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Endocrinology, doi:10.1210/en.2004-1667
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Endocrinology Vol. 146, No. 5 2406-2414
Copyright © 2005 by The Endocrine Society

Adenoviral-Mediated Transduction of Human Pancreatic Islets: Importance of Adenoviral Genome for Cell Viability and Association with a Deficient Antiviral Response

Andreea R. Barbu, Göran Akusjärvi and Nils Welsh

Departments of Medical Cell Biology (A.R.B., N.W.) and Medical Biochemistry and Microbiology (G.A.), Uppsala University, S-751 23 Uppsala, Sweden

Address all correspondence and requests for reprints to: Dr. Andreea Barbu, Department of Medical Cell Biology, Biomedical Center, P.O. Box 571, S-751 23 Uppsala, Sweden. E-mail: andreea.barbu{at}medcellbiol.uu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
As adenoviral vectors are extensively used for genetic manipulation of insulin-producing cells in vitro, there is an increasing need to evaluate their effects on the function, morphology, and viability of transduced pancreatic islets. In the present study we observed that specific adenoviral genotypes, carrying E4 and E1/E3 deletions, correlate with differential induction of necrosis in pancreatic islet cells. In particular, the adenovirus death protein encoded from the E3 region of the adenoviral genome was able to modulate the changes induced in the morphology and viability of the transduced cells. We also propose a putative role for the transcriptional regulator pIX. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell toxicity, our results showed that they were unable to build up an efficient antiviral response after transduction and that their survival was dependent on the exogenous addition of {alpha}-interferon. An intact and fully functional ß-cell is crucial for the successful application of gene therapy approaches in type 1 diabetes, and therefore, the implications of our findings need to be considered when designing vectors for gene transfer into pancreatic ß-cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE 1 DIABETES IS an autoimmune disease that eventually causes complete destruction of insulin-secreting ß-cells in the pancreas. Recently, pancreatic islet transplantation has been validated as a promising alternative to correct for the insulin deficiency in type 1 diabetes (1). However, the requirement for islets derived from two to four donors to treat a single patient is a barrier to the use of this technique on a larger scale. As a result, much effort is going into developing new therapeutic strategies to increase durable functional islet mass. Gene therapy offers a potential means to protect against ß-cell death by induction of immunoregulatory, cytoprotective, or antiapoptotic genes (2, 3) as well as to stimulate ß-cell differentiation and regeneration by induction of different transcriptional regulators and growth factors (4, 5, 6).

The ability to engineer pancreatic ß-cells is a prerequisite for successful application of gene therapy approaches in type 1 diabetes. However, pancreatic islets are terminally differentiated cell clusters that are difficult to reach in vivo and troublesome to maintain in tissue culture. Therefore, the use of nonviral transfection strategies for genetic modification of insulin-producing cells, including lipofection, electroporation, and biolistic particles, mediate only low transfection efficiencies (7, 8). In contrast, due to their capacity to mediate highly efficient gene transfer in nondividing cells, viral vectors have emerged as the first choice for engineering ß-cells. Together with lentiviruses (9) and adeno-associated viruses (10), adenoviruses are the most commonly used viral vectors in current ß-cell research (11, 12). However, despite their advantages, such as high titer production and the diminished risk for insertional mutagenesis due to the epichromosomal location within the target cell, adenoviral transduction and gene transfer may interfere with ß-cell function and/or induce cell death. Adenoviruses contain genes that are able to initiate and modulate cell death by inducing or suppressing specific processes within the target cell, and these effects are likely to be dependent on both the genotype of the viral vector used and the cell system studied (13). Adenoviral genes are transcribed in a complex temporal manner. Early genes are transcribed from five different promoters, in the order E1A, E4, E3, E1B, and E2. These genes are mainly concerned with the regulation of viral gene expression and DNA replication and the modulation of cell death. The E3–11.6K protein, also known as the adenovirus death protein (ADP), is encoded by the E3 transcription unit of the adenoviral genome, but its expression is turned on preferentially by a strong adenovirus major late promoter. In contrast to the other antiapoptotic genes located in the same region, ADP has been shown to facilitate viral exit from the nucleus of the infected cells by inducing cell death (14). The early production of interferons (IFNs) is believed to be an important host response to many viral infections, including adenoviruses (15), and, in pancreatic islets, IFN production has been associated with hyperexpression of major histocompatibility complex (MHC) class IA antigens (16, 17).

Previously, we reported that E1/E3-deleted adenoviral vectors induce ß-cell cytotoxicity (12). In the present study we investigate the effects of various adenoviral genome deletions on rat and human islet cell viability to determine whether a specific adenoviral genotype correlates with ß-cell death. We also try to determine whether islet cells are able to establish an antiviral state in response to adenoviral transduction and the role of type I IFNs during this process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
RPMI 1640, fetal calf serum (FCS), L-glutamine, penicillin/streptomycin, trypsin-EDTA, bisbenzimide (Hoechst-33342), and propidium iodide were purchased from Sigma-Aldrich Corp. (St. Louis, MO). All other reagents used, unless otherwise specified, were obtained from E. Merck (Darmstadt, Germany).

Cell culture
Rat pancreatic islets of Langerhans were isolated from 3-month-old Sprague Dawley rats (local Uppsala colony) by a collagenase digestion procedure and cultured in groups of 150/50-mm well in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, benzylpenicillin (100 U/ml), and streptomycin (0.1 mg/ml), containing 11.1 mM glucose (18). Human pancreatic islets, provided by Prof. Olle Korsgren (Uppsala University, Uppsala, Sweden), were cultured in the medium described above, containing 5.6 mM glucose, at 37 C in humidified air with 5% CO2. Culture medium was replaced every 48 h. The use of rat and human pancreatic islets was in accordance with international guidelines (NIH publication 85-23, revised 1985) and was approved by the local ethics committees for Uppsala University.

Adenoviral vectors and viruses
The following human adenovirus (Ad) serotype 5 mutants were used in this study: 1) AdCMVProg, from a progesterone-inducible system, in which the cytomegalovirus (CMV) promoter drives the cDNA for an inactive transactivator protein (genotype E1/E3-deleted {Delta}455–3328 and {Delta}30005–30750) (19); 2) Adeno-X-Tet-Off (BD Clontech Laboratories, Inc., Palo Alto, CA), encoding a tetracycline-controlled transactivator protein (genotype E1/E3-deleted, {Delta}342–3528 and {Delta}27865–30995, with bigger deletions both in the E1 region, encompassing a portion of the pIX promotor, and in the E3 region of the adenoviral genome, encompassing ADP) (20); and 3) dl366, E4-deleted, expressing none of the products of the early region 4 (21). The Adeno-X-Tet-Off viral vector is similar to the AdEasy-1 vector, but not identical, because AdEasy-1 is deleted in nucleotides 480-3533 and 28130–30820 (22). All virus stocks were purified by cesium chloride density gradient centrifugation (L-80 ultracentrifuge, Beckman Coulter, Fullerton, CA) and plaque-titrated by serial dilution and agar overlay on their respective transcomplementing cell lines. Typical titers were 109 plaque-forming units (PFU)/ml or higher, representing 1–5% of the total viral particles as determined by readings of the ODs.

The E3-ADP variant adenoviruses used in this study, rec700 and pm734.1, were provided by Prof. William S. M. Wold (St. Louis University School of Medicine, St. Louis, MO). rec700 is an Ad5-Ad2-Ad5 recombinant (23) and is the parental virus for pm734.1 (with ADP residues 1–48 deleted) (14).

Adenoviral transduction, viral transduction, and assessment of transfection efficiency
Rat and human islets were dispersed into individual cells by treatment with trypsin (5 mg/ml) for 5 min, in Ca2+- and Mg2+-free Hanks’ solution and were cultured for an additional 1 h at 37 C. Single cells from dispersed islets were transduced with adenoviral vectors or wild-type viruses in a minimum volume of 0.1 ml RPMI 1640 supplemented with 2% FCS, containing various adenoviral concentrations (PFU per cell). After 1-h incubation at 37 C, islet cells were washed with RPMI 1640 medium and plated in 96- or 24-well plates, coated with 0.1% gelatin. The plates were incubated at 37 C, and medium was changed every second day for up to 1 wk post transduction. In some cases, 24 h before transduction and during the experiments, islet cells were treated with 1000 U/ml rat or human IFN-{alpha} (PBL Biomedical Laboratories, Piscataway, NJ).

To assess the efficiency of the adenoviral-induced ß-cell transduction, we coincubated intact islets and dispersed rat islet cells with 100 PFU/cell of an adenoviral vector expressing the ß-galactosidase gene under control of the CMV promoter. The next day islets were dispersed, cells were fixed, and enzymatic activity and transduction efficiency were determined using light microscopy studies as previously described (24). To evaluate the capacity of wild-type adenoviruses to infect dispersed islet cells, we performed fluorescence microscopy studies after staining for the E2–72kD adenovirus DNA binding protein (25).

Sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) assay
Adenoviral-induced cell death was assessed using the colorimetric assay (XTT-based) for cell viability (12). Adenoviral-transduced and nontransduced rat and human islet cells were plated in 96-well plates in 100 µl complete RPMI 1640 culture medium. Three days after transduction when using E3-ADP variant adenoviruses or 7 d after transduction when using recombinant adenoviral vectors, 50 µl 1 mg/ml XTT (Roche, Mannheim, Germany) was added to each well. After 5 h of incubation at 37 C, the ODs were read in a Microplate autoreader (Bio-Tek Instruments, Inc., Watford, UK) at 490 nm.

Fluorescence microscopy
To investigate the effect of adenoviruses on islet cell viability, we performed fluorescence microscopy studies after 3–7 d (rat islet cells) and 5–7 d (human islet cells) of culture post transduction. Rat and human islet cells were incubated in RPMI 1640 complete medium containing 5 µg/ml bisbenzimide and 10 µg/ml propidium iodide for 10 min at 37 C. The cells were then washed, detached by mild trypsination, and examined by fluorescence microscopy.

RNA extraction and cDNA synthesis
Twenty to 24 h after transduction of rat and human islets with adenoviruses, total cellular RNA was isolated by a single-step method, using a commercial system (Ultraspec RNA isolation system, Biotecx Laboratories, Inc., Houston, TX), followed by digestion with ribonuclease-free deoxyribonuclease (Qiagen, Chatsford, CA). The total RNA for each sample was reverse transcribed with Moloney murine leukemia virus reverse transcriptase using an oligo(deoxythymidine) primer in the presence of 5 mM MgCl2, 1 mM of each deoxy-NTP, and 1 U/µl recombinant RNasin ribonuclease inhibitor. The RT reaction was performed at 42 C for 40 min, followed by inactivation of the enzyme at 95 C for 5 min.

Real-time PCR analysis
On the basis of the respective rat and human cDNAs, primers were designed to amplify specific regions of Ifn-{alpha}, class IA MHC antigen-processing and antigen presentation genes [ß2-microglobulin (ß2m), low molecular mass polypeptide 2 (Lmp-2), and transporter associated with antigen processing 1 (Tap-1) for human islet cells; ß2m, Lmp-7, and Tap-1 for rat islet cells], and the ß-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs. PCR amplifications were carried out in a real time LightCycler (Roche) using the temperature-time sequence: denaturation for 15 sec at 95 C, annealing for 13 sec at 55 C, and extension for 6 sec at 72 C, and the primer sequences described in Table 1Go.


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  		TABLE 1. PCR primers used to assess gene expression in human and rat islet cells

 
To obtain relative mRNA expression values, PCRs were performed with ß-actin for rat samples and GAPDH or ß-actin for human samples for each of the experimental groups. The amount of PCR product relative to these housekeeping genes was calculated as previously described (26). For each experiment the data are presented as normalized values against the relative mRNA levels of the control samples. All PCR products were verified by agarose gel electrophoresis and SYBR Gold (Molecular Probes, Eugene, OR) staining.

Statistical analysis
Data are summarized as the mean ± SEM. The significances of differences between the groups were determined by one- or two-way ANOVA for repeated measurements and the Bonferroni or Dunnett’s test. Differences were considered significant at P < 0.05. Statistical analysis was performed using SigmaStat (SPSS Science Software, Erkrath, Germany).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To evaluate the efficacy of adenoviral vectors to transduce islet cells, we stained for ß-galactosidase activity and counted cells in a light microscope. In a typical experiment we observed more than 90% ß-galactosidase-positive cells in the dispersed islet cell group and only 20–30% ß-galactosidase-positive cells in the intact islet cell group (results not shown). In all subsequent transduction experiments, only dispersed islet cells were used to ensure high transduction efficiency. The adenoviral vector AdCMVProg belongs to an inducible system that we have previously described (12). We showed that increasing concentrations of the virus resulted in a corresponding increase in protein expression levels, with more than 70% ß-cells transduced at 100 PFU/cell. We obtained similar transduction efficiencies (>80%) with another adenoviral vector expressing green fluorescent protein that has the same backbone as the AdTetOFF vector used in the present study.

Effects of different adenoviral vector genomes on rat and human islet cell viability
To determine whether the adenoviral-induced cytotoxicity correlates to a particular adenoviral genotype, we transduced dispersed rat and human islet cells with different replication deficient adenoviral vectors carrying deletions in one or more of the E1, E3, or E4 regions of the adenoviral genome.

XTT-based cell viability assays performed 7 d after adenoviral vector/islet cell incubation, revealed that transduction of rat islet cells with dl366 virus (carrying intact E1 and E3 regions, but none of the genes of the E4 region) significantly diminished islet cell viability (to 61.8 ± 0.038%) at 1 PFU/cell and that the adenoviral-induced cell death occurred in a dose-dependent manner (with no viable cells left at 100 PFU/cell; Fig. 1AGo,

{cjs2113}

). To investigate whether this cytotoxicity was due to nonspecific effects of proteins belonging to the adenoviral capsids we coincubated islet cells with heat-inactivated dl366 viruses. One week post transduction, no effect on the cell viability was observed (results not shown), suggesting that adenovirus-induced cell death is secondary to the internalization of functional vector particles into the cells. Adenovirus-mediated toxicity was also seen after transduction of islet cells with the E1/E3-attenuated viral vector, AdCMVProg (Fig. 1BGo,

{cjs2113}

), although to a lesser extent. Thus, 1–10 PFU/cell of the {Delta}E4 dl366 virus induced 50% cytotoxicity in rat islet cells, whereas in AdCMVProg-transduced cells the same effect was obtained only at concentrations as high as 10–100 PFU/cell. Interestingly, transduction of rat islet cells with AdTetOFF vector, another E1/E3-deleted viral vector that carries additional deletions in the E1 region (encompassing a portion of the pIX promotor) and in the E3 region (encompassing ADP), resulted in a lesser induction of islet cell death and only at a high concentration of the vector (Fig. 1CGo,

{cjs2113}

).



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  		FIG. 1. Decreased islet cell viability in response to dl366 (A), AdCMVProg (B), and AdTetOFF (C) adenoviral transduction. Dispersed human ({blacksquare}) and rat ({cjs2113}) islet cells were transduced with various concentrations of the adenoviral vectors, and cellular viability was assessed 7 d post transduction using an XTT-based assay. Individual colorimetric readings are normalized to the value of nontransduced islet cells. Results are the mean ± SEM of eight (rat islet cells) or 10 (human islet cells) separate experiments. * and #, P < 0.05; ** and ##, P < 0.01; *** and ###, P < 0.001 [vs. nontransduced human (*) or rat (#) cells, by one-way ANOVA, Bonferroni test]. D, ADP and pIX mRNA expression in rat and human islet cells after adenoviral exposure was analyzed by RT-PCR as described in Materials and Methods. The reaction was stopped during the exponential phase (23 cycles for ADP and pIX and 20 cycles for ß-actin), and the products were analyzed by ethidium bromide staining and agarose gel electrophoresis. E, Schematic of the adenoviral genome and the specific deletions of the adenoviral vectors used in this study: E1–E4, early transcribed adenoviral genes; MLP, major late promotor; VA-RNA, adenoviral RNA.

 
In human islet cells, transduction with dl366 vector induced a similar pattern of cytotoxicity, but at considerably higher viral concentrations (100 PFU/cell reduced cellular viability to 55.3 ± 10.9% in human islet cells vs. 0% in rat islet cells; Fig 1AGo, {blacksquare}). At high concentrations (100 PFU/cell) both E1/E3-attenuated adenoviruses reduced islet cell viability to 89.4 ± 3.9% when cells were transduced with AdTetOFF (Fig. 1CGo, {blacksquare}) and 54 ± 13.3% when cells were transduced with AdCMVProg (Fig. 1BGo, {blacksquare}). These results are in line with our previous observation that an adenoviral vector induces cytotoxicity in pancreatic islet cells (12) and suggest that this effect is modulated by the adenoviral genome and the particular characteristics of the target cells.

Effects of ADP on wild-type adenovirus-induced islet cell toxicity
RT-PCR analysis performed on mRNA isolated from transduced islet cells demonstrated a differential expression of the adenoviral genes IX and ADP. As anticipated, we could not detect ADP and IX cDNA in control or AdTetOFF cells (Fig. 1DGo). In addition, expression of the ADP and IX genes was clearly stronger in cells transduced with dl366 than in those transduced with AdCMVProg (Fig. 1DGo). Taken together, these data (Fig. 1Go) raise the possibility that the cytotoxic effect of adenoviruses in islet cells may be in part correlated to ADP expression. Indeed, after transduction of rat islet cells with an ADP– virus (pm734.1), we observed a milder effect on islet cell viability, as assessed by the XTT-based metabolic assay (Fig. 2AGo). Although not statistically significant, there was also a trend for a milder effect of the ADP– virus in human islet cells (Fig. 2BGo).



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  		FIG. 2. Estimation of cellular cytotoxicity induced in dispersed islet cells by adenoviruses carrying differential expression of ADP. Rat islet cells (A) and human islet cells (B) were transduced with various concentrations of rec700 or pm734.1 virus. Cell viability was assessed on d 3 (rat islet cells) or d 5 post transduction (human islet cells) using the XTT-based assay. Individual colorimetric readings are normalized to the value of nontransduced islet cells. Results are the mean ± SEM of six separate experiments. Statistical significance was determined by two-way ANOVA and Dunnett’s test.

 
Fluorescence microscopic analysis after double staining with bisbenzimide/propidium iodide indicated that viral exposure promoted necrosis in both human (Fig. 3AGo) and rat (Fig. 3BGo) islet cells. The nuclei of the transduced cells were extremely distended, and this specific morphology preceded the rupture of the plasma membrane. Note that no increase in typical apoptotic features was observed in the transduced cells.



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  		FIG. 3. Micrographs showing bisbenzimide/propidium iodide fluorescence of human (A) and rat (B) islet cells. Cells were transduced with 30 PFU/cell (rat cells) or 50 PFU/cell (human cells) of rec700 (wild type) and pm734.1 (ADP–), and cell morphology was evaluated by fluorescence microscopy at 5 d for human islet cells (a) and 3 d post transduction for rat islet cells (B). Nuclear blue bisbenzimide staining reflects viable cells, whereas red propidium/iodide staining reflects necrotic cells. Magnification, x40.

 
Effects of IFN-{alpha} on the morphology and viability of adenovirus-transduced rat and human islet cells
To determine whether the capacity to respond to IFNs is important for islet cell survival during mild adenoviral transduction, we pretreated both rat and human islet cells with 1000 U/ml IFN-{alpha} for 24 h. Using fluorescence microscopy, we examined rat islet cells at 3 d post transduction and human islet cells at 5 d post transduction with rec700 and pm731.4 viruses and at 7 d post transduction with the recombinant adenoviral vectors. IFN-{alpha} treatment significantly decreased adenovirus-induced necrosis in both human and rat islet cells to control levels (Fig. 4Go, A, C, and E). Moreover, the typical distended nuclear morphology induced by adenovirus transduction was significantly reduced in both human and rat islet cells after IFN-{alpha} treatment (Fig. 4Go, B and D). To exclude the possibility that IFN-{alpha} treatment affected adenovirus internalization into islet cells, we analyzed the expression of the 72-kDa DNA binding protein encoded by the E2 region. No effect of IFN-{alpha} was seen, as assessed by fluorescence microscopic studies (data not shown). These results suggest that type I IFNs are able to induce and/or enhance the transition of pancreatic islet cells to an antiviral state and thereby promote cell survival.



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  		FIG. 4. Effects of IFN-{alpha} treatment (1000 U/ml) on the viability (A, C, and E) and morphology (B and D) of adenovirus-transduced human (A, B, and E) and rat (C and D) islet cells. Dispersed human and rat islet cells were transduced with various adenoviruses [30 PFU/cell for rat islet cells and 50 PFU/cell for human islet cells when transduced with rec700 and pm734.1 (A–D) and 500 PFU/cell for human islet cells when transduced with dl366, AdCMVProg, and AdTetOFF (E)]. Half the groups were treated for 24 h before and during the transduction with 1000 U/ml IFN-{alpha} ({square}). Cell viability was assessed on d 5 post transduction (rec700 and pm734.1) or d 7 post transduction (dl366, AdCMVProg, and AdTetOFF) for human islet cells or on d 3 post transduction (rec700 and pm734.1) for rat islet cells, using fluorescence microscopy. At least 200 cells were counted for each group. Results are the mean ± SEM of four (human islet cells) or three (rat islet cells) different experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. nontransduced cells). #, P < 0.05; ##, P < 0.01; ###, P < 0.001 (vs. corresponding groups not treated with IFN-{alpha}). Statistical significance was determined using a two-way ANOVA and the Bonferroni test.

 
Evaluation of the antiviral response of rat and human islet cells after adenovirus transduction
The capacity of islet cells to induce an antiviral response as well as the ability of IFN-{alpha} to modulate this response were investigated by RT-PCR analysis. In particular, we compared the effects of adenovirus exposure and IFN-{alpha}-treatment on the mRNA expression of Ifn-{alpha} and on the expression of class IA MHC antigen processing and antigen presentation genes as follows: ß2m, Lmp-2, and Tap-1 for human islet cells, and ß2m, Lmp-7, and Tap-1 for rat islet cells.

RT-PCR analysis demonstrated that Ifn-{alpha} mRNA was expressed under basal conditions by both human and rat islet cells (Fig. 5AGo). At 24 h post transduction, rec700 significantly decreased the expression of Ifn-{alpha} mRNA in rat islet cells (Fig. 5AGo,

{cjs2113}

), whereas in human islet cells no effect on Ifn-{alpha} mRNA expression was observed (Fig. 5AGo, {blacksquare}). Ifn-{alpha} mRNA expression in human islet cells was not affected by the three deleted adenoviral vectors (Fig. 5EGo).



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  		FIG. 5. Effects of IFN-{alpha} treatment and adenoviral exposure on Ifn-{alpha} (A and E), Lmp-2, Lmp-7 (B and F), Tap-1 (C and G), and ß2m (D and H) mRNA expression in human ({blacksquare}) and rat ({cjs2113}) islet cells. After adenoviral transduction [30 PFU/cell for rat islet cells and 50 PFU/cell for human islet cells when transduced with rec700 and pm734.1 (A–D) and 500 PFU/cell for human islet cells when transduced with dl366, AdCMVProg, and AdTetOFF (E–H)] or IFN-{alpha} (1000 U/ml) treatment, cells were cultured for 24 h in normal RPMI 1640 medium, and total RNA was extracted and analyzed by RT-PCR as described in Materials and Methods. GAPDH or ß-actin (for human islet cells) and ß-actin (for rat islet cells) mRNA expression was assayed as a constitutive control. Results are normalized against nontreated cells and expressed as the mean ± SEM of three different experiments. * and #, P < 0.05; ** and ##, P < 0.01; *** and ###, P < 0.001 [vs. nontreated human (*) or rat (#) cells, using one-way ANOVA and Dunnett’s test].

 
We also observed basal expression of both low molecular mass polypeptide 7 of the proteasome complex (Lmp-7) for rat islets and Lmp-2 for human islets. However, although in rat islet cells, Lmp-7-mRNA was significantly suppressed by pm734.1 and rec700 (Fig. 5BGo,

{cjs2113}

), none of the adenoviral constructs affected Lmp-2 expression in human islets (Fig. 5BGo, {blacksquare}, and Fig. 5FGo).

A similar pattern was observed when we evaluated the expression of transporter associated with antigen processing 1 (Tap-1) mRNA. Adenoviruses (rec700 and pm734.1) induced a clear inhibition in rat islets (Fig. 5CGo,

{cjs2113}

), whereas in human islets, there was only a nonsignificant trend to lower Tap-1 mRNA in response to AdCMVProg and AdTetOFF (Fig. 5CGo, {blacksquare}, and Fig. 5GGo).

In both rat and human islet cells, no alteration of ß2m mRNA expression was observed 24 h after adenovirus transduction (Fig. 5DGo) or transduction with the deleted adenoviral vectors (Fig. 5HGo). Moreover, in human islet cells, IFN-{alpha} pretreatment significantly up-regulated mRNA expression of all genes belonging to MHC class IA (ß2m, Lmp-2, and Tap-1) and Ifn-{alpha} mRNA (Fig. 5Go, E–H). In rat islet cells, IFN-{alpha} treatment resulted only in up-regulation of Lmp-7-mRNA expression with no other significant alteration of the MHC class IA mRNAs evaluated (Fig. 5Go,

{cjs2113}

).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In a previous study we showed that E1/E3-attenuated adenoviral vectors mediate highly efficient transgene expression. However, the adenovirus-mediated transduction was followed by a significant induction of cellular death, mainly necrosis (12). It is likely that ß-cells located in intact islets are less prone to die in response to adenoviral transduction, because the general viability of free islet cells is diminished compared with that of nondispersed islet cells. Nevertheless, although higher adenoviral titers might be required in the intact islet, the same cell death-inducing events are probably occurring in both cases. Therefore, it is likely that the present results are also relevant to whole islet transduction.

We observed that all adenoviral vectors and adenoviruses, albeit with different efficiencies, induce the death of human and rat islet cells. This is at variance with previous studies in which no adverse effect of adenoviral vectors was observed (3). However, in these studies, intact islets were transduced, which, in our experience, results in transfection of only the cells located at the periphery (Barbu, A., J. Olerud, L. Jansson, M. Welsh, G. Akusjärvi, and N. Welsh, manuscript in preparation). In addition, in some of these reports, ß-cell function was assessed after only 2–3 d and not after 7 d as in the present investigation. Interestingly, not only are ß-cells negatively affected by adenoviral transduction, but adrenocortical cells have also recently been shown to lose function after transduction with adenoviral vectors (27).

The majority of adenoviral vectors used for gene transfer studies have deletions in their early genes to render the vector replication incompetent. Proteins encoded in these regions are known to induce (E1A and E4-orf 6) or inhibit (E1B, E3, and E4-orf 4) cellular death in transduced host cells (28, 29, 30, 31, 32, 33, 34, 35). Transduction of dispersed rat and human islet cells with dl366 virus, which has intact E1 and E3 regions, but no E4 genes, diminished islet cell viability below the already low concentrations of the viral vector (1 PFU/cell), whereas transduction of islet cells with adenoviruses attenuated by deletions in the E1 and E3 regions (AdCMVProg) promoted cell death to a lesser extent. Moreover, transduction of islet cells with the E1/E3-deleted adenoviral vector, AdTetOFF, which has more comprehensive deletion of E1 and E3 regions than AdCMVProg, resulted in the weakest induction of rat and human islet cell death.

The pronounced cytotoxicity in response to the E4-deleted adenoviral vector is not surprising considering that vectors with intact E1 regions have higher transcription of adenoviral genes than vectors without E1 (36). Thus, there is probably a larger de novo synthesis of viral proteins from the E4-deleted than from the E1/E3-deleted vector. Indeed, we observed high ADP and pIX mRNA levels in dl366-transduced cells compared with AdCMVProg-transduced cells.

Our observations also point out the possibility that the E3 gene product ADP and the E1 located adenoviral protein IX, which are usually expressed at late stages of transduction, might participate in adenovirus-induced islet cell death. The expression of genes located in the E3 and E4 regions of the adenoviral genome is usually low in the absence of E1A genes (37). However, other studies have shown residual expression of E4 genes in cells transduced with E1A-deleted vectors (35). In line with these results, we presently demonstrate residual expression of two genes of the adenoviral genome ADP and the adenoviral gene IX in cells transduced with AdCMVProg. This basal residual expression seems to be sufficient for modulating cell death in islet cells.

To explore the effect of adenovirus death protein on islet cell viability, we transduced rat and human islet cells with adenoviruses with differential expression of ADP. As expected, both adenoviruses (rec700 and pm734.1) were potent and rapid inducers of islet cell death (2–3 d for rat cells and 4–5 d for human cells), and the adenovirus expressing ADP (rec700) induced cell death more potently than the vector lacking ADP (pm734.1). Thus, the expression of ADP negatively affects the viability of adenovirus-transduced islet cells.

As in our previous study (12), adenovirus transduction of islet cells resulted mostly in islet cell necrosis, which was paralleled by a distended morphology of the nuclei. This feature of the adenoviral transduction was previously reported and might be related to the capacity of the E1B-19K protein to alter the organization of the intermediary filaments and nuclear lamina in transduced cells (38). The fact that recombinant adenoviral vectors with intact E1 regions (dl366), but not recombinant adenoviral vectors with E1 deletions (AdCMVProg), induce a morphology (results not shown) similar to that observed with the rec700 and pm734.1 adenoviruses also supports this hypothesis.

As discussed above, our results support a modulating role of ADP in adenovirus-mediated islet cell death. In addition, it is possible that the product of the adenoviral gene IX, pIX, could enhance this cytotoxic effect. The adenovirus protein pIX is expressed after the early adenoviral genes and has been shown to be incorporated into the viral capsid, where it is in part responsible for virion stability. More recently, pIX has been identified as a transcriptional activator (39) with the capacity to enhance gene expression from E1A, E4, and the major late adenoviral promoters. With this function, pIX might very well be able to modulate death pathways in targeted cells. However, in the few studies addressing this issue, its capacity to enhance gene transcription varies considerably (from 1.4- to 70-fold increase in gene expression) (39, 40) and may be dependent on the cellular system studied. Therefore, the specific effects of IX+/– recombinant adenoviral vectors on the viability of transduced islet cells should be investigated.

From another perspective, the study of islet cell-adenovirus/recombinant adenovirus interactions can reveal important features concerning the pathogenesis of type 1 diabetes, because viral transduction, although mostly of enteroviral origin, has been implicated as an important environmental factor that may trigger the subsequent autoimmune reaction against ß-cells in genetically susceptible individuals (41, 42). In this context, two scenarios have been proposed: 1) the ß-cell defense against viral transduction is in some cases low, which results in ß-cell necrosis and also autoimmunity (43); and 2) the ß-cell IFN response is strong, which leads to the survival of the ß-cell after viral transduction, but also to autoimmunity (44). The second hypothesis is supported by the finding that ß-cells may constitutively express high levels of IFN-{alpha} (44).

Type I IFNs (IFN-{alpha}/ß) are produced very rapidly (within hours) in direct response to viral transductions, and they are believed to strongly induce the antiviral state in target cells (45). In our system, however, the IFN response in pancreatic islets was not triggered by adenoviral transduction or adenoviral vectors. RT-PCR studies of mRNA expression of the Ifn-{alpha} and MHC class IA genes revealed no major effect of virus exposure on human islets, whereas in rat cells a significant down-regulation of MHC class IA gene expression was observed, consistent with down-regulation of the Ifn-{alpha} gene. These data suggest that pancreatic islet cells are not able to build up an antiviral state after adenoviral transduction and/or that the adenoviral proteins (E1A) are very efficient blockers of the IFN signal transduction pathway (46) in this type of cell. Furthermore, in our experience, exogenous addition of IFN-{alpha} was vital for preventing adenovirus-induced islet cell death. Indeed, treatment of rat and human pancreatic islets with IFN-{alpha} before and during transduction blocked adenovirus-induced cytolysis and prevented the nuclear structural modifications of the transduced cells. The protective effect of type I IFNs may be related to their capacity to suppress virus replication and inhibit early viral gene expression (47). The ultimate outcome of IFN signaling is activation of the transcription of target genes, such as 2',5'-oligoadenylate synthetase, MxA, MxB genes (48) and MHC class IA antigens (16, 17). Consistent with these reports, we found that IFN-{alpha} treatment was able to up-regulate expression of MHC class IA genes (ß2m, Lmp-2, and Tap-1) in human islets. In rat islet cells, exogenous addition of IFN-{alpha} stimulated expression of the proteasome subunit Lmp-7 and ß2m mRNA, although the later was not statistically significant. Thus, these combined observations are not compatible with the second scenario, described in the previous paragraph, stating that the ß-cell IFN response is strong. Instead, they support the idea that the virus-induced ß-cell IFN response is suboptimal, leading to ß-cell death and possibly autoimmune triggering. It may be that the pancreatic ß-cell is exceptional in this aspect, because other cell types are known to induce an IFN response when transduced with adenovirus (49, 50).

Previous studies with isolated pancreatic islets have revealed that human ß-cells are clearly more resistant against toxin- and cytokine-induced damage than rodent cells (51, 52). Our present results suggest that this is also the case in adenovirus-induced islet cell death. Moreover, in response to IFN-{alpha} treatment, human islet cells seem to be more efficient in inducing genes belonging to the antigen processing and antigen presentation MHC class IA family, which might be a critical event in the context of viral transduction.

Genetic engineering of ß-cells for use in gene therapy approaches aims at enhancing the resistance against poor grafting, rejection, and autoimmune attack in type 1 diabetes. For this purpose as well as for basic ß-cell research, viral-derived gene transfer tools should not have any impact by themselves on the function, morphology, or viability of target cells. We showed in this study that prolonged culture of adenoviral-transduced islet cells leads to significant cytotoxicity and that pancreatic islet cells do not have the capacity to induce an IFN response. This process is more evident when high concentrations of the virus are used, and the outcome seems to depend upon the extent of the deletion of the viral genome. If inappropriate viral vectors are chosen in an in vivo situation, pancreatic ß-cells could be directly destroyed by adenovirus-induced cytolysis, or alternatively, the transduction process could cause an inflammatory reaction, leading to or exacerbating the ß-cell-targeted autoimmune response. Considering the wide use of adenoviral vehicles for genetic modification of nondividing cells, these findings should be considered in the design and application of such vectors in experimental and clinical ß-cell research.


   Acknowledgments
 
We gratefully acknowledge the excellent technical assistance of Ing-Marie Mörsare. We also thank Prof. William S. M. Wold (Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine, St. Louis, MO) for providing us with the wild-type adenoviruses.


   Footnotes
 
This work was supported in part by Swedish Medical Research Council Grants 72P-12995, 12X-11564, and 12X-109; the Swedish Diabetes Association; and the Family Ernfors Fund.

First Published Online February 10, 2005

Abbreviations: Ad, Adenovirus; ADP, adenovirus death protein; CMV, cytomegalovirus; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN, interferon; MHC, major histocompatibility complex; PFU, plaque-forming unit; XTT, sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate.

Received December 27, 2004.

Accepted for publication January 31, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman, NM, Rajotte RV 2000 Islet transplantation in seven patients with type I diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230–238[Abstract/Free Full Text]
  2. Rabinovitch A, Suarez-Pizon W, Strynadka K, Ju Q, Edelstein D, Brownlee M, Korbutt GS, Rayotte RV 1999 Transfection of human pancreatic islets with an anti-apoptotic gene (Bcl-2) protects ß-cells from cytokine-induced destruction. Diabetes 48:1223–1229[Abstract]
  3. Giannoukakis N, Rudert WA, Ghivizzani SC, Gambotto A, Ricordi C, Trucco M, Robbins PD 1999 Adenoviral gene transfer of the interleukin-1 receptor antagonist protein to human islets prevents IL-1ß-induced ß-cell impairment and activation of islet cell apoptosis in vitro. Diabetes 48:1730–1736[Abstract]
  4. Taniguchi H, Yamato E, Tashiro F, Ikegami H, Oghihara T, Miyazaki J 2003 ß-Cell neogenesis induced by adenovirus-mediated gene delivery of transcription factor pdx-1 into mouse pancreas. Gene Ther 10:15–23[CrossRef][Medline]
  5. Garcia-Ocana A, Takane KK, Syed MA, Philbrick WM, Vasavada RC, Stewart AF 2000 Hepatocyte growth factor overexpression in the islet of transgenic mice increases ß cell proliferation, enhances islet mass, and induces mild hypoglycemia. J Biol Chem 275:1226–1232[Abstract/Free Full Text]
  6. Hui H, Wright C, Perfetti G 2001 Glucagon-like peptide 1 induces differentiation of islet duodenal homeobox-1-positive pancreatic ductal cells into insulin-secreting cells. Diabetes 50:785–796[Abstract/Free Full Text]
  7. Bartlett R, Denis M, Inverardi L, Ricordi C 1998 Introduction of immunomodulatory genes into isolated pancreatic islets via biolistic particle bombardment. Transplant Proc 30:452–459[CrossRef][Medline]
  8. Lakey JRT, Young ATL, Pardue D, Calvin S, Albertson TE, Jacobson L, Cavanagh TJ 2001 Nonviral transfection of intact pancreatic islets. Cell Transplant 10:697–708[Medline]
  9. Ju Q, Edelstein D, Brendel MD, Brandhorst D, Bretzel RG, Brownlee M 1998 Transduction of non-dividing adult human pancreatic ß cells by an integrating lentiviral vector. Diabetologia 41:736–739[CrossRef][Medline]
  10. Flotte T, Agarwal A, Wang J, Song S, Fenjves ES, Inverardi L, Chesnut K, Afione S, Loiler S, Wasserfall C, Kapturczak M, Ellis T, Nick H, Atkinson M 2001 Efficient ex vivo transduction of pancreatic islet cells with recombinant adeno-associated virus vectors. Diabetes 50:515–520[Abstract/Free Full Text]
  11. Bilbao G, Contreras JL, Dmitriev I, Smyth CA, Jenkins S, Eckhoff D, Thomas F, Thomas J, Curiel DT 2002 Genetically modified adenovirus vector containing an RDG peptide in the HI loop of the fiber knob improves gene transfer to nonhuman primate isolated pancreatic islets. Am J Transplant 2:237–243[CrossRef][Medline]
  12. Barbu AR, Akusjarvi G, Welsh N 2002 Adenoviral-induced islet cell cytotoxicity is not counteracted by Bcl-2 overexpression. Mol Med 8:733–741[Medline]
  13. Braithwaite AW, Russell IA 2001 Induction of cell death by adenoviruses. Apoptosis 6:359–370[CrossRef][Medline]
  14. Tollefson AE, Scaria A, Hermiston TW, Ryerse JS, Wold LJ, Wold WSM 1996 The adenovirus death protein (E3–11.6K) is required at very late stages of transduction for efficient cell lysis and release of adenovirus from transduced cells. J Virol 70:2296–2306[Abstract]
  15. Guidotti LG, Chisari FV 2000 Cytokine-mediated control of viral transductions. Virol 273:221–227
  16. Pujol-Borrel R, Todd I, Doshi M, Gray D, Feldman M, Bottazzo GF 1986 Differential expression and regulation of MHC products in the endocrine and exocrine cells of the human pancreas. Clin Exp Immunol 65:128–139[Medline]
  17. Vives-Pi M, Armengol MP, Alcalde L, Costa M, Somoza N, Vargas F, Jaraquemada D, Pujol-Borrel R 1996 Expression of transporter associated with antigen specific processing-1 in the endocrine cells of human pancreatic islets: effects of cytokines and evidence of hyperexpression in IDDM. Diabetes 45:779–788[Abstract]
  18. Andersson A 1978 Isolated mouse pancreatic islets in culture: effects of serum and different culture media on the insulin production of the islets. Diabetologia 14:397–404[CrossRef][Medline]
  19. Molin M, Shoshan MC, Öhman-Forslund K, Linder S, Akusjärvi G 1998 Two novel adenovirus systems permitting regulated protein expression in gene transfer experiments. J Virol 72:8358–8361[Abstract/Free Full Text]
  20. Gossen M, Bujard H 1992 Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–5551[Abstract/Free Full Text]
  21. Weiden MD, Ginsberg HS 1994 Deletion of the E4 region of the genome produces adenovirus DNA concatemers. Proc Natl Acad Sci USA 91:153–157[Abstract/Free Full Text]
  22. He T-C, Zhou S, Da Costa LT, Yu J, Kinzler KW, Vogelstein B 1998 A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95:2509–2514[Abstract/Free Full Text]
  23. Bhat BM, Wold WS 1986 Genetic analysis of mRNA synthesis in adenovirus region E3 at different stages of productive transduction by RNA-processing mutants. J Virol 60:54–63[Abstract/Free Full Text]
  24. Saldeen J, Curiel DT, Eizirik DL, Andersson A, Strandell E, Buschard K, Welsh N 1996 Efficient gene transfer to dispersed human pancreatic islet cells in vitro using adenovirus-polylysine/DNA complexes or polycationic liposomes. Diabetes 45:1197–1203[Abstract]
  25. Linne T, Jornvall H, Philipson L 1977 Purification and characterization of the phosphorylated DNA-binding protein from adenovirus-type-2-transduced cells. Eur J Biochem 76:481–490[Medline]
  26. Gibson UE, Heid CA, Williams PM 1996 A novel method for real time quantitative RT-PCR. Genome Res 6:995–1001[Abstract/Free Full Text]
  27. Alesci S, Ramsey WJ, Bornstein SR, Chrousos GP, Hornsby PJ, Benvenga S, Trimarchi F, Ehrhart-Bornstein M 2002 Adenoviral vectors can impair adrenocortical steroidogenesis: clinical implications for natural transductions and gene therapy. Proc Natl Acad Sci USA 99:7484–7489[Abstract/Free Full Text]
  28. Horwitz MS 2001 Adenovirus immunoregulatory genes and their cellular targets. Virology 279:1–8[CrossRef][Medline]
  29. Putzer B, Stiewe T, Parssanedjad K, Rega S, Esche H 2000 E1A is sufficient by itself to induce apoptosis independent of p53 and other adenoviral gene products. Cell Death Differ 7:177–188[CrossRef][Medline]
  30. Marcellus RC, Teodoro JG, Wu T, Brough DE, Ketner G, Shore GC, Branton PE 1996 Adenovirus type 5 early region 4 is responsible for E1A-induced p53-independent apoptosis. J Virol 70:6207–6215[Abstract]
  31. Querido E, Blanchette P, Yan Q, Kamura T, Morrison M, Boivin D, Kaelin W, Conaway R, Conaway JW, Branton PE 2001 Degradation of p53 by adenovirus E4 orf6 and E1B 55K proteins occurs via a novel mechanism involving a Cullin-containing complex. Genes 15:3104–3117
  32. Routes JM, Ryan S, Clase A, Miura T, Kuhl A, Potter TA, Cook JL 2000 Adenovirus E1A oncogene expression in tumor cells enhances killing by TNF-related apoptosis-inducing ligand (TRAIL). J Immunol 165:4522–4527[Abstract/Free Full Text]
  33. Lesokhin AM, Delgado-Lopez F, Horwitz MS 2002 Inhibition of chemokines expression by adenovirus early region three (E3) genes. J Virol 76:8236–8243[Abstract/Free Full Text]
  34. Krajcsi P, DimitrovT, Hermiston TW, Tollefson AE, Ranheim TS, VandePol SB, Stephenson AH, Wold WS 1996 The adenovirus E3–14.7K protein and the E3–10.4/14.5K complex of proteins, which independently inhibit tumor necrosis factor (TNF)-induced apoptosis, also independently inhibit TNF-induced release of arachidonic acid. J Virol 70:4904–4913[Abstract/Free Full Text]
  35. Zhang F, Cheng J, Hackett NR, Lam G, Shido K, Pergolizzi R, Jin DK, Crystal RG, Rafii S 2003 Adenovirus E4 gene promotes selective endothelial cell survival and angiogenesis via activation of the VE-cadherin/Akt signaling pathway. J Biol Chem 279:11760–11766
  36. Russell WC 2000 Update on adenovirus and its vectors. J Gen Virol 81:2573–2604[Free Full Text]
  37. Nevins JR 1981 Mechanisms of activation of early viral transcription by the adenovirus E1A gene product. Cell 26:213–220[CrossRef][Medline]
  38. White E, Cipriani R 1989 Specific disruption of intermediate filaments and the nuclear lamina by the 19-kDa product of the adenovirus E1B oncogene. Proc Natl Acad Sci USA 86:9886–9890[Abstract/Free Full Text]
  39. Lutz P, Rosa-Calatrava M, Kedinger C 1997 The product of the adenovirus intermediate gene IX is a transcriptional activator. J Virol 71:5102–5109[Abstract]
  40. Sargent KL, Meulenbroek RA, Parks RJ 2004 Activation of adenoviral gene expression by protein IX is not required for efficient virus replication. J Virol 78:5032–5037[Abstract/Free Full Text]
  41. Bach JF 1994 Insulin-dependent diabetes mellitus as an autoimmune disease. Endocr Rev 15:516–542[Abstract]
  42. Yoon JW, Jun HS, Santamaria P 1998 Cellular and molecular mechanisms for the initiation and progression of ß-cell destruction resulting from the collaboration between macrophages and T cells. Autoimmunity 27:109–122[Medline]
  43. Flodström M, Maday A, Balakrishna D, Malo Cleary M, Yoshimura A, Sarvetnick N 2002 Target cell defence prevents the development of diabetes after viral transduction. Nature Immunol 3:373–382[CrossRef][Medline]
  44. Stewart TA 2003 Neutralizing interferon {alpha} as a therapeutic approach to autoimmune diseases. Cytokine Growth Factor Rev 14:139–154[CrossRef][Medline]
  45. Goodbourn S, Didcock L, Randall RE 2000 Interferons: cell signaling, immune modulation, antiviral responses and virus contrameasures. J Gen Virol 81:2341–2364[Free Full Text]
  46. Kalvakolanu DVR, Bandyopadhyay SK, Harter ML, Sen GC 1991 Inhibition of the interferon inducible gene expression by adenovirus E1A proteins: block in the transcriptional complex formation. Proc Natl Acad Sci USA 88:7459–7463[Abstract/Free Full Text]
  47. Taniguchi T, Takaoka A 2002 The interferon-{alpha}/ß system in antiviral responses: a multimodal machinery of gene regulation by the IRF family of transcription factors. Curr Opin Immunol 14:111–116[CrossRef][Medline]
  48. Der SD, Zhou A, Williams BRG, Silverman RH 1998 Identification of genes differentially regulated by the interferon {alpha}, ß or {gamma} using oligonucleotide arrays. Proc Natl Acad Sci USA 95:15623–15628[Abstract/Free Full Text]
  49. Dong Z, Yan, X, Fidler IJ 1997 Neutralization of endogenous interferon-ß increases the efficiency of adenoviral vector-mediated gene transduction. J Interferon Cytokine Res 17:401–407[Medline]
  50. Vilquin JT, Guerette B, Kinoshita I, Roy B, Goulet M, Gravel C, Roy R, Tremblay JP 1995 FK506 immunosuppression to control the immune reactions triggered by first-generation adenovirus-mediated gene transfer. Hum Gen Ther 6:1391–1401[Medline]
  51. Eizirik DL, Pipeleers DG, Ling Z, Welsh N, Hellerstrom C, Andersson A 1994 Major species differences between humans and rodents in the susceptibility to pancreatic ß-cell injury. Proc Natl Acad Sci USA 91:9253–9256[Abstract/Free Full Text]
  52. Welsh N, Margulis B, Borg LA, Wiklund HJ, Saldeen J, Flodstrom M, Mello MA, Andersson A, Pipeleers DG, Hellerstrom C 1995 Differences in the expression of heat shock proteins and antioxidant enzymes between human and rodent pancreatic islets: implications for the pathogenesis of insulin dependent diabetes mellitus. Mol Med 1:806–820[Medline]



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