler
of encapsulated
from
cavity
of freshly
28
islets was
isolated
.tU/mL, the
islets
encapsulated
peritoneal
not
islets:
507
respectively.
insulin
to
of
265
50
2
versus
6 days
encapsulated
±
for different
81 iU/mL
±
However,
release
decreased
implanted
significantly
after
im-
islets
was
(P < 0.01;
iiU/mL
5). co-culture
showed
an
glucose
of 26
sulated
islets.
eantly
modify
et a!.
after
murine
The
Macrophage
28%, addition
the
38
±
and
response
for
and
free antibodies
macrophages activity
RU/mL
rat
insulin
of anti-IL-113
with
functional
500 (Fig.
in
respectively,
islets
their
implantation
macrophages
decrease
and
of free
j.tU/mL versus IL-1 antibody
6 days
of
important
co-culture
AN69
islets
with
the
of murine
of
structure
with
covered
preservation
an
the
examination
of the
However, eral
the
by
3,
modifications
Histological
The
respee-
whereas
ofthe
bound
of IL-i
was necrosis
aspect
an
Indeed,
Furthermore,
presence
The by
disintegrated.
cellular
by
bound
membrane
in the
shown
of islets
presence
Fig.
islets
However,
as
mod-
3).
(Fig.
structures
3C).
exhibited
of culture
unaltered
structure
of the (Fig.
completely ones
totally
morphological
significantly
Fur-
increased
morphological
was
was
almost
cells
islets
was
as round
surface
2 days the
freshly
were
inner
of the
the
appeared
of isolated
plantation,
com-
15%,
6 days
of encapsulated
encapsulated
of only
and
islet-
ones.
coverage
devices
2 days of
empty
65
in-
of encap-
macrophages
of the
to reach
islet-
surface
by
surface
and
After
islets isolated
but
Functional
cells
cells
presence
of encapsulated with
the
the
other
reaction.
colonized
of the
studies after
the the
of of
empty
membrane
duration and
mem-
regardless 80%
cellular the
percentage
implantation for
of
were
5%
to the
of the
However, this
45%
only
that,
macrophages,
fibroblasts.
be-
six days
devices.
surface
were
devices
with
adhered
entire
4B).
modified
and
outline
exhibited
demonstrated not
intact
the
adhesion.
.
cells
im-
membrane,
1 ) Two
approximately
increased
implantation,
was
revealed
membrane
devices
the
network
(Fig.
a few
study
duration
of
longer
phages,
cellu-
cellular
no
After
the
important
aspect
islets
part
preserved. of
an
membrane
empty
observed
was
any
fiber
adhesion
only of
of
typical
of the
cytological
containing
surface
devoid
of cell
was
compared
layers
implantation modifica-
the
ified
the
fossa.
nor
examination
external
islet-
ileal
The
device
showed
of the
areas
of the
devices were
structure the
The
the
rings
and
right
omentum
microscope
of
the
the necrosis
the
aspect
islet-containing
whereas
tissue of
electron
empty in
omentum.
Neither
macroscopic
device
implantation,
devices
the
planted lar
macroencapsulation
days,
did reaching
in the
islets to eneapin the
not 422
absence
signifi±
83
of anti-
6).
in mice
cytotoxicity
(magnification:
against
A. x314;
encapsulated
B. x5000).
islets
731
Fig.
2.
after
implantation
Phagocytosis
Similar in the the
32%
culture Study
patterns
presence
insulin for
were
test
of munine
were ofIL-i3.
macnophages
observed After
release decreased the encapsulated
significantly
higher
of islets of the
in the viability
for 2 days
the
culture
Journal
of
Leukocyte
surface
of islets
ofexposure
of empty
(A,
B) and
nat islets
containing
(C,
D) devices
2 and
6 days
than free
those
observed
Biology
(Fig.
encapsulated
Volume
islets
60,
and to values
in the
of macrophages and
IL-i3
release
and
diffusion
to IL-1
tured in the presence of IL-i 3 showed a similar in the islet viability to 31 and 23%, respectively
732
membrane
to 44% for free islets islets. However, these
presence of
at the
x 125).
(magnification:
co7A). cul-
decrease (Fig. 7B).
December
1996
After
2 days
of co-culture,
the
significantly influence macrophages. Indeed, creted by the macrophages ±
15 pg/mL
and
118
the 160 ±
presence
of rat islets
did
not
release
of IL-i 3 from murine ± 41 pg/mL of IL-i3 were Sein the absence of islets and 110
24 pg/mL,
macrophages were cultivated in the sulated or encapsulated islets.
respectively, presence
when of non-encap-
the
Fig. 3. Phase-contrast x80; D, x200).
Fig.
4.
(B, x80;
Phase-contrast 1000
and
light
micrograph
micnographs
of encapsulated
of freshly
rat
isolated
islet
after
(A,
x 80;
2 days
B, x 250
of culture
) and
in the
encapsulated
presence
of murine
rat islets
6 days
macnophages
after
(A,
implantation
x
80)
and
(C,
IL- 113
pg/mL).
Kessler
et al.
Macrophage
cytotoxicity
against
encapsulated
islets
733
800 * **
* * *
1
macrophage membrane phological
Cl)
were
600
q,
Cl)
400
reaction observed on the surface of the during xenotransplantation. Furthermore, and functional alterations of the implanted
observed
concurrently
in vitro
study
showed
unable phages
to protect and IL-i3.
be involved islets
The
200 Cl)
2
6
time(days)
of Fig.
5.
Insulin
freshly
isolated
tation.
Results
the
release and
secretion
response
to
encapsulated
were
mean
of free
and
glucose
rat s
±
islets
stimulation 2 and
islets
(4
6 days
of 6 experiments.
implanted
g/L)
after
< 0.05;
of
implan-
Comparison
(*P
sence
24
h of incubation
in the
between
**P
presence
of the membrane, only 40% remained measurable (Fig.
IL.fl3 sion,
IL-iD
did
20%
of the
initial
partments
not 24
(Fig.
AN69
of
the
the
or in the
membrane.
ab-
However,
found
diffusion
in both
human
of
the
study
that device
the
rat
were
IL-i3
islets
at
equilib-
placed
involved
The proved
activity
of macromight
of encapsulated
vivo.
encapsulated
islets
in
in
the
in
vitro,
and
in
for
their
a
the
lar
reaction
islets
5J. However, observed only
in
that of
macro-
effect,
of
by
encapsulated
reaction in allografts were hyperglycemic,
reaction
induced
low-
poly-lysine 1171. absence of cellu-
transplantation
cellular
exin the
found
islets. It has recently been of rat islets by UVB irra-
the macrophage when the animals
and/or
reaction
1161. The
immunomodulation
syngenie
the
islets
fibrotic
in vivo might be
from encapsulated that a pretreatment
known
was
involved
the
altered
in the
ne-
islets.
Em-
bedding creates
tissue found around the macrocapsules not a physical barrier to the passage of nutrients
only and
insulin
but
consumer.
The
effect of both intact islets and insulin macrophages has been demonstrated
on the in vitro
also
ehemotactic peritoneal demonstrates
reaction.
membrane
ered the fibrotic reaction around alginate Furthermore, Horeher et al. showed the
erosis
8B).
macroencapsulation
cellular
implanted microcapsules of such cellular reaction
suggesting
This
DISCUSSION This
of
macrophages
around planation
was
com-
experiment.
concentration
in
involvement
antigen release demonstrated
< 0.001).
of the initial amount of 8A). After 1 h of diffu-
of IL-i 3 were
h
represented
rium
the
amount
after
value
cross
of functional
implantation
diation, After
this artificial
phage reaction was determinant. The fact that implanted empty devices remained free from cellular adhesion mdieated that the material used for their preparation was not responsible for the activation of the macrophages, whereas alginate poly-lysine microcapsules triggered the activation
0 0
the
the islets against the toxicity This suggests that macrophages
in the loss
after
with that
AN69 morislets
behaves
as
a
competing
118, 191. Although collagen is also known as a ehemotactic factor, it did not influence the cellular reaction in our study. Implanted devices void of islets but containing collagen remained free of cellular deposits. Furthermore, this ab-
the
murine
sence
of cellular
implantation. munocompetent pate Cl)
of the
both
I
free
the and
insulin
release
served
in both
use
cytotoxicity
of islets.
in the Using
2 and
6 days
depending
on the
after
and impartici-
permeabil-
membrane.
of murine
encapsulated
of resident
of IL-iD
observed
effect,
artificial
In vitro,
0
was
the products of islet necrosis located inside the islets could
in the chemotaxis
ity
.
reaction
Finally, cells
islets
while
their
cases.
This
macrophages
induced
morphological
result
macrophages, macrophages
aspect
could as
supernatant
in the was
be explained
illustrated
by the
of co-culture
in the
activated
against
a decrease
pre-
by the low
level
presence
by intraperitoneal
in-
jection of Corynebacterium parvum, Kr#{246}ncke et al. showed an increase in the macrophage cytotoxicity [20J. Wiegand Fig.
6.
Insulin
freshly
isolated
absence
