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Reference
Modulation of an early and lethal autoimmune diabetes in transgenic mice by IL-4 overexpression and treatment with anti-IFN-γ antibody
HERRERA, Pedro Luis, et al.
HERRERA, Pedro Luis, et al . Modulation of an early and lethal autoimmune diabetes in transgenic mice by IL-4 overexpression and treatment with anti-IFN-γ antibody. Transgenics , 1999, vol. 3, p. 97-106
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the
B-cells bear theB7-1
(CD80) co-stimulator on their surface[3].
Transgenic mice bearing one or theother
transgenedo not
develop diabetes;the
TNF transgenic mice do, however, display a severe insuli- tis affectingall
their islets, but they conserve normal B-cell numbers [2]. In contrast, all double transgenic mice develop diabetes by 5 to 6 weeksof
age (lo07oof
more than 100 such mice obtainedin
successive breeding experiments were diabeticby
the 6th weekModulation of an Early and Lethal Autoimmune Diabetes
in Transgenic Mice by IL-4 Overexpression and Tfeatment with Anti-IFN-y Antibody
pEDRO LUIS HERRERAU*, LILIANE FOSSATIb, SHOZO ZUIb, DAVID M. HARLANc, JEAN-DOMINIQUE VASSALLIA, LELIO ORCIA ANd PIERRE VASSALLID
àDepartmefis of Morphology andb Pathologl,, University of Genevtt Metlical Sclrcol,
I
rue Michel-Servet, l2II
Genève 4, Stvitzerlantl and-
clnnwtobiology Departnrcn; Naval Medical Research Institute, Betltesda, Marylcutd, USA (Receivetl Decetnber 23, 1998)
Transgenic mice constitutively expressing TNF and
87-l
tt'ansgenes in their islet B-cells, both under the control of insulin gene promoter, develop insulin-dependent diabetes mellitus (IDDM) at 5 or 6 weeks of age. We have previously shown that the diabetes can be ptevented by treatment of these mice with anti-CD8 rnonoclonal antibody [1], indicating the Th1-type nature of this disease. We now report thât increased expression of cilculating IL-4 in these mice (through a third transgene expressed in B lymphocytes [17]) or decreased useofIFNy
(through injection of anti-IFNy antibody during four weeks) retarded by several weeks or months the occurrence of this diabetes. The effect of these two procedures appeared to be cumulative, leading occasionally to complete protection from diabetes, but always with per- sistence of the insulitis. Diabetes in TNF /
87-l
transgenic mice is accompanied by destruc- tion of both B-cells and non B-cells. In contrast, histologic analysis of pancreata from IL-4 overexpressing mice revealed intact islet non B-cells. These data suggest that partial deviation fi-om a Th I to a Th2 l'esponse can modify both diabetes occurrence and islet lesions observed in this form of early severe autoimmune IDDM.Ke1tw6fi"' mouse, transgenic, islet, IDDM, autoimmunity, immune deviation, IL-4, IFN-1
INTRODUCTION
We
have developeda
transgenic mouse modelof
autoimmune diabetes that is characterized by an early ageof
onset, rapid progression, and complete pene- trance by crossing two different strainsof
transgenic mice. TNFI B1-l
double transgenic mice[l]
weregenerated
by
crossingmice
overexpressingTNF
in their insulin-producing B-cells[2] to
micein
which* Mailing address: Dr. Pedro Herrera, Département de Morphologie, Centre Medical Universitaire, 1, rue Michel-Servet, CH-121 i
Genève 4, Switzerland, tel.: +41221O2 5225,fax +4122702 5260, e-mail: Pedro.Herrera@medecine.unige.ch 9'7
98 PEDRO LUIS HERRERA er a/.
of life), with the clinical signs of type I,
rnsu-lin-dependent, diabetes
mellitus (IDDM)
including:hyperglycemia (>> 300 mg/dl), polyuria, polydipsia, polyphagia, and weight loss.
In
these mice, disease progresses rapidly, such that the mice are infertile andtheir longevity is
considerably reduced(10 to
20 weeks). Histologically, the severe insulitis is accom- paniedby
rnassive destructionof
insulin-producing cells.An
identical modelof
diabetes has also been developedby
others,with similar
observations [4].This
acute diabetes has been shownto
be CD8+ T lymphocyte-dependent,since anti-CD8 (but
not anti-CD4) antibody prevented its appearance [1].During a normal immune response, CD4+
T
lym- phocytes differentiateinto two different
groupsof
effector cells, the so-calledThl
and Th2 subpopula- tions,which
are characterizedby
the productionof distinct
setsof
cytokines andby
thefulfillment of
specific functions [5].Thl
lymphocytes play a majorrole in
delayed-type hypersensitivity responses by secretingIL-2
andIFN-y,
amongother
cytokines;antibody-mediated responses are modulated
by
Th2 cells through the productionof IL-4, IL-5, IL-6
andIL-10, in
particular.lt is
considered, although lesswell
established, that CD8+T
lymphocytes can also differentiate into comparable categoriesof
cytotoxic Tc1 and Tc2 cells[6-8].
Different studies have sug- gestedthat one
possible therapeutic approach forautoimmune inflammatory
diseases,like
IDDM,could be the
inductionof
antigen-specific immune deviation by means of cytokine treatment [9]. As Thl and Th2 cells regulate each other[5], Thl
cytokines down-regulatingthe
emergenceof Th2 cells
andvice-versa, it was
suggestedthat
inflammatory autoimmune diseases,in which Thl cells
appear to play a dominant role, could be treatedwith
Th2{ype cytokines, mainlyIL-4,
and some successful results have been obtainedin
experimental animal models[0].
Thereis
indeed strong experimental evidence supporting the idea that the ratio between the releaseof
IFN-y and ofIL-4
determines the degreeof
tissue damage mediatedby
auto-reactiveT
lymphocytes (reviewedin [0]).
Lymphocytic co-stimulatory signals, such as those conveyed by the T lymphocyte receptor CD28 and its
B7-1 (CD80)
andB7-2
(CD86) ligands, are neces-sary
for
optimal activationof
naiveT
cells: they also appear to regulate differentially Th cell commitment, so that selective involvementof B7-l or B7*2
may becritical for
the developmentof
autoimmune dis- ease[11].
Some experiments indicate thatthe 87- I|CD29
pathway favorsThl-type
responses, which are also favoredby low
antigen doses[2]. In
thiscontext, transgenic expression
of B7-l on
p-cellsleads to autoimmune diabetes, considered to be a Thl
or Tcl type of
disease,in
various typesof
double transgenic mice [1, 3,4, l3l,
and accelerates diabetes onsetin
nonobese diabetic (NOD) mice[4].
Never-theless, there is no a simple view of "Th2 equals pro- tection"; for instance, NOD mice deficient in CD28 or transgenic
for IL-10
have a more rapid onsetof
the disease, and transferof
Th2-like cells rnay induce a more severe damagein
the islets thanThl-like
cells1il, ls,
161.In
the presentwork
we have investigated whether the early diabetes onset and its rapid progression can be modulatedin
doubly transgenic TNF/ 87-1
mice by altering the circulating cytokine balance. We show that constitutiveIL-4
over-expression(in
mice bear- ing a third transgene), or that treatment with a neutral- izing anti-IFN-y antibody, can delay or even prevent theIDDM
development.MATERIALS AND METHODS
MiceThe production
of TNF I B7-l
transgenic mice has been described previously [1]. Transgenic mice con-stitutively
expressingIL-4 in
B-lymphocytes were obtained from Dr.\ff.
Mueller (Kôln, Germany)[7];
transgene presence and expression was explored by FACS analysis
on
peripheral blood lymphocytes to detectMHC
classII
expression as described [17].Blood samples were collected by orbital sinus bleed- ing after a 4-hour fasting period. Glycemia was mea-
sured using the haemoglukotest strips
fromBoehringer-Manheim (catalog
#
1119420).For
neu- tralization of IFN-y, new-born mice were tolerized at birth by injectingi.
p. 20pl
of normal rat serum. Rat anti-mouseIFN-y
neutralizing monoclonal antibodyR4642 [18]
was then administered intraperitoneallyCYTOKINE MODULATION OF DIABETES PROGRESSION 99
(i.p.), twice weekly at a dose of 0.25 mg, to mice from
2
weeksto 4
weeksof
age,when the
dose was increased to 0.5 mg until the 6th week, at which point antibody treatment was discontinued. Murine recom- binantIL-4
was obtainedfrom
Ono Pharmaceutical Co. Ltd., Osaka (lot #881213). 100 ng ofrIL-4
(1.65x
106 U/mg) was given i.p. twice weekly, from birth until the 6th week of life.Immunostaining
Pancreata
were fixed either in Bouin's or in
47oparaformaldehyde, dehydrated and embedded
in
par- affin. Alternatively, the pancreata were frozen in cold methylbutane and kept subsequentlyin liquid
nitro- gen. The four different anti-islet hormone antibodies usedfor
immunofluorescence (anti-insulin, anti-glu-cagon, anti-somatostatin, and
anti-pancreatic polypeptide), as well as those used to stain T (GK1.5;H35-11.2) and B cells (p Ig
chains),have
been described elsewhere[2]. When
indicated, paraffin sectionswere
stainedusing the
aldehyde fuchsin merhod fl9l.
Morphometry
and statistical analysisQuantification of pancreatic islet endocrine cells was performed using three
3 to 8
week-old animals in each group, computing a minimumof 3l
islets per hormone and experimental group. The analysis wascarried out using
sectionsof splenic
pancreata, derivedfrom the
dorsal pancreaticbud,
where the islets arerich in
glucagon cells but poorin
PP cells.Data
from TNF / B7-l
double transgenics and non transgenic control mice were comparedby
ANOVA (data were transformed into their square roots in order tofit
a normal distribution).100
90
80
70
E .g
60Eo,
950
Ëto
Ê40
Lo
t30
lL4-TNF-87 mice, anti-lFN1-treated
anti-lFN1, either alone or combined
with lL-4
L
lL4-TNF-87 mice
I I
I
\
lo Ëo
:
Ê 2010
0\t (, (t i- @ oNltTrô(o
FFfFFf
age (weeks)
FIGURE I Incidence of diabetes in TNF / 87-l doubly transgenic mice, and in IL-4 / TNF / B7-l tfiply transgenic mice. Doubly transgenic mice wele either untreated or injected with IL-4, with anti-IFN-y antibodies, or both. Triple transgenics were either untreated or treated with ant!IFN-1 antibodies
untreated or lL4{reated
100 PEDRO LUIS HERRERA er a/.
RESULTS
Generation of
triple
transgenic mice: TNF/r_7-l
mice
secretinglL-4 from their
B lymphocytesIn pilot
studies,two TNF / B1*l
doubly transgenic mice receivedtwo
weeklyi. p.
injectionsof
100 ng murinerIL-4,
starting from birth. This dose has been shown to be protectivein NOD
mice [20]; however, these mice became diabetic as their control untreated transgenic littel'-mates, i.e., at5-6
weeks of age. Con- sequently, transgenic rnice constitutively expressingIL-4
under the control of theIgH
enhancer-promoter[l7]
were crossedto
theTNF
andB7-l
transgenic mice. Double transgenicIL-4 / TNF andlL-4 lBl-l
mice, which never develop diabetes, were then inter-
bred, and twenty-two triple
transgenicmice
were obtainedfor
explorationof
diabetes occurrence.All TNF i B7-l
double transgenic rnice obtained as a result of such cl'ossings, either females or males, were diabeticat 5 or 6
weeks.Strikingly
however,at
9 weeks, 18/22lL-4 / TNF / B7-l triple
transgenicmice
(82o/o) remained normoglycemic, ancl 12122(54%) remained normoglycemic at 12
weeks (Figurel). At
15 weeks,only
three mice(of
which two were fernales) werestill
normoglycemic, and twoof
them became diabetic at6
rnonths. The last triple transgenic mouse (a fernale) remained normoglyce-mic for the entire period of study,
II
months (Figure 1), when the animal was sacrificedfor
histo- logical analysis.The insulitis
in
triple transgenic mice, evenin
the absence of diabetes, wasof
similar cellular composi- tion and severity as compared to thatof
TNFI B1-I
double transgenic mice (Figure 2).
In
particular, theCD4/CD8 ratio among islet-infiltrating cells
was comparable both in double and triple transgenics. His- tologic and immunofluorescent studies performed on TNF/ B7-1
double transgenic animalsin
the weeksfollowing the
occunenceof
diabetesshowed
a decreaseof
the islet lymphocyticinfiltrate,
as com- paredto
whatis
observedat the time of
diabetes occulTence, accompanied by a massive reduction notonly of the
insulin-producing B-cells, as described previously,but
also remarkablyof the
three otherendocrine cell types, the so-called non
B-cells(Figure 3, panels
E to H;
TableI).
Interestingly, the frequency of endocrine cells (of the four types) found outside the islets, i.e. present within the epitheliumof
the pancreatic ducts or isolatedin
the interstitial tis- sue, was increased (128 cells out of904 cells counted,i.e.
147o) comparedto
what we observedin
normal mice (0.6Vo; 29/5101).In
contrastto
these observa- tions, histological analysis of the diabetic IL-4 / TNF/ B7-1 triple
transgenic mice showed that islet non-Bcells
remained abundant (Figure3,
panelsI to L;
Table I). In addition, the pancreata of some
of
thetri-
ple transgenic mice, including the mouse that escaped diabetes and had nolmal islet B-cell content, showed a markedinsulitis
and fibrosis,with
ductal structures and adipose tissue replacing mostof the
exocline acini in several lobes (Figure 4, A to C).Injections of
neutralizing
anti-IFN-yantibody Nine TNF / B7-l double
transgenicmice
were injectedi.p. either
anti-IFNy alone (threemice)
or anti-IFNy andrIL-4 in
combination(six
mice) untiltheir 6th week. Both
treatmentsyielded
sirnilar results: no diabetes was observed at 6 weeks, but all mice were diabeticby
the 9th week (Figurel).
Thehistological appearance of the islets from the diabetic
mice
wasidentical to
thatof
the untreated double transgenic diabetic mice,with
disappearanceof
all four types ofendocrine cells.Since treatment with anti-IFN-T antibody appeared to delay diabetes onset
in
the TNF/ B1-1
mice, six IL-4 / TNF/ B1-l
triple transgenic mice we1'e treatedwith
anti-IFN-y antibodyfollowing
the same proto-col. At
the endof
antibody treatment(6
weeksof
age),all were
normoglycemic (Figurel) and
one mouse, sacrificed at this time for histologic examina-tion,
showed the presenceof
a lymphocytic insulitisbut
abundant B-cells (Figure4, D
andE). Of
the remainingfive
mice, three showed a late occurrenceof
diabetes andtwo
remained normoglycemic until sacrifice, atI
months (Figure 1); the two non diabetic mice carriedout
normal pregnanciesgiving birth
to normal litters. Histological analysis of pancreata fromthe
anti-IFNy-treatedmice, either
doubleor
tripleCYTOKINE MODULAIION OF DIABETES PROGRESSION 101
FIGURE 2 8 to 9 pm-thick cryostat sections, stained by immunofluorescence, of pancreata from TNF / B7-1 doubly transgenic mice (4, C, and E), and IL-4 / TNF / B7-l triply transgenic mice (8, D, and F). Panels A and B show islets stained for CD4+ cells; CD8+ cells are labeled in C and D, whereas B cells are demonstrated in E and F. The ratio CD4+/CD8+ is sirnilar in doubly and triply transgenic mice. The islet shown in panel F contains only few peripheral B lyrnphocytes. All mice were 5.5 weeks-old: the TNF / B7-1 were ah'eady hyperglyce- mic, whereas the IL-4 / TNF / B7-1 was normoglycemic. Bar represents 50 ptm (same magnification in all panels) (See Color Plate V at the back of this issue)
transgenics, either normoglycemic or diabetic, always showed the presence of an insulitis (Figure 4, F), indi- cating that delay
or
preventionof
diabetesdid
notresult
from
variationin
the incidenceof
isletinfiltra-
t1on.
102 PF.DRO LIIIS HERRERA et al.
FIGURE 3 5 pm-thick paraffin sections, stained by immunofluorescence, of pancreata from control (non transgenic) mice (A-D), TNF / B7 1 double transgenics (E-H), and IL-4 / TNF /
87-l
triply transgenic mice (I-L). A, E, and I, anti-insulin staining; B, F, and J, anti-glucagon staining; C, G, and K, anti-PP family staining; D, H, and L, anti-somatostatin staining. Overtly diabetic TNF / B7-l mice (E to H) show an almost complete loss of all islet endocrine cell types, wheleas diabetic IL-4 / TNF / B7-1 mice (I to L) have abundant non F-cells (J-L). Mice were 8 to 10 weeks-old. Bar represents 50 pm (same magnification in all panels) (See Color Plate VI at the back of this issue)DISCUSSION
The present
work
shows that constitutive productionof IL-4 by
a transgene expressedin B
lymphocytesinfluences the early and severe autoimmune diabetes
resulting from
combined transgenic expressionof
TNF andB7-1
by pancreatic B-cells, by modifying its course, incidence and even islet lesioned pattern. TheCYTOKINE MODULATION OF DIABETES PROGRESSION 103
FIGURE 4 5 pm+hick paraffin sections of pâncreata from IL-4 / TNF / B7-1 triply transgenic mice. A, B, and C are from a triple transgenic mouse that wâs normoglycemic at sacrifice, when I months-old. The islets appear heavily infiltrated and distorted (A, B), with abundant col- lagen deposition (gleen, in B), while many acini undergo ductular metaplasia (A, C), so that entire pancreatic lobes appear as fibrotic, with abundant adipocytes filling the interstitia (C). In C, adipocytes appear as large white holes, whereas exocrine acini are mostly replaced by altered ducts (small holes, also visible in A). B-cells are still abundant (8, a cluster of cells stained violet). D and E are consecutive sections
from a normoglycemic triple transgenic mouse killed when anti-IFN-y trcatment was ended (6 weeks-old), showing severe insulitis (D) together with abundant remaining B-cells (violet, in E). F, insulitis in an anti-IFN-T-treated triple transgenic mouse having escaped diabetes (10.5 month-old). A, C, D, and F are sections stained by the hematoxylin and eosin procedure, whereas B and E are colored by the aldehyde fuchsin (Gomori's) method, which allows identification of insulin-producing B-cells (violet) as well as of collagen and elastin fibers (green and violet, respectively). Bars represent 50 pm (magnification is the same for A, D, E, and F) (See Color Plate VII at the back of this issue)
influence of
IL-4
is increased when the effect of IFNyis
simultaneously (but only transiently) decrcased, as the result of anti-IFNy antibody injection.While
1007oof
the TNFI B1-l
double transgenicmice
developed diabetesby the
6thweek of
age (whatever the genetic background resulting from vari- ous crossings, and independently of gender), diabetes was uniformly delayedby
weeks or months in triple transgenic mice, and even did not occur before sacri-fice at almost one year of age in one case. The obser- vations are different
from
those madewith IL-4
inNOD
mice,which
were protectedby
administrationof
recombina\tIL-4 [20]
(inefficientin
the caseof
TNF /B7-1
transgenic mice) and in which expression of an IL-4 transgene within the islets prevented insuli- tis, and thus diabetesl2ll;in
contrast, the triple trans- genic micain
the present study had insulitis, even in the case that never developed diabetes.A
plausible104 PEDRO LUIS HERRERA et al.
interpretation
of
these datais
that theThl
autoim- mune response(or
probablyTcl,
sinceit
does notrequire the
presenceof CD4 T
lymphocytes[])
develop within the TNF-induced insulitis because
of the
antigen plusB7-1
stimulation has been "devi- ated" by theIL-4
released byB
lymphocytes presentin the insulitis [2],
towardsa Th2 or Tc2
typeof
response, thus favoring auto-antibody formation but decreasing
the
strengthof the T cell
cytotoxic response. This explanation remains nevertheless spec- ulative, since we could not demonstrate any shift inthe cytokine profile of islet infiltrating cells
(asassayed by semi-quantitative RT-PCR from total pan- creas
RNA,
not shown),nor in
theThl /
Th2 ratioamong the islet-infiltrating lymphocytes (as assayed
by
immunofluorescence on pancreatic sections usingnewly
availableThl-
and Th2-specific monoclonal antibodies, not shown). The effectof
anti-IFNy anti- body treatment on this typeof
diabetes may have a comparable explanation, although excessof IL-4
is more directly instrumental than decreased availabilityof
IFNyin
differentiationof
stimulated naïve T lym-phocytes toward a pattern favoring a
humoral response t5, 101. In the case of NOD mice, prolonged treatmentwith
anti-IFNy antibody reduces the inci- denceof
diabetes 122), andin RIP-LCMV
transgenic mice bearing an IFNy null mutation, both insulitis and diabetesdo not
appear[23]. Anti IFN-y
antibodytreatment did
appearto
enhancethe
protectiveanti-diabetic effect of IL-4 transgene expression in the
TNF / B7-1
transgenic mice, sincetwo
mice outof
nine never developed diabetes.A
potential explana- tion for the protective effect conferred by anti-IFNy is that the lack of IFNy may prevent an adequate expres- sionof
MHC classI
andII
antigens on B-cells [24],thus preventing lymphocyte stimulation,
without influencing the TNF-driven insulitis.TABLE I Average numbel ol endocrine cells per islet section in TNF /
B7-1 and in IL-4 / TNF / B7-1 transgenic mice (See "Morythometry and Statistical Analysls" paragraph in the Methods section)
Instrlin
GlucctgonPP
Sonnlo-family
stûtitr13 13*
10 22
We observed that elevated circulating
IL-4 in
the transgenicmice not only
decreased diabetes inci- dence,but also modified the
natureof the
islet lesions. We were surprised to observe and now reportfor
thefirst time that all four
endocrinecell
types were destroyed in the TNF /B7-1
transgenic mice.It
is thus the onlyIDDM
model so far reported, either spontaneousor
experimental,in
which the islet non B-cells (i.e. those producing either glucagon, somato- statinor
pancreatic polypeptide) are destroyed. This non p-cell destruction takes placein
spiteof
the fact that in these transgenic mice only B-cells, and not the other islet cells, bear theB7-1
molecule, and would therefore be predicted to deliver the required co-stim- ulatory signalto
activate B-cell-specificT
cells. One possible explanation is that non p-cell specific T lym- phocytes could be co-stimulated intrans;
that is from aneighboringBT-l
expressing p-cell. Indeed, CTL precursorswith TCR
displayingaffinity for
peptide presented by the various islet cells may be, becauseof
the strong insulitis and local lymphocyte traffic result-
ing from
the local expressionof
theTNF
transgene [2], in contact with both an islet non B-cell, that some of them may recognize, andB7-l
molecule borne by adjacent B-cells, thus providing the required second signal (Figure 5). Strikingly, the presenceof IL-4
in triple transgenic mice, in particular byB
cells known to be presentin
the lymphocyticinfiltrate [2],
modi- fies the patternof
islet cells remainingin
the diabetic animals, since non B-cells were preserved. This canbe explained by a
decreasedcytotoxic
response resultingfrom
the proposedshift
towardsa
type 2 lymphocyte response discussed above.In this
case,only the cytotoxic
response expressed against the most antigenic cells, those expressing self-peptides togetherwith
theB7-1
molecule (i.e., B-cells) would persist.Finally, another alteration observed as the result
of
the expression of the IL-4 transgene was the pl'esence of fibrosis
in
the exocrine pancreas.IL-4
is known to enhance the synthesis of extracellular matrix proteins by fibroblasts [25]. Peri-insular fibrosis has also been observed in transgenic mice producinglL-4 under the control of an insulin promoter [26].Control TNF / B7_1
IL-4lTNF/87_1
25 19
2
3'o 1
I9 l3
" p<0.001, CONTROL vs. TNF / B7-l double transgenics.
A
o o o o
|t o 9O
o
B
class I MHC protein
"anti a-cell"
T cell receptor
CD-28
"anti p-cell"
T cell receptor FIGURE 5 Drawings depicting how an "anti-non B-cell" CD8+ T lymphocyte (labeled "CTL- 1") might be, within the islets of TNF / B7-l doubly transgenic mice, simultaneously in contact with a non B-cell (only glucagon cells are represented) and with a B-cell, and be activated by both. T cells as the one labeled *CTL-2" would be responsible of the destruction of B-cells. Panel A was drawn from an electron micrography of an infiltrated islet
In conclusion, the TNF
lB7-l
transgenic mice rep- resentan
unique modelof
autoimmuneIDDM,
in which the pathogenic mechanismof T
cell-mediated105
B-cell destruction can be more precisely studied and influenced, since these events take place
in a
very limited period of time. Thus, they may afford a better opportunity to study how changes in cytokine expres- sion may be used to alter the cause of diabetes, as has been done in the present study. They should also facil-itate the
studyof islet cell
regeneration followingrapid and
massive destruction (reviewedin
[27D, since an increased presenceof
extra-islet endocrine cells was found in these diabetic mice.Acknowledgements
We are most grateful to
Mr
JorgeRitz for
handlingand breeding mice, to Miss Gissela Gallardo and Mrs Ileana Condacci
for
genotyping by PCR, and to Mrs Danielle Ben Nasr andMrs
Gyslaine Moussardfor
paraffin sectioning and staining. Thiswork
was sup- portedin
partby a
grantfrom
the Fonds National Suisse pour la Recherche Scientifique, andin
part by Naval Medical Research and Development CommandGrant
EW.0O95.OO3.|4I2.The
RIP-mCD80 trans- genicmice
describedin this
study were generated according to the principles set forthin
the Guide for the Care and Use of Laboratory Animals (Kommitteeon Care and Use of Laboratorl, Animals
(1985)Guide
for
the Care and Use of Laboratory Animals(Natl. Inst. Health,
Bethesda),DHHS. PubL
No.(NIH) 86-23). The views expressed
in
this article are those of the authors and do not reflect the official pol-icy or position of the
Departmentof the
Navy, Departmentof
Defense, nor the United States Gov- ernment.References
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o o
o o o
oo o
o
aa
a
O3a
a
aa
o(-cell
O
c
c oco
CTL-1
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