Therapy (2000) 7, 2015–2022  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt CELL-BASED THERAPY RESEARCH ARTICLE Prevention of beta cell dysfunction and apoptosis activation in human islets by adenoviral gene transfer of the insulin-like growth factor I

N Giannoukakis1,ZMi1, WA Rudert2, A Gambotto1, M Trucco2 and P Robbins1 1Department of Molecular Genetics and Biochemistry, and 2Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Interleukin-1␤ is a potent pro-inflammatory that has escent as controls. Adenoviral gene transfer of been shown to inhibit islet ␤ cell function as well as to acti- human IGF-I prevented IL-1␤-mediated nitric oxide pro- vate Fas-mediated apoptosis in a nitric oxide-dependent duction from human islets in vitro as well as the suppression manner. Furthermore, this cytokine is effective in recruiting of ␤ cell function as determined by glucose-stimulated insu- lymphocytes that mediate ␤ cell destruction in IDDM onset. lin production. Moreover, IGF-I gene transfer prevented IL- The insulin-like growth factor I (IGF-I) has been shown to 1␤-induced, Fas-mediated apoptosis. These results suggest block IL-1␤ actions in vitro. We hypothesized that gene that locally produced IGF-I from cultured islets may be ben- transfer of the insulin-like growth factor I to intact human eficial in maintaining ␤ cell function and promoting islet sur- islets could prevent IL-1␤-induced ␤ cell dysfunction and vival before and following islet transplantation as a potential sensitization to Fas-triggered apoptosis activation. Intact therapy for type I diabetes. Gene Therapy (2000) 7, 2015– human islets were infected with adenoviral vectors encoding 2022. IGF-I as well as ␤-galactosidase and enhanced green fluor-

Keywords: gene therapy; IL-1␤; IGF-I; islets; adenovirus

Introduction vent IL-1␤-induced suppression of glucose-stimulated insulin release and nitric oxide (NO) production in vitro The immunopathological characteristics of insulin-depen- in rat and human islets.6,7,10,11 However, there are con- dent diabetes mellitus (IDDM) in the well-defined non- flicting data on the effects of IL-1␤ on human islets.11–15 obese diabetic (NOD) mouse model consist of apoptosis We have demonstrated that islets from recently diag- 1 as early as 3 weeks of age 1, followed by a progressing nosed IDDM patients undergo apoptosis in an IL-1␤- ␤ 2 insulitis culminating in overt cell destruction. This dependent manner involving the up-regulation of Fas sequence of events also appears to occur in humans as (CD95) on ␤ cells.16 Similarly, IL-1␣ can promote the cell- 3,4 + + well. Although CD4 and CD8 T lymphocytes and surface localization of Fas in mice.17 Blockade of nitric their production of pro-inflammatory such as oxide (NO) production by chemical inhibition of iNOS ␣ ␣ ␥ ␥ (TNF ) and interferon (IFN ) prevented Fas up-regulation and Fas-triggered apoptosis ␤ play a role in cell destruction, the trigger for insulitis in human islets in vitro.16 The importance of 3,4 onset is unknown. Resident macrophages within islets and -derived IL-1␤ in IDDM onset derives ␤ ␤ may initiate cell damage by secreting interleukin-1 from several in vivo observations. In the BioBreeding rat, ␤ (IL-1 ) which in turn can lead to the activation of auto- a rat model of IDDM, silica particle depletion of islet reactive lymphocytes in the peripheral lymphoid system macrophages could prevent insulitis onset and IDDM.18 ␤ 5–7 ␤ upon presentation of cell-specific antigens. IL-1 Furthermore, recombinant IL-1Ra infusion into streptozo- treatment of murine islets in vitro results in the sup- tocin-treated NOD mice transplanted with syngeneic ␤ pression of cell function as determined by static glu- islets, prevented insulitis of the transplanted islets as well cose-stimulated insulin release assays, associated with IL- as IDDM.19 Recent data from our laboratory indicate that ␤ 1 -induced activation of both the expression as well as high, local production of IL-1Ra from human islets, can 5–9 the activity of inducible nitric oxide synthase (iNOS). suppress IL-1␤-induced, Fas-triggered apoptosis induc- ␤ Inhibition of IL-1 binding to the type I IL-1 signaling tion in addition to protecting against ␤ cell dysfunction , using the naturally occurring interleukin-1 in a NO-dependent manner.20 receptor antagonist protein (IL-1Ra) was shown to pre- Beta cell development and survival is, in part, con- trolled by the availability of growth factors and, in parti- cular, the insulin-like growth factors (IGFs).21–24 The IGFs Correspondence: P Robbins, Department of Molecular Genetics and Bio- chemistry, W1246 BST, University of Pittsburgh School of Medicine, consist of two potent mitogens, IGF-I and IGF-II. IGF-I Pittsburgh, PA 15261 USA expression progressively increases in liver during devel- Received 26 April 2000; accepted 29 August 2000 opment ultimately to mediate the growth-promoting Adenoviral IGF-I gene transfer to human islets N Giannoukakis et al 2016 effects of growth hormone postnatally. IGF-II plays a transduced.20 We have demonstrated that infection of prominent role during fetal growth in a wide variety of human islets with recombinant, replication-defective mammals including humans and is expressed in many adenoviruses deleted for E1 and E3 results in very high tissues postnatally, albeit at lower levels. Although the levels of transgene expression without affecting the liver is the major site of IGF-I synthesis, many other ability of the ␤ cells to respond normally to a glucose tissues are capable of secreting IGF-I in a growth challenge.20 We routinely infected groups of 200–300 hormone-independent manner. In these tissues, IGF-I islets within 48–72 h following islet isolation from acts in a paracrine and/or autocrine fashion and its cadaveric donor pancreas. To examine the effects of IGF- actions are modulated by six characterized binding pro- I on islets following gene transfer, islets were infected teins.25 The predominant site of IGF production in rodent with Ad-IGF-I as well as Ad-eGFP and Ad-LacZ at ident- and human islets is the ␤ cell, which can also respond ical p.f.u. (1 × 106) in a minimal volume of serum-free to their actions as it possesses the type I IGF signaling medium. IGF-I protein was detected in the culture super- receptor.21,24 IGFs can act in an autocrine and paracrine natant using a commercially available ELISA in which manner during islet development as mitogens and they IGF-I was measured following acid–ethanol extraction to may modulate insulin release postnatally, directly or remove IGF binding . IGF-I in the media of unin- indirectly through changes in ␤ cell mass or glucose fected islets was detectable (28.4 ± 2.6 ng/ml), however, responsiveness.21,24 Human fetal pancreas contains IGF-I in Ad-IGF-I-infected islets, a significantly higher level of and appears to secrete IGF-I in a glucose-dependent man- IGF-I (102.8 ± 34.2 ng/ml; Table 1) was detected. ner in vitro.21,24 In addition to their growth-promoting effects, the IGFs, and IGF-I in particular, display anti- Prevention of IL-1␤-induced suppression of glucose- apoptotic characteristics, including hematopoietic and stimulated insulin release as well as nitric oxide myeloid progenitor cells, neurons, cardiomyocytes, and production with adenoviral gene transfer of IGF-I to a variety of tumor cell lines.24,26–28 human islets in vitro Recently, it was suggested that apoptosis in islets was To determine if IGF-I is able to block the effects of IL-1␤ developmentally regulated and that the peak of apoptosis on islet ␤ cell function, intact infected islets were treated coincides with low IGF-II and increased iNOS levels.29 with 50 units of recombinant IL-1␤ . This amount of IL- Incubation of islets during the period of peak apoptosis 1␤ is sufficient to impair the ability of islets to respond to in the presence of IGF-I or IGF-II was able to increase a high glucose concentration (18 mm), both in uninfected the rate of ␤ cell survival.29 Although the mechanism of islets as well as in those infected with the Ad-LacZ con- apoptosis was not extensively studied, other data support trol virus (Figure 1). Islets infected with Ad-IGF-I the involvement of Fas activation. Neonatal rat islets cul- secreted higher insulin relative to uninfected and Ad- tured with IL-1␤, TNF␣ and IFN␥, underwent rapid LacZ-infected islets when exposed to glucose at 5 mm apoptosis when co-incubated with an agonistic anti-Fas final concentration. The difference was statistically sig- antibody.30 Preincubation with IGF-I, however, pre- nificant (430 ± 18.4% from Ad-IGF-I-infected islets versus vented IL-1␤-induced, Fas-triggered apoptosis. In a simi- 113 ± 80% in Ad-LacZ-infected islets where uninfected lar study, pretreatment of rat islets with recombinant islets exposed to 5 mm glucose are taken to represent IGF-I resulted in a decrease of IL-1␤-mediated NO forma- 100% as control, P Ͻ 0.05). As shown in Table 2a,1 Ad- tion by inhibition of iNOS expression and synthesis as IGF-I-infected islets were refractory to the effects of IL- well as restoration of glucose-stimulated insulin release.31 1␤ in the presence of high glucose and secreted signifi- Interestingly, IGF-I treatment of early age NOD mice cantly more insulin at 18 mm glucose than islets exposed could decrease the incidence of IDDM as well as the insu- to 5 mm. The difference was statistically significant (P Ͻ litis grade.32 Administration of recombinant IGF-I to 0.05). In Figure 1b and Table 2B, we show that the same NOD mice was also able to promote the integrity of ␤ protective effect can be achieved with lower titers of Ad- cell mass as well as to reduce the incidence of adoptive IGF-I, however, there is a lower limit at which no protec- transfer of IDDM with T cells from diabetic NOD tion is conferred (1 × 104 p.f.u. and less). mice.33,34 As NO has been shown to be an important determinant In this article, we have examined the ability of IGF-I of the suppression of IL-1␤-induced impairment of glu- gene transfer to islets to confer protection against IL-1␤. cose-stimulated insulin secretion, we evaluated the level We demonstrate that adenoviral gene transfer of the of nitrite in the culture supernatant of mock-infected, Ad- human IGF-I gene into human islets in vitro can prevent LacZ- and Ad-IGF-I-infected islets in the presence or IL-1␤-mediated ␤ cell dysfunction, NO production and absence of 50 units of IL-1␤ using the Griess reagent. Fas-triggered apoptosis induction. These results suggest Mock-infected islets as well as those infected with Ad- that gene transfer of immunoregulatory cytokines LacZ produced a basal amount of NO in the absence of together with growth factors like IGF-I, which promote any IL-1␤ (Figure 2). There was a significant increase of the integrity and survival of ␤ cells, may be one means of NO production when islets were exposed to 50 units of facilitating the survival of islet transplants as a potential IL-1␤ (213 ± 15.3% relative to control, P Ͻ 0.05). Ad-IGF- therapy for IDDM in humans.35 I-infected islets did not produce NO at significantly higher levels than mock-infected or Ad-LacZ-infected islets which were not exposed to IL-1␤ (Ad-IGF-I: 100 ± Results 5%, Ad-LacZ: 116 ± 10% relative to uninfected control islets). In the presence of IL-1␤, however, Ad-LacZ- Ad-IGF-I gene transfer to islets results in high IGF-I infected islets displayed a significant increase in NO pro- protein secretion duction relative to uninfected islets exposed to IL-1␤ (375 Adenoviral gene transfer to human and murine islets is ± 40% versus 213 ± 15.3% IL-1␤-treated uninfected islets, very efficient36–38 with greater than 70% of islet cells being P Ͻ 0.05). Finally, Ad-IGF-I-infected islets exposed to IL-

Gene Therapy Adenoviral IGF-I gene transfer to human islets N Giannoukakis et al 2017 Table 2A Glucose-stimulated insulin release assay

Islet Insulin release % ± Insulin release % ± status s.e.m. (5 mm glucose) s.e.m. (18 mM glucose)

Control 100 866 ± 200 Ad-LacZ 113 ± 80 900 ± 133** IL-1␤ 66 ± 53 80 ± 13 Ad-LacZ + IL-1␤ 93 ± 680± 26 Ad-IGF-I 430 ± 18.4 676 ± 22.5** Ad-IGF-I + IL-1␤ 316 ± 4.1 553 ± 20.5**

**P Ͻ 0.05.

Table 2B Glucose-stimulated insulin release assay

Islet status Insulin release ␮IU/ml Insulin release ± s.e.m. (5 mM glucose) ␮IU/ml ± s.e.m. (18 mM glucose)

Control 35.13 ± 14 100.92 ± 50** 1 × 105 p.f.u. Ad- 51.02 ± 2 106.31 ± 47** IGF-I 1 × 104 p.f.u. Ad- 55.2 ± 11.3 142.74 ± 57** IGF-I 1 × 103 p.f.u. Ad- 90.6 ± 18 104.6 ± 7 IGF-I + 50 U IL-1␤ 1 × 105 p.f.u. Ad- 60.5 ± 19.7 118.1 ± 14** IGF-I Figure 1 (a) Adenoviral gene transfer of IGF-I to human islets prevents 1 × 104 p.f.u. Ad- 69 ± 3 100 ± 9 ␤ the IL-1 -induced impairment of glucose-stimulated insulin release. Two IGF-I 6 hundred to 300 islets were infected with 1 × 10 p.f.u. of Ad-LacZ, Ad- × 3 ± ± ␤ 1 10 p.f.u. Ad- 54 4234 IGF-I or mock-infected and exposed to 50 units of IL-1 for a period IGF-I between 18 and 24 h. Islets were then subjected to a static glucose-stimu- lated insulin release assay as described in Materials and methods and Ͻ insulin was measured in the supernatant by ELISA. The bars indicate the **P 0.05. means of triplicate experiments with triplicate determinations each and the error bars denote the s.e.m. The data are presented as percent above control where the insulin secretion by untreated, uninfected islets exposed to 5 mm glucose is taken as 100%. (b) Effect of different Ad-IGF-I titers on insulin secretory response of human islets in the presence and absence of IL-1␤. Two hundred to 300 islets were infected with Ad-IGF-I at titers shown in the graph. IL-1␤ was added at 50 units 24 h following infection and static glucose-stimulated insulin secretion was evaluated in the super- natant by ELISA. The bars indicate the means of triplicate wells and the error bars the s.e.m. Data are shown as actual ␮IU/ml insulin.

Table 1 Human IGF-I levels (ng/ml)

Control Ad-IGF-I

28.4 ± 2.6 102.8 ± 34.2

Detection of human IGF-I by ELISA in the conditioned media of human islets. Groups of 200 islets were infected with 1 × 106 p.f.u. of Ad-IGF-I or mock-infected as described in Materials and methods. The media were collected 48 h following infection and IGF-I was measured. Figure 2 Adenoviral gene transfer of IGF-I to human islets can prevent IL-1␤-induced NO production. Groups of 200–300 islets were infected 1␤ produced NO at levels no higher than mock-infected with 1 × 106 p.f.u. of Ad-LacZ, Ad-IGF-I or mock-infected and then islets unexposed to IL-1␤ (Ad-IGF-I: 100 ± 5.8% versus exposed to 50 units of IL-1␤ for a period between 18 and 24 h. The culture control; IL-1␤-treated islets: 213% ± 15.3% versus control, media were then collected and assayed for nitrite levels using the Griess P Ͻ 0.05 Ad-IGF-I versus uninfected islets in the presence reagent. Bars indicate the means of three different experiments each perfor- ␤ med in triplicate and the error bars denote the s.e.m. Islets used were of IL-1 ). These results demonstrate that IGF-I is able to obtained from at least five different donors. Values are shown as percent block completely the production of NO in response to of control where the nitrite level in mock-infected, untreated islets is taken IL-1␤. as 100%.

Gene Therapy Adenoviral IGF-I gene transfer to human islets N Giannoukakis et al 2018 Human islets infected with Ad-IGF-I are protected from IL-1␤-stimulated, Fas-triggered apoptosis activation in vitro To examine the effect of IGF-I on Fas-mediated apoptosis, an agonistic Fas antibody was used to induce apoptosis in untreated mock-infected and infected islets in the absence or presence of IL-1␤. We assessed caspase-3 activity as a marker of apoptosis induction since recent reports demonstrate that it is specifically activated in response to the same Fas antibody.39–42 Ad-eGFP was used as a control virus that we have shown does not affect function or apoptosis of human islets in vitro, simi- lar to Ad-LacZ.20,43 IL-1␤ was added to mock-infected, Ad-eGFP and Ad-IGF-I-infected islets and the agonistic antibody was then added for 1 h. Caspase-3 activity was normalised to the number of cells in the lysate by an indirect quantitative method whereby the amount of DNA is evaluated using a fluorescent intercalating agent. As shown in Figure 3a, caspase-3 activity was signifi- cantly suppressed in islets infected with Ad-IGF-I com- pared with uninfected islets (23 ± 5% relative to control, where 100% represents the caspase-3 activity in mock- infected, untreated islets, P Ͻ 0.05). There were no sig- nificant differences in caspase-3 activity among untreated, uninfected islets and those exposed to IL-1␤ or the agonistic antibody CH-11. Pretreatment of uninfected islets with IL-1␤ followed by exposure to CH-11 resulted in a significant increase in caspase-3 activity (307 ± 10.7% in IL-1␤ + CH-11-treated islets versus 137 ± 1.3% in IL- 1␤-treated islets alone and 101 ± 3.7% in islets treated Figure 3 (a) Infection of human islets in culture with an adenoviral vector Ͻ ␤ + expressing human IGF-I can suppress IL-1␤-stimulated, Fas-triggered only with CH-11, P 0.05 for IL-1 CH-11 versus × control). The reduction in caspase-3 activity in Ad-IGF-I- activation of apoptosis. Two hundred to 300 islets were infected with 1 106 p.f.u. Ad-eGFP, Ad-IGF-I or mock-infected and then treated with 50 infected islets compared with control, was abolished in ␤ ␤ units of IL-1 for a period between 18 and 24 h. Immediately thereafter, Ad-IGF-I-infected islets exposed to IL-1 , however, the 250 ng of an agonistic anti-Fas antibody were added to each group and level was almost identical to that in control islets. The the islets were lysed 1 h later. Caspase-3 activity was detected as described most striking observation was the suppression of IL-1␤- in Materials and methods and the value was corrected for cell number stimulated, Fas-triggered caspase-3 activation in islets indirectly, by assessing the amount of DNA present in the lysate. The infected with Ad-IGF-I (Figure 3a). Compared with unin- bars indicate the means of three independent experiments performed in triplicate and the error bars denote the s.e.m. We show the means as per- fected islets and those infected with Ad-eGFP that were ␤ cent of control where the ratio of caspase-3 activity to DNA content in exposed to IL-1 and the agonistic CH-11 antibody, IGF- uninfected, untreated islets represents 100%. (b) Glucose-stimulated insu- I-expressing islets exhibited statistically significant lower lin secretion of islets following sequential exposure to 50 units IL-1␤ and levels of caspase-3 (40 ± 20% compared with 307 ± 10.7% CH-11 antibody. Two hundred to 300 islets were treated identically as in control islets or 313 ± 14% in Ad-eGFP-infected islets; indicated above and then assayed for insulin release by ELISA following P Ͻ 0.05 in both cases). We also examined the insulin glucose addition. The results are derived from triplicate determinations and are presented as actual ␮IU/ml of insulin secreted into the culture secretion of Ad-IGF-I-infected islets in response to glu- supernatant. Bars indicate the means and error bars the s.e.m. cose following the sequential addition of IL-1␤ and CH- 11 to the cultures. In Figure 3b we show that there was no significant effect on insulin response following the to block the inhibitory effects of IL-1␤ on glucose- combined cytokine/antibody treatment. stimulated insulin secretion, NO production and Fas- dependent apoptosis activation. Discussion Although several reports suggest that IGF-I inhibits insulin secretion in vivo and in certain instances in In this article, we demonstrate that adenoviral gene trans- vitro,46–49 one study suggests that IGF-I actually fer of the human IGF-I gene to human islets in vitro can potentiates glucose-stimulated insulin release from cul- suppress the IL-1␤-induced impairment of glucose- tured human islets.50 Another study suggests that IGF-I stimulated insulin secretion. It is important to note has a dual effect on glucose-stimulated insulin release that adenoviral infection of intact human islets in culture from isolated islets in culture that is IGF-I- and glucose does not change their viability or functional charac- concentration-dependent.51 Based on those observations, teristics at multiplicities of infection we and others have we calculate that the IGF-I output between the time of used20,37,38,43,44 and in vivo, murine islets infected with Ad-IGF-I infection and static glucose-stimulated insulin adenoviral vectors have survived for up to 20 weeks release assay was well below the concentration able to without any evidence of impairment.45 At low glucose inhibit insulin release significantly. In fact, the in vivo concentration (5 mm), islets infected with Ad-IGF-I pro- suppression of insulin secretion by IGF-I administration duce significantly more insulin than uninfected islets or may be secondary to IGF-I effects at the peripheral level those infected with Ad-LacZ. Ad-IGF-I infection was able on liver and adipose tissue.50

Gene Therapy Adenoviral IGF-I gene transfer to human islets N Giannoukakis et al 2019 We observed no increase in NO production in Ad-IGF- ditions of the islets following isolation. It is possible that I-infected islets exposed to IL-1␤ compared with the different culture conditions may promote ␤ cell resistance increase seen in the mock-infected islet cultures treated to the effects of IL-1␤ by inducing the down-regulation with IL-1␤. Ad-IGF-I infection alone did not change the (internalization) of the type I IL-1 receptor. This would NO levels compared with uninfected, untreated islets. In have the effect of suppressing basal NO production and contrast, Ad-LacZ infection followed by IL-1␤ treatment, increasing the threshold for IL-1␤ sensitivity. Conse- resulted in a dramatic increase in NO production that quently, the relocation of the type I IL-1 receptor to the was paralleled by an increase in IL-1␤-induced, Fas-trig- cell surface would require stimulation by cytokines like gered apoptosis activation in Ad-eGFP-infected islets, as TNF␣ or IFN␥. Both human and rodent islets have been measured by caspase-3 levels (see Figure 3a). The well-characterized in their ability to produce IGFs and increased nitrite accumulation by Ad-LacZ-infected islets their binding proteins.21,22,24,61 In particular, IGF-I acts as in the presence of IL-1␤ suggests that the adenoviral a paracrine and autocrine factor to stimulate DNA syn- infection triggers intrinsic islet antiviral defense mech- thesis and may act as a survival factor, in addition to IGF- anisms that are perhaps mediated by resident macro- II, which has been suggested to prevent apoptosis during phages and potentiated by exogenously added IL-1␤. islet maturation, at least in rats.29 The anti-apoptotic More importantly, adenoviral infection has been shown effects of IGF-I have been documented in transformed to stimulate NO production.52,53 It is possible that the cell lines, neurons, and hematopoietic progenitor cells.62–68 nitrite detected in the Ad-LacZ-infected islets derived Furthermore, recent evidence suggests that IGF-I pre- from islet-resident antigen presenting cells that were acti- treatment of islets in culture can prevent ␤ cell impair- vated in response to the viral proteins. Antigen ment as well as Fas-triggered cell death induced by a presenting cells respond to viral infection by producing combination of IL-1␤, TNF␣ and IFN␥.30 Although Ad- NO,54–57 however, their low numbers (less than 15 macro- IGF-I gene transfer to islets can protect them from the phages and dendritic cells per islet) could explain the effects of IL-1␤, we have shown that it is unable to pro- small increase we observed compared with control. vide protection from Fas-induced apoptosis activation The basal NO production by uninfected, untreated mediated by a cytokine cocktail consisting of TNF␣, IL- islets as well as the detectable caspase-3 activity in paral- 1␤ and IFN␥ (data not shown). However, given that there lel, may reflect a natural response to trauma that the islets is evidence demonstrating a central role of IL-1␤ and only were subjected to at the time of pancreas procurement potentiating roles of TNF␣ and IFN␥ on mediating islet and/or during the islet isolation procedure. Furthermore, dysfunction in vitro, it is likely that IGF-1 expression will the removal of islets from the pancreas may deprive them be beneficial in vitro and in vivo.6,7 of trophic factors which naturally suppress proapoptotic Apoptosis is a process involving the activity of casp- events, including nitric oxide formation. Ad-IGF-I infec- ases. To date, caspase-3 activation commits all cells exam- tion, however, was able to prevent the IL-1␤-induced, ined, including ␤ cells to apoptosis and is an early step Fas-triggered activation of caspase-3 activity. Basal cas- in mediating Fas signalling.17,69–72 In response to Fas lig- pase-3 activity in islets was also significantly reduced fol- ation by the CH-11 monoclonal antibody, caspase-3 acti- lowing Ad-IGF-I infection, demonstrating the anti-apop- vation is seen as early as 5 min in a fibrosarcoma cell totic property of IGF-I. Finally, Ad-IGF-I-infected islets line.39 While we have not formally ruled out the possi- appear to be sensitive to IL-1␤-stimulated, but not to Fas- bility that caspase-3 activity in our cultures partly derives triggered caspase-3 activation, although the enzyme from non-␤ cells, that caspase-3 activity is at control lev- activities in both groups are lower than those observed els in islets expressing IGF-I demonstrates the utility of in untreated, uninfected islets. Why the level of caspase- IGF-I to protect all islet cells from the apoptotic Fas 3 is higher in Ad-IGF-I-infected islets exposed to IL-1␤ trigger. than that in infected islets treated only with the agonistic Although it is unclear exactly how IGF-1 expression in CH-11 antibody is not known at present. Clearly, how- islets regulates apoptosis, IGF-1 has been implicated in ever, there are differences in the signal transduction path- blocking apoptosis through several different pathways. ways between the IL-1 and the Fas pathways. IGF-I might In particular, IGF-I binding to the type I IGF receptor acti- act on either of the two pathways (IL-1 or Fas) more vates phosphatidylinositol-3 kinase (PI3K) which has effectively than the other. been shown to be involved in the suppression of Although there is strong support for IL-1␤ alone apoptosis.73 IGF-I signaling through PI3K apparently can inducing NO production and ␤ cell dysfunction in rat prevent the processing of ICE/LAP3 protease as well as islets, this does not always appear to be the case for that of procaspase-3.74 Furthermore, IGF-I can attenuate human beta cells.5,12–14,58,59 There are instances, however, Bax induction as well as caspase-3 activation in cardi- where in human islets, IL-1␤ alone promotes significant omyocytes, thereby preventing apoptosis.75 IGF-I could NO production and ␤ cell impairment in agreement with also prevent apoptosis in PC12 pheochromocytoma cells our findings.12–14,16,20,60 While it is not readily apparent as well as interleukin-3-deprived BaF3 cells by up-reg- 76,77 why there is such variability between the data between ulating bcl-xL production. Caspase-3 activation was different investigators and experiments, factors that also inhibited in IL-3-deprived, IGF-I-treated BaF3 cells.78 could have influenced our findings that we can rule out IGF-I is able to suppress c-myc-sensitized, Fas-activated include: (1) the degree of islet contamination by non- apoptosis downstream of the Fas antigen, perhaps at the endocrine, exocrine or other pancreatic cells is minimal; level of bcl-279 and rat fibroblasts can be protected from (2) variability of islet viability and function; and apoptosis with IGF-I through the Akt kinase (protein kin- (3) isolation-associated cell damage. One important dif- ase B)/PI3K pathway.80 Akt kinase is expressed in rat ference between our procedures and those in some other pancreatic islets as well as in a variety of rat ␤ cell lines studies where IL-1␤ alone could not promote NO pro- and more importantly, IGF-I leads to a phosphorylation duction or ␤ cell impairment involves the culture con- and activation of Akt kinase in these cells.81

Gene Therapy Adenoviral IGF-I gene transfer to human islets N Giannoukakis et al 2020 In this article, we have demonstrated the feasibility of Detection of secreted transgene products and using an adenoviral vector encoding IGF-I to infect evaluation of ␤ cell function following IL-1␤ treatment human islets as a means of preventing IL-1␤-induced Secreted IGF-I protein was detected in the culture super- impairment of ␤ cell function. Moreover, we have dem- natants 2 days following infection using commercially onstrated that IGF-I production from the genetically available ELISA kits (IDL Laboratories, San Leandro, CA, modified islets can suppress NO production in the pres- USA). This also coincided with the day of IL-1␤ treatment ence of IL-1␤ as well as Fas-triggered caspase-3 acti- in the designated cultures. To assess the effects of IL-1␤ vation, an early marker of apoptosis induction. Based on on ␤-cell function of genetically modified and unmodi- these results, we suggest that IGF-I gene transfer to islets fied islets, we used glucose-stimulated insulin secretion may be a means of preserving their integrity and promo- as a functional assay. Islets were first treated with 50 U ting their survival and function before and following of recombinant human IL-1␤ (Sigma, St Louis, MO, USA) transplantation into diabetic hosts as a potential therapy for a period between 18 and 24 h immediately following for type I diabetes. a pre-incubation in fresh media between 16 and 24 h. The IL-1␤-containing medium was removed and the islets Materials and methods were washed twice with Krebs-Ringer-Hepes buffer (KRH buffer; 25 m, Hepes pH 7.4, 115 mm NaCl, 24 mm Generation of recombinant adenoviruses NaHCO3,5mm KCl, 2.5 mm CaCl2,1mm MgCl2). Incu- ° E1-deleted adenoviruses containing cassettes encoding bation was carried out at 37 C in KRH buffer for 30 min the reporter ␤ galactosidase (Ad-LacZ) and followed by an additional incubation for 30 min in the enhanced green fluorescent protein (Ad-eGFP), and the presence of 5 and 18 mm glucose (final concentration). cDNA of IGF-I (Ad-IGF-I), were generated as The buffer was subsequently removed and its insulin described82,83 by Cre–lox recombination in CRE8 cells. content was determined by a commercially available The LacZ transgene encodes ␤ galactosidase. Briefly, the ELISA kit (Dako Chemicals, Carpinteria, CA, USA) which transgene cDNAs (␤ galactosidase, eGFP or IGF-I) were specifically recognizes processed human insulin. inserted into the pAdlox shuttle plasmid containing the In order to evaluate NO production, the islet culture CMV promoter. E1-substituted recombinant adenovirus supernatants were collected between 18 and 24 h follow- ␤ was generated by cotransfection of SfiI-digested pAdlox- ing the addition of IL-1 and an aliquot was subjected to transgene and ␺5 helper virus DNA into the adenoviral the Griess reaction. packaging cell line CRE8, propagated and purified as described.82,83 Assessment of apoptosis activation in vitro Uninfected islets, as well as those infected with Ad-LacZ Isolation and culture of human islets or Ad-IGF-I were treated with 50 units IL-␤ for 24 h as Human islets were obtained from the Diabetes Research described above. Furthermore, a subset of islets Institute of the University of Miami (Dr Camillo Ricordi) (pretreated or not with IL-1␤) were challenged with the as well as from the University of Minnesota (Dr Bernhard agonistic human Fas antibody (clone CH-11; Upstate Hering) which are part of the Juvenile Diabetes Foun- Biotechnology) for 1 h at 37°C. At the end of the incu- dation International Islet Distribution Program. Pancreata bation, islets were lysed and processed for the detection were obtained from at least 20 cadaveric donors of differ- of caspase-3 (CPP32) activity using a commercially avail- ent ages and sex and subjected to digestion, isolation and able kit (ApoAlert; Clontech, Palo Alto, CA, USA). As an purification as described.84 For each experiment, islets indirect means of correcting for cell number, the CPP32 from at least five donors were used. The purity of islets activity was corrected by the number of nanograms of was usually greater than 80% (mantled islets). Viability DNA in the lysate assayed, using the PicoGreen reagent, of the cultured islets as assessed by vital dye exclusion, an intercalating DNA fluorogenic compound (Molecular insulin staining and morphology was routinely greater Probes, Eugene, OR, USA). than 85%. Statistics Gene transfer of ␤-galactosidase, eGFP and human Statistics were performed using the SPSS for Windows v. IGF-I to islets in culture using E1-E3-deleted adenoviral 8.0 package and a P value less than 0.05 by ANOVA was vectors taken to indicate statistically significant differences. Islets were washed twice in serum-free RPMI 1640 (Gibco-BRL, Rockville, MD, USA) supplemented with a Acknowledgements 1% penicillin/ streptomycin solution (Gibco-BRL) before infection. Two hundred to 300 islets were infected with We would like to thank the Juvenile Diabetes Foundation Ad-LacZ, Ad-eGFP or Ad-IGF-I at a p.f.u. (plaque for- International Islet Distribution Program; Elina Linetsky, ming units) of 1 × 106 in a minimal volume of serum-free Alessandra Ranuncoli and Jeff Ansite for the isolation RPMI 1640 for 2–4 h at 37°C. To assess the effect of viral and purification of human islets as well as Christy Bruton titer on protective effects, we also infected islets with 1 and Bruce Baldwin for technical assistance. This work × 103–1 × 106 p.f.u./200 islets. Following the infection, the was supported in part by Public Health Service grant AR- islets were washed twice in serum-free medium and then 6–2225 (PDR) and a program project grant from the Juv- once with medium containing 10% heat-inactivated fetal enile Diabetes Foundation International (MT and PDR). calf serum (Gibco-BRL). Islets were then incubated at NG is the recipient of a post-doctoral fellowship from the 37°C in medium with serum for 2 days after which all Juvenile Diabetes Foundation International and a prize the assays were carried out. All the functional assays from the Fonds pour la formation de chercheurs et a described below were performed in triplicate on at least l’aide a la recherche (Fonds FCAR) from the provincial three different occasions unless otherwise indicated. government of Quebec, Canada.

Gene Therapy Adenoviral IGF-I gene transfer to human islets N Giannoukakis et al 2021 References ment in rat, and modulation of IGF actions on rat islet DNA synthesis by IGF BPs. Adv Exp Med Biol 1992; 321: 113–120. 23 Hill DJ et al. Somatomedin-C in human fetal pancreas. Cellular 1 Kurrer MO, Pakala SV, Hanson HL, Katz JD. Beta cell apoptosis localization and release during organ culture. Diabetes 1987; 36: in -mediated autoimmune diabetes. Proc Natl Acad Sci USA 465–471. 1997; 94: 213–218. 24 Hill DJ, Petrik J, Arany E. Growth factors and the regulation of 2 Wicker LS, Todd JA, Peterson LB. Genetic control of auto- Diabetes Care 21 immune diabetes in the NOD mouse. Annu Rev Immunol 1995; fetal growth (in process citation). 1998; (Suppl. 13: 179–200. 2): B60–69. 3 Bach JF. Insulin-dependent diabetes mellitus as an autoimmune 25 Jones JI Clemmons DR. Insulin-like growth factors and their disease. Endocr Rev 1994; 15: 516–542. binding proteins: biological actions. Endocr Rev 1995; 16: 3–34. 4 Eisenbarth GS. Molecular aspects of the etiology of type I dia- 26 Russell JW, Windebank AJ, Schenone A, Feldman EL. Insulin- betes mellitus. J Diabetes Complications 1993; 7: 142–150. like growth factor-I prevents apoptosis in neurons after nerve 5 McDaniel ML et al. Cytokines and nitric oxide in islet inflam- growth factor withdrawal (in process citation). J Neurobiol 1998; mation and diabetes. Proc Soc Exp Biol Med 1996; 211: 24–32. 36: 455–467. 6 Arnush M et al. Potential role of resident islet macrophage acti- 27 Kelley KW et al. Insulin growth factor-I inhibits apoptosis in vation in the initiation of autoimmune diabetes. J Immunol 1998; hematopoietic progenitor cells. Implications in thymic aging. 160: 2684–2691. Ann NY Acad Sci 1998; 840: 518–524. 7 Arnush M et al. IL-1 produced and released endogenously 28 Minshall C et al. IL-4 and insulin-like growth factor-I inhibit the within human islets inhibits beta cell function. J Clin Invest 1998; decline in Bcl-2 and promote the survival of IL-3-deprived 102: 516–526. myeloid progenitors. J Immunol 1997; 159: 1225–1232. 8 Southern C, Schulster D, Green IC. Inhibition of insulin 29 Petrik J, Arany E, McDonald TJ, Hill DJ. Apoptosis in the pan- secretion by interleukin-1 beta and tumour necrosis factor-alpha creatic islet cells of the neonatal rat is associated with a reduced via an l-arginine-dependent nitric oxide generating mechanism. expression of insulin-like growth factor II that may act as a sur- FEBS Lett 1990; 276: 42–44. vival factor. Endocrinology 1998; 139: 2994–3004. 9 Welsh N, Eizirik DL, Bendtzen K, Sandler S. Interleukin-1 beta- 30 Harrison M et al. Growth factor protection against cytokine- induced nitric oxide production in isolated rat pancreatic islets induced apoptosis in neonatal rat islets of Langerhans: role of requires gene transcription and may lead to inhibition of the Fas. FEBS Lett 1998; 435: 207–210. Krebs cycle enzyme aconitase. Endocrinology 1991; 129: 3167– 31 Mabley JG, Belin V, John N, Green IC. Insulin-like growth factor 3173. I reverses interleukin-1beta inhibition of insulin secretion, 10 Welsh N, Bendtzen K, Welsh M. Expression of an induction of nitric oxide synthase and cytokine-mediated insulin/interleukin-1 receptor antagonist hybrid gene in insulin- apoptosis in rat islets of Langerhans. FEBS Lett 1997; 417: producing cell lines (HIT-T15 and NIT-1) confers resistance 235–238. against interleukin-1-induced nitric oxide production. J Clin 32 Kaino Y, Hirai H, Ito T, Kida K. Insulin-like growth factor I Invest 1995; 95: 1717–1722. (IGF-I) delays the onset of diabetes in non-obese diabetic (NOD) 11 Eizirik DL, Tracey DE, Bendtzen K, Sandler S. An interleukin-1 mice. Diabetes Res Clin Pract 1996; 34: 7–11. receptor antagonist protein protects insulin-producing beta cells 33 Bergerot I, Fabien N, Maguer V, Thivolet C. Insulin-like growth against suppressive effects of interleukin-1 beta. Diabetologia factor-1 (IGF-1) protects NOD mice from insulitis and diabetes. 1991; 34: 445–448. Clin Exp Immunol 1995; 102: 335–340. 12 Eizirik DL et al. Cytokines suppress human islet function irres- 34 Bergerot I, Fabien N, Thivolet C. Effects of insulin like growth pective of their effects on nitric oxide generation. J Clin Invest factor-1 and insulin on effector T cells generating autoimmune 1994; 93: 1968–1974. diabetes. Diabetes Metab 1996; 22: 235–239. 13 Rabinovitch A et al. Human pancreatic islet beta-cell destruction 35 Giannoukakis N, Rudert WA, Robbins PD, Trucco M. Targeting by cytokines is independent of nitric oxide production. J Clin autoimmune diabetes with gene therapy. Diabetes 1999; 48: Endocrinol Metab 1994; 79: 1058–1062. 2107–2121. 14 Hostens K et al. Exposure of human islets to cytokines can result 36 Csete ME et al. Adenoviral-mediated gene transfer to pancreatic in disproportionately elevated proinsulin release. J Clin Invest islets does not alter islet function. Transplant Proc 1994; 26: 1999; 104: 67–72. 756–757. 15 Corbett JA et al. Nitric oxide mediates cytokine-induced inhi- 37 Csete ME et al. Efficient gene transfer to pancreatic islets bition of insulin secretion by human islets of Langerhans. Proc mediated by adenoviral vectors. Transplantation 1995; 59: 263– Natl Acad Sci USA 1993; 90: 1731–1735. 268. 16 Stassi G et al. Nitric oxide primes pancreatic beta cells for Fas- 38 Weber M et al. Adenoviral transfection of isolated pancreatic mediated destruction in insulin-dependent diabetes mellitus. J islets: a study of programmed cell death (apoptosis) and islet Exp Med 1997; 186: 1193–1200. function. J Surg Res 1997; 69: 23–32. 17 Yamada K et al. Mouse islet cell lysis mediated by interleukin- 39 Vercammen D et al. Dual signaling of the : initiation 1-induced Fas. Diabetologia 1996; 39: 1306–1312. of both apoptotic and necrotic cell death pathways. J Exp Med 18 Hanenberg H, Kolb-Bachofen V, Kantwerk-Funke G, Kolb H. 1998; 188: 919–930. Macrophage infiltration precedes and is a prerequisite for lym- 40 Fernandes-Alnemri T, Litwack G Alnemri ES. CPP32, a novel phocytic insulitis in pancreatic islets of pre-diabetic BB rats. human apoptotic protein with homology to Caenorhabditis ele- Diabetologia 1989; 32: 126–134. gans cell death protein Ced-3 and mammalian interleukin-1 19 Sandberg JO, Eizirik DL, Sandler S. IL-1 receptor antagonist beta- converting enzyme. J Biol Chem 1994; 269: 30761–30764. inhibits recurrence of disease after syngeneic pancreatic islet 41 Casciola-Rosen L et al. Apopain/CPP32 cleaves proteins that are transplantation to spontaneously diabetic non-obese diabetic essential for cellular repair: a fundamental principle of apoptotic (NOD) mice. Clin Exp Immunol 1997; 108: 314–317. death. J Exp Med 1996; 183: 1957–1964. 20 Giannoukakis N et al. Adenoviral gene transfer of the interleu- 42 Lazebnik YA et al. Cleavage of poly(ADP-ribose) polymerase by kin-1 receptor antagonist protein to human islets prevents IL- a proteinase with properties like ICE. Nature 1994; 371: 346–347. 1beta-induced beta-cell impairment and activation of islet cell 43 Giannoukakis N et al. Infection of intact human islets by a lenti- apoptosis in vitro. Diabetes 1999; 48: 1730–1736. viral vector. Gene Therapy 1999; 6: 1545–1551. 21 Hill DJ, Hogg J. Growth factor control of pancreatic hyper- 44 Becker TC et al. Overexpression of hexokinase I in isolated islets plasia. Bailli`eres Clin Endocrinol Metab 1991; 5: 689–698. of Langerhans via recombinant adenovirus. Enhancement of 22 Hill DJ, Hogg J. Expression of insulin-like growth factors (IGFs) glucose metabolism and insulin secretion at basal but not stimu- and their binding proteins (IGF BPs) during pancreatic develop- latory glucose levels. J Biol Chem 1994; 269: 21234–21238.

Gene Therapy Adenoviral IGF-I gene transfer to human islets N Giannoukakis et al 2022 45 Muruve DA, Manfro RC, Strom TB, Libermann TA. Ex vivo 65 Bondurant MC et al. C-myc expression affects proliferation but adenovirus-mediated gene delivery leads to long-term not terminal differentiation or survival of explanted erythroid expression in pancreatic islet transplants. Transplantation 1997; progenitor cells. J Cell Physiol 1996; 168: 255–263. 64: 542–546. 66 Feldman EL, Sullivan KA, Kim B, Russell JW. Insulin-like 46 Zhao AZ et al. Attenuation of insulin secretion by insulin-like growth factors regulate neuronal differentiation and survival. growth factor 1 is mediated through activation of phosphodies- Neurobiol Dis 1997; 4: 201–214. terase 3B. Proc Natl Acad Sci USA 1997; 94: 3223–3228. 67 Granerus M, Schofield P, Bierke P, Engstrom W. Growth factors 47 Mauras N, Horber FF, Haymond MW. Low dose recombinant and apoptosis in development. The role of insulin like growth human insulin-like growth factor-I fails to affect protein anab- factor I and TGFbeta1 in regulating cell growth and cell death olism but inhibits islet cell secretion in humans. J Clin Endocrinol in a human teratocarcinoma derived cell line. Int J Dev Biol 1995; Metab 1992; 75: 1192–1197. 39: 759–764. 48 Van Schravendijk CF, Heylen L, Van den Brande JL, Pipeleers 68 Kelley KW et al. Growth hormone, growth factors and hemato- DG. Direct effect of insulin and insulin-like growth factor-I on poiesis. Horm Res 1996; 45: 38–45. the secretory activity of rat pancreatic beta cells. Diabetologia 69 de Craen MV et al. The proteolytic procaspase activation net- 1990; 33: 649–653. work: an in vitro analysis. Cell Death Differ 1999; 6: 1117–1124. 49 Katz LE et al. Suppression of insulin oversecretion by subcutane- 70 Slee EA, Adrain C, Martin SJ. Serial killers: ordering caspase ous recombinant human insulin-like growth factor I in children activation events in apoptosis. Cell Death Differ 1999; 6: 1067– with congenital hyperinsulinism due to defective beta-cell sul- 1074. fonylurea receptor. J Clin Endocrinol Metab 1999; 84: 3117–3124. 71 McIlroy D, Sakahira H, Talanian RV, Nagata S. Involvement of 50 Eizirik DL, Skottner A, Hellerstrom C. Insulin-like growth factor caspase 3-activated DNase in internucleosomal DNA cleavage I does not inhibit insulin secretion in adult human pancreatic induced by diverse apoptotic stimuli. Oncogene 1999; 18: 4401– islets in tissue culture. Eur J Endocrinol 1995; 133: 248–250. 4408. 51 Hill DJ Sedran RJ, Brenner SL, McDonald TJ. IGF-I has a dual 72 Wolf BB, Schuler M, Echeverri F, Green DR. Caspase-3 is the effect on insulin release from isolated, perifused adult rat islets primary activator of apoptotic DNA fragmentation via DNA of Langerhans. J Endocrinol 1997; 153: 15–25. fragmentation factor-45/inhibitor of caspase-activated DNase 52 Haddad IY et al. Modulation of adenovirus-mediated gene inactivation. J Biol Chem 1999; 274: 30651–30656. transfer by nitric oxide. Am J Respir Cell Mol Biol 1997; 16: 73 O’Connor R. Survival factors and apoptosis. Adv Biochem Eng 501–509. Biotechnol 1998; 62: 137–166. 53 Beckman JS, Crapo JD. The role of nitric oxide in limiting gene 74 Singleton JR, Dixit VM, Feldman EL. Type I insulin-like growth transfer: parallels to viral host defenses. Am J Respir Cell Mol factor receptor activation regulates apoptotic proteins. J Biol Biol 1997; 16: 495–496. Chem 1996; 271: 31791–31794. 54 Benencia F, Courreges MC. Nitric oxide and macrophage anti- 75 Wang L et al. Insulin-like growth factor I modulates induction viral extrinsic activity. Immunology 1999; 98: 363–370. of apoptotic signaling in H9C2 cardiac muscle cells. Endocrin- 55 Bogdan C. Of microbes, macrophages and nitric oxide. Behring ology 1998; 139: 1354–1360. Inst Mitt 1997; 99: 58–72. 76 Parrizas M, Saltiel AR, LeRoith D. Insulin-like growth factor 1 56 Kreil TR, Eibl MM. Nitric oxide and viral infection: NO antiviral inhibits apoptosis using the phosphatidylinositol 3Ј-kinase and activity against a flavivirus in vitro, and evidence for contri- mitogen-activated protein kinase pathways. J Biol Chem 1997; bution to pathogenesis in experimental infection in vivo. Virology 272: 154–161. 1996; 219: 304–306. 77 Parrizas M, LeRoith D. Insulin-like growth factor-1 inhibition of 57 Pertile TL, Karaca K, Sharma JM, Walser MM. An antiviral effect apoptosis is associated with increased expression of the bcl-xL of nitric oxide: inhibition of reovirus replication. Avian Dis 1996; gene product. Endocrinology 1997; 138: 1355–1358. 40: 342–348. 78 Suzuki J, Kaziro Y, Koide H. Synergistic action of R-Ras and 58 Corbett JA et al. Nitric oxide mediates IL-1 beta-induced islet IGF-1 on Bcl-xL expression and caspase-3 inhibition in BaF3 dysfunction and destruction: prevention by dexamethasone. cells: R-Ras and IGF-1 control distinct anti-apoptotic kinase Autoimmunity 1993; 15: 145–153. pathways. FEBS Lett 1998; 437: 112–116. 59 Corbett JA et al. Interleukin 1 beta induces the formation of nitric 79 Hueber AO et al. Requirement for the CD95 receptor-ligand oxide by beta-cells purified from rodent islets of Langerhans. pathway in c-Myc-induced apoptosis (see comments). Science Evidence for the beta-cell as a source and site of action of nitric 1997; 278: 1305–1309. oxide. J Clin Invest 1992; 90: 2384–2391. 80 Kulik G, Weber MJ. Akt-dependent and -independent survival 60 Eizirik DL, Welsh N, Hellerstrom C. Predominance of stimu- signaling pathways utilized by insulin-like growth factor I. Mol latory effects of interleukin-1 beta on isolated human pancreatic Cell Biol 1998; 18: 6711–6718. islets. J Clin Endocrinol Metab 1993; 76: 399–403. 81 Holst LS et al. Protein kinase B is expressed in pancreatic beta 61 Hogg J, Hill DJ, Han VK. The ontogeny of insulin-like growth cells and activated upon stimulation with insulin-like growth factor (IGF) and IGF-binding protein in the rat factor I. Biochem Biophys Res Commun 1998; 250: 181–186. pancreas. J Mol Endocrinol 1994; 13: 49–58. 82 Hardy S et al. Construction of adenovirus vectors through Cre- 62 Baserga R. The insulin-like growth factor I receptor: a key to lox recombination. J Virol 1997; 71: 1842–1849. tumor growth? Cancer Res 1995; 55: 249–252. 83 Mittereder N, March KL, Trapnell BC. Evaluation of the concen- 63 Baserga R et al. The IGF-I receptor in cell growth, transformation tration and bioactivity of adenovirus vectors for gene therapy. and apoptosis. Biochim Biophys Acta 1997; 1332: F105–126. J Virol 1996; 70: 7498–7509. 64 Baserga R, Resnicoff M, D’Ambrosio C, Valentinis B. The role 84 Linetsky E et al. Improved human islet isolation using a new of the IGF-I receptor in apoptosis. Vitam Horm 1997; 53: 65–98. enzyme blend, liberase. Diabetes 1997; 46: 1120–1123.

Gene Therapy