Adenovirus (Ad) gene therapy has been proposed as a drug-delivery system for the targeted administration of protein-based therapies, including growth factors and biological response modifiers. However, inflammation associated with Ad transduction has raised concern about its safety and efficacy in acute inflammatory diseases. In the present report, intratracheal and i.v. administration of a first-generation adenoviral recombinant (E1,E3 deleted) either containing an empty cassette or expressing the anti-inflammatory cytokines viral or human IL-10 (IL-10) was administered to mice subjected to zymosan-induced multisystem organ failure or to acute necrotizing pancreatitis. Pretreatment of mice with the intratracheal instillation of Ad expressing human IL-10 or viral IL-10 reduced weight loss, attenuated the proinflammatory cytokine response, and reduced mortality in the zymosan-induced model, whereas pretreatment with a control adenoviral recombinant did not significantly exacerbate the response. Pretreatment of mice with pancreatitis using adenoviral vectors expressing IL-10 significantly reduced the degree of pancreatic and liver injury and liver inflammation when administered systemically, but not intratracheally. We conclude that adenoviral vectors can be administered prophylactically in acute inflammatory syndromes, and expression of the anti-inflammatory protein IL-10 can be used to suppress the underlying inflammatory process.

Gene therapy is currently under clinical investigation for the treatment of several genetic diseases, including cystic fibrosis and deficiencies in α1-antitrypsin, adenosine deaminase, and ornithine transcarbamoylase (1). Gene therapy also is being applied to acquired diseases with a genetic component, such as p53 mutations in ovarian and head and neck cancers. In these cases, the goal of gene therapy is the restoration of normal gene function and protein expression (2).

However, gene therapy has several potential uses beyond the restitution of normal gene function. In 1995, Crystal (3) proposed the use of gene therapy as a novel drug delivery system to express proteins in individual tissues. This approach offers several theoretical and practical advantages over the administration of recombinant proteins. With adenoviral constructs, a single intratracheal or i.v. administration could result in sustained protein expression for periods generally lasting between 7 and 14 days, but occasionally for longer periods (4, 5). In addition, by using tissue-specific promoters or taking advantage of the natural tropism of adenovirus (Ad)3 for bronchial epithelium or for the liver and pancreas, targeted expression in these organs, but not in other tissues, could be achieved. This type of local administration and expression may improve therapeutic efficacies of some proteins, particularly cytokines.

However, clinical development of adenoviral vectors has been limited by their inflammatory potential and the induction of an acquired immune response that limits repeat dosing (6). Previous studies have shown that first-generation adenoviral vectors activate the innate immune response and stimulate the synthesis of the proinflammatory cytokine TNF-α (4, 5). This increased TNF-α production not only reduces the duration and magnitude of transgene expression (4, 7), but also is a concern in acute inflammatory conditions where exaggerated TNF-α expression may contribute to organ injury. There have been only limited studies evaluating the safety and efficacy of adenoviral constructs in the context of an ongoing inflammatory process.

In the present report, we have explored the feasibility of adenoviral gene therapy as a drug delivery system for the treatment of two acute inflammatory diseases, necrotizing pancreatitis induced by a choline-deficient, ethionine supplemented (CDE) diet, and zymosan-induced multisystem organ failure (MSOF). We have specifically asked whether adenoviral expression of the anti-inflammatory cytokines human (h) or viral (v) IL-10 could be successfully used in these acute inflammatory processes. We chose these two models because previous studies have shown that administration of IL-10 protein can alter the progression of disease and improve outcome. In particular, Deviere and colleagues (8, 9) have shown that exogenously administered and endogenously produced IL-10 reduces the systemic inflammatory response to experimental pancreatitis. These same investigators have shown that a single administration of IL-10 in patients before endoscopic retrograde cholangiopancreatography reduces the risk of pancreatitis (10). Similarly, Jansen and colleagues (11) have shown that repeated administration of IL-10 also can improve outcome in zymosan-induced MSOF.

The findings presented here suggest that adenoviral transduction can be accomplished during existing inflammatory processes without significant exacerbation of organ injury, and Ad-induced expression of IL-10 is associated with reduced inflammation and organ injury and improved outcome.

A derivative of hAd serotype 5 (12) was used as the source of viral DNA backbone. This modified adenoviral vector backbone contains a deletion of base pairs 355-4021, resulting in a deletion of the E1a, E1b, and protein IX polypeptides. In addition, there is a deletion of base pairs 28,592–30,470 that results in a loss of 1.9 kb of DNA from the E3 region. rAd expressing hIL-10 and vIL-10 cDNA transgenes were constructed by standard homologous recombination methods as described by Graham and Prevec (13). Briefly, hIL-10 and vIL-10 cDNAs containing the full-length translated regions (from pDSRG-IL10 and pcDSR-BCRF1 plasmids, respectively, obtained from K. Moore at DNAX Research Institute, Palo Alto, CA; Ref. 14) were subcloned into the BamHI/XbaI cloning site of the pACN transfer plasmid. The pACN transfer plasmid is based on pBR322 and contains, from 5′ to 3′, the Ad5 inverted terminal repeat and packaging signal (Ad5 bp 1–358), the hCMV immediate early enhancer/promoter, the Ad2 tripartite leader sequence, a multiple cloning site, and Ad2 bp 4,021–10,457. This plasmid was cotransfected into 293 cells, along with a claI-linearized fragment of the plasmid described above containing the modified hAd5 adenoviral vector backbone (12). Additionally, a rAd containing an empty expression cassette was constructed for use as a control. All of the viral constructs were similar with the exception of the transgene, and the production and purification procedures were identical.

Relative expression levels of hIL-10 and vIL-10 have been directly compared after in vitro transduction of cell lines as well as in vivo transduction with the respective adenoviral vectors (15). Transduction of cell lines in vitro with the adenoviral vectors expressing hIL-10 and vIL-10 resulted in similar amounts of protein being secreted into the supernatant. Likewise, i.v. injection of adenoviral vectors expressing hIL-10 and vIL-10 resulted in similar amounts of protein being released into the serum. However, there was a comparatively larger portion of vIL-10 that was retained in the cell lysate portion of in vitro transduced cells and in vivo transduced tissues.

Zymosan A (lot no. 49H0557), a cell wall component of Saccharomyces cerevisiae, and endotoxin-free paraffin oil were obtained from Sigma (St. Louis, MO). Zymosan A was irradiated with 5 kGy γ radiation over 5 h. Endotoxin testing of 1 mg/ml zymosan A in sterile H2O demonstrated that zymosan A contained < 5 EU/mg. The irradiated zymosan A was suspended in sterile paraffin oil at a concentration of 0.6 mg/mouse g body weight using a sterile technique. The zymosan-paraffin oil solution then was placed in a Fisher sonicating water bath (50/60 Hz) for 30 min and a high-frequency sonicator (Fisher Sonic Dismembrator model 300; Fisher Scientific, Pittsburgh, PA) was used at a setting of 70% for 15 min to further resuspend the zymosan in the paraffin oil. The zymosan suspension then was placed in a 100°C water bath for 80 min. After this, the solution was vortexed and drawn into sterile syringes.

Specific pathogen-free female C57BL/6 mice (20–25 g; The Jackson Laboratory, Bar Harbor, ME) were housed in a Bio-Safety Level 2 barrier facility with unlimited chow and water for the duration of the experiments. All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Florida. The use of adenoviral recombinants in mice was approved by the Institutional Biosafety and the Recombinant DNA Committees of the University of Florida. The laboratory adheres to the “Guiding Principles of Laboratory Animal Care,” as promulgated by the American Physiological Society.

Mice were anesthetized with 35 mg/kg body weight of i.p. sodium pentobarbital. A mid-line incision was made in the neck, the trachea was visualized and cannulated with a sterile 30-gauge needle, and 32 μl of buffer (PBS, pH 7.5, containing 3% (w/v) sucrose, 2 mM MgCl2) or buffer containing adenoviral vector (107 particles per animal) was delivered. Mice received an intratracheal instillation of either 107 particles of a recombinant adenoviral construct expressing hIL-10 (Ad/hIL-10), vIL-10 (Ad/vIL-10), or an empty expression cassette (Ad/empty), or buffer alone on day −1 or day 5 in the zymosan model, and on day −1 in the CDE pancreatitis model. In the CDE pancreatitis model, i.v. tail vein injections of 100 μl of buffer or buffer containing 1010 particles of either Ad/hIL-10, Ad/vIL-10, or Ad/empty (n = 15 per group) also were performed on day −1 using a sterile 30-gauge needle.

On day 0, the mice underwent an i.p. injection of 1 ml of the zymosan suspension with a sterile 20-gauge needle or were fed a CDE diet (100 g choline-deficient murine chow supplemented with 0.5 g dl-ethionine). On day 4, the CDE diet was exchanged for normal murine chow. A single i.p. injection of sterile zymosan suspended in paraffin oil leads to the development of a triphasic illness characterized by an initial septic shock-like phase (days 1–3) with an associated mortality of 25–30%, followed by a recovery period (days 4–8), and then ultimately the development of MSOF with an additional 60–70% mortality (days 9–14; Refs. 16 and 17). The CDE diet induces a severe necrotizing pancreatitis in mice as described previously (18, 19).

In the zymosan model, Ad/hIL-10-, Ad/vIL-10-, and buffer-treated animals were weighed daily and followed for survival for 18 days (n = 15–24 per group). Additional mice (n = 9 per group) were sacrificed by cervical dislocation 24 h after zymosan administration. Blood was collected via a retroorbital venipuncture with a capillary tube. Liver and lungs were removed and snap-frozen in liquid nitrogen.

In the CDE pancreatitis model, Ad/hIL-10-, Ad/vIL-10-, and buffer-treated animals were followed for survival for 10 days (n = 10). Additional mice (n = 15) were sacrificed by cervical dislocation 66 h after induction of pancreatitis (19). Blood was again collected via retroorbital venipuncture, and liver and lungs were removed en bloc and snap-frozen in liquid nitrogen.

Murine IL-6, IL-1α, hIL-10, and vIL-10 levels in the organ homogenates and in the serum were measured by sandwich ELISA with commercially available reagents (mIL-6, hIL-10, and vIL-10 by Endogen, Woburn, MA, and murine IL-1α and IL-10 by R&D Systems, Minneapolis, MN). Bioactive TNF-α was measured in serum and lung homogenates with the TNF-α-sensitive WEHI 164 clone 13 murine fibrosarcoma cell line (20). A standard curve was generated with hTNF-α, and the sensitivity of the assay was 5–25 pg/ml for serum and 75–375 pg/gram wet weight (gww) for lung.

Pulmonary and hepatic neutrophil sequestration were quantitated in the pancreatitis studies by measuring tissue myeloperoxidase content (5). Pancreatic injury was assessed by measuring serum amylase and lipase with a Vitros system analyzer (Shands Hospital Diagnostic Laboratories, Gainesville, FL).

Total RNA was extracted from ∼100 mg of lung tissue with Tri-Reagent (Molecular Research Center, Cincinnati, OH) per the manufacturer’s protocol. Total RNA was treated with DNase I (Boehringer-Mannheim, Indianapolis, IN) to remove residual DNA. The complete removal of DNA in all RNA samples was confirmed with murine gapdh PCR. RNA concentrations were determined with OD 260/280 absorbance ratios, and ∼0.1 μg of total RNA was used for each RT-PCR. Quantification of mRNA and viral DNA were performed with real-time quantitative RT-PCR, which uses the 5′ nuclease activity of Taq polymerase (Taqman; PerkinElmer Applied Biosystems, Norwalk, CT) to detect PCR amplicons. Briefly, in addition to primers, a target sequence-specific oligonucleotide probe labeled with a reporter fluorescent dye (FAM (6-carboxy-tetramethyl-rhodamine)) at the 3′ end was added to the PCR. When the probe is intact, the fluorescence emission of the reporter is quenched because of the physical proximity of the reporter and quencher fluorescent dyes. The resulting relative increase in reporter fluorescent dye emission is monitored in real time during PCR amplification by a sequence detector, the 7700 sequence detector (PerkinElmer Applied BioSystems). In addition to Ad/hIL10 and Ad/vIL10, relative quantification of murine GAPDH RNA and DNA also were performed to ensure the quality of RNA and DNA.

RT-PCR was performed with the Taqman EZ RT-PCR kit (PerkinElmer Applied BioSystems). Reactions were performed in a total volume of 50 μl containing 1.0 μl of total RNA extracted from lungs or cells, 300 mM each of dATP, dGTP, or dCTP, and 600 mM of dUTP, 10 U of recombinant Thermus thermophilus DNA polymerase, 0.5 U of AmpErase uracil N-glycosylase (all from PerkinElmer Applied BioSystems) to eliminate carry-over PCR product contamination, 10 mM of each primer, and 10 mM of probe. The following thermal cycler parameters were optimized for IL10 transgene detection: 2 min at 50°C, 30 min at 60°C, 10 min at 95°C, followed by 40 cycles of 20 s at 95°C and 1 min at 61°C. The primer and probe sequences for RT-PCR and PCR were: Ad/hIL-10 and Ad/vIL-10 forward primer, 5′-AACGGTACTCCGCCACC-3′; Ad/hIL-10 reverse, 5′-CGGCCGCTCGAGTCTAGAC-3′; Ad/vIL-10 reverse, 5′-ATGATGGAGCTCTAGACTCGAGA3′; Ad/hIL-10 and Ad/vIL-10 probe, FAM-TCCGCATCGACCGGATCGGTAMRA; murine GAPDH forward, 5′-GAAGGTGAAGGTCGAGTC-3′; GAPDH reverse, 5′-GAAGATGGTGATGGGATTTC-3′; GAPDH probe, FAM-CAAGCTTCCCGTTCTCAGCC-TAMRA. The PCR thermal profile was 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative quantification of gene expression was performed with a serially diluted RNA isolated from either Ad/vIL-10- or Ad/hIL-10-infected 293 cells. MEQ is a relative quantification unit, using an arbitrarily assigned number. For viral DNA quantification, serially diluted viral DNA was isolated from Ad/hIL-10 or Ad/vIL-10 viruses. All RT-PCR and PCR were done in duplicate and the detection limit of RT-PCR assay is 20 MEQ/mg of tissue and PCR assay is 10 copies/mg of tissue.

Data are presented as the mean ± SEM (n = 9–18 animals/group). Student’s t test was used for analyses comparing healthy controls to buffer-treated animals in the zymosan and pancreatitis models. A one-way ANOVA was used to compare animals at the same time point receiving different treatments (buffer, Ad/hIL-10, or Ad/vIL-10), and post hoc comparisons were performed with Dunn’s multiple range test. A one-way ANOVA with repeated measures was used to compare the animals’ weight changes over time in the zymosan model, and pair-wise comparisons were performed by the Fisher least significant differences method. Survival was assessed with Kaplan-Meier analysis. Statistical differences were considered to be significant at p < 0.05.

In the initial studies, C57BL/6 mice underwent intratracheal instillation with either 107 particles of Ad/hIL-10 or Ad/vIL-10 in 32 μl of buffer or 32 μl of buffer alone at day −1. On day 0, the animals then were challenged with i.p. zymosan A in paraffin oil as described. As shown in Fig. 1 A, ∼30% of the buffer-treated mice died within the first 3 days, and then survival plateaued for an additional 3–5 days. Thereafter, mortality in the buffer group was progressive with only 25% of the animals surviving 18 days.

FIGURE 1.

Survival after zymosan challenge with intratracheal Ad/hIL-10, Ad/vIL-10, Ad/empty, and buffer pretreatment. A, Eighteen-day survival was significantly improved in mice pretreated intratracheally with Ad/hIL-10 (▿; 47%; #, p < 0.05) and Ad/vIL-10 (▪; 73%; ∗, p < 0.001), as compared with mice treated with buffer alone (•; 25%). B, Early mortality (days 1–4) was modestly increased in the mice pretreated with the Ad/empty, as compared with buffer alone (p > 0.05 by Fisher’s exact test). However, 18-day survival was not significantly different in the animals pretreated with Ad/empty (▾; 22%) as compared with mice pretreated with buffer alone (•; 33%; p = 0.3).

FIGURE 1.

Survival after zymosan challenge with intratracheal Ad/hIL-10, Ad/vIL-10, Ad/empty, and buffer pretreatment. A, Eighteen-day survival was significantly improved in mice pretreated intratracheally with Ad/hIL-10 (▿; 47%; #, p < 0.05) and Ad/vIL-10 (▪; 73%; ∗, p < 0.001), as compared with mice treated with buffer alone (•; 25%). B, Early mortality (days 1–4) was modestly increased in the mice pretreated with the Ad/empty, as compared with buffer alone (p > 0.05 by Fisher’s exact test). However, 18-day survival was not significantly different in the animals pretreated with Ad/empty (▾; 22%) as compared with mice pretreated with buffer alone (•; 33%; p = 0.3).

Close modal

Survival was significantly improved in both the Ad/hIL-10- and Ad/vIL-10-treated animals, as compared with buffer only-treated mice (47%; p < 0.05 and 73%; p < 0.001, respectively, vs 25% in the buffer group; Fig. 1,A). To ensure that the survival benefits that were seen in the Ad/hIL-10 and Ad/vIL-10 treatment groups were attributable to the expression of IL-10 and not secondary to adenoviral transduction per se, the experiment was repeated, and buffer-treated mice were compared with mice that received the intratracheal instillation of an identical adenoviral vector containing an empty cassette. Mice (n = 18 per group) were again pretreated with the intratracheal delivery of 32 μl of buffer containing 107 particles of Ad/empty or buffer alone, and survival was measured. In the Ad/empty treatment group, there was an insignificant trend toward decreased survival in the first phase of the zymosan-induced illness (44% survival by day 4), as compared with mice treated with buffer only (77% survival by day 4). However, 18-day survival was similar between the mice treated with the Ad/empty and buffer (22 vs 33%, respectively; Fig. 1 B).

Mice receiving the intratracheal administration of Ad/hIL-10 and Ad/vIL-10 also demonstrated less initial weight loss and more rapid weight gain, a sensitive clinical marker of murine illness, after zymosan challenge, as compared with buffer only-treated animals (Fig. 2). Interestingly, the animals treated intratracheally with Ad/hIL-10 appeared to lose benefit in the third phase of illness, as the mice began to demonstrate weight losses that mirrored the buffer only-treated mice. In contrast, mice receiving the intratracheal administration Ad/vIL-10 continued to experience stable weights, or actually demonstrated weight gain, from days 9 to 18, when MSOF usually develops in this model (Fig. 2). Both the loss of survival protection as well as the weight loss seen in the third phase of illness in the Ad/hIL-10 treatment group occurred around days 9–10.

FIGURE 2.

Weight change after zymosan challenge with intratracheal Ad/hIL-10 and Ad/vIL-10 pretreatment. Weight gain was significantly increased in the mice pretreated with Ad/hIL-10 (▿) and Ad/vIL-10 (▪) on days 3 and 4 after zymosan challenge, as compared with mice pretreated with buffer alone (•; ∗, p < 0.05). The mice pretreated with Ad/hIL-10 and buffer demonstrated weight loss during days 9–18, the third phase of the zymosan-induced illness, whereas the Ad/vIL-10-pretreated mice demonstrated stabilization of their weight during this time (∗, p < 0.05).

FIGURE 2.

Weight change after zymosan challenge with intratracheal Ad/hIL-10 and Ad/vIL-10 pretreatment. Weight gain was significantly increased in the mice pretreated with Ad/hIL-10 (▿) and Ad/vIL-10 (▪) on days 3 and 4 after zymosan challenge, as compared with mice pretreated with buffer alone (•; ∗, p < 0.05). The mice pretreated with Ad/hIL-10 and buffer demonstrated weight loss during days 9–18, the third phase of the zymosan-induced illness, whereas the Ad/vIL-10-pretreated mice demonstrated stabilization of their weight during this time (∗, p < 0.05).

Close modal

Despite the improved outcomes in the IL-10-treated animals, neither hIL-10 nor vIL-10 protein could be readily recovered from the lungs of Ad/IL-10-treated mice at 24 h, 7 days, or at 14 days after zymosan instillation, by a sandwich ELISA. Levels in the tissues were below the sensitivity of the immunoassay (200 pg protein/gww tissue). Therefore, real-time PCR (Taqman) also was used to determine the presence of vIL-10 and hIL-10 DNA and mRNA in the lungs of these animals at 24 h, when expression is usually at its peak. The ability to detect this virally delivered DNA was highly variable, ranging from undetectable in some animals to concentrations in excess of 4 × 104 copies/mg of tissue. In general, mRNA levels were at least three logs lower than DNA levels, ranging from undetectable (<1 × 101 MEQ) to 7 × 101 MEQ/mg of sample. Thus, the therapeutic effects were mediated by very low levels of IL-10 achieved in the target tissue.

As shown in Table I, a step-wise reduction in serum IL-1α and IL-6 was seen in mice treated intratracheally with Ad/hIL-10 and Ad/vIL-10, as compared with buffer alone, with the greatest reductions seen in the Ad/vIL-10 treatment group (p = 0.05 for IL-6; Table I). TNF-α could not be detected in the serum at any time point in any animal. Lung IL-6 levels also were significantly reduced in the Ad/vIL-10 treatment group at 24 h (1.54 ± 0.12 ng/gww in the Ad/vIL-10 animals vs 6.55 ± 2.97 ng/gww in the buffer only animals; p < 0.05). Similarly, lung and liver murine IL-10 levels were significantly reduced in the Ad/hIL-10 and Ad/vIL-10 treatment groups (2.09 ± 0.04 ng/gww for lung Ad/vIL-10 vs 2.72 ± 0.17 ng/gww for lung buffer (p < 0.05) and 10.51 ± 0.37 ng/gww for liver Ad/hIL-10, 10.42 ± 1.3 ng/gww for liver Ad/vIL-10 vs 14.19 ± 0.92 ng/gww for liver buffer (p < 0.05)).

Table I.

Serum cytokine levels 24 h following intratracheal delivery of Ad/IL-10- and zymosan-induced generalized inflammation

Treatment GroupIL-1α (ng/ml)IL-6 (ng/ml)TNF-α (ng/ml)
Healthy controls 0.06 ± 0.01 <0.03 <0.05 
Buffer only 0.26 ± 0.10a 40.93 ± 19.11a <0.05 
Ad/hIL-10 0.19 ± 0.09 13.04 ± 4.82 <0.05 
rAd/vIL-10 0.08 ± 0.02 2.40 ± 0.40b <0.05 
Treatment GroupIL-1α (ng/ml)IL-6 (ng/ml)TNF-α (ng/ml)
Healthy controls 0.06 ± 0.01 <0.03 <0.05 
Buffer only 0.26 ± 0.10a 40.93 ± 19.11a <0.05 
Ad/hIL-10 0.19 ± 0.09 13.04 ± 4.82 <0.05 
rAd/vIL-10 0.08 ± 0.02 2.40 ± 0.40b <0.05 
a

, p ≤ 0.05 as compared to healthy controls; determined by t test.

b

, p = 0.05 as compared to buffer-treated animals; determined by one-way ANVOA.

Lung histology was examined in all groups 48 h after zymosan administration. As demonstrated in Fig. 3, significant neutrophil infiltration (dense, multilobular, nucleated, cellular infiltrate) of the lung parenchyma was seen in both the buffer alone- (Fig. 3,A) and Ad/hIL-10-treated (Fig. 3,B) mice. However, in stark contrast, the Ad/vIL-10-treated (Fig. 3 C) mice had more modest pulmonary neutrophil infiltration with preservation of more normal lung architecture.

FIGURE 3.

Histological analysis of lungs from mice after zymosan treatment. Lungs were obtained 2 days after zymosan treatment and were fixed in buffered formalin. Hematoxylin and eosin staining was performed. Magnification, ×400. A significant neutrophil infiltration was seen in the lungs of mice pretreated with the buffer (A) and the Ad/hIL-10 (B), whereas the lungs from the Ad/vIL-10-treated animals (C) had minimal neutrophil infiltration with preservation of the normal lung architecture.

FIGURE 3.

Histological analysis of lungs from mice after zymosan treatment. Lungs were obtained 2 days after zymosan treatment and were fixed in buffered formalin. Hematoxylin and eosin staining was performed. Magnification, ×400. A significant neutrophil infiltration was seen in the lungs of mice pretreated with the buffer (A) and the Ad/hIL-10 (B), whereas the lungs from the Ad/vIL-10-treated animals (C) had minimal neutrophil infiltration with preservation of the normal lung architecture.

Close modal

Additional studies were conducted to determine whether posttreatment of mice with the recombinant adenoviral vectors expressing IL-10 also would confer a survival benefit. Day 5 was chosen for the posttreatment, as it represented the second phase of the model when survival and weight gain were stable (see Fig. 2). In contrast to the pretreatment studies, mice receiving the intratracheal instillation of Ad/hIL-10 or Ad/vIL-10 on day 5 after zymosan administration did not demonstrate any improvement in weight gain (data not shown) or survival (36% survival (5 of 14) for Ad/hIL-10 and 21% survival (3 of 14) for Ad/vIL-10) as compared with buffer controls (36% survival (5 of 14); p > 0.05).

Given the encouraging results seen with an intratracheal pretreatment approach in the zymosan model, similar studies were initially conducted in the CDE pancreatitis model. C57BL/6 mice underwent intratracheal instillation with 107 particles of either Ad/hIL-10, Ad/vIL-10, or buffer alone on day −1, as described. Pancreatitis then was induced and mice were followed for survival, with additional mice sacrificed at 66 h for the measurement of tissue cytokine production and serum amylase and lipase concentrations. Intratracheal pretreatment in this model with hIL-10- or vIL-10-expressing vectors did not lead to an improvement in survival (22% (2 of 9 in the buffer-treated animals), vs 36% (4 of 11 in the Ad/hIL-10-treated animals) and 22% (2 of 9 in the Ad/vIL-10-treated animals); p > 0.05), nor was there any reduction in lung and liver cytokine production or serum amylase and lipase in the Ad/hIL-10 and Ad/vIL-10 treatment groups as compared with buffer controls (data not shown).

Unlike the zymosan model, which has a well-described associated hemorrhagic lung injury (16, 17), the pathology seen in the CDE model of pancreatitis is primarily restricted to the pancreas and liver (18, 19). With lung myeloperoxidase levels as an indicator of neutrophil infiltration, there was not an associated lung infiltration in mice with CDE-induced pancreatitis (0.41 ± 0.07 U/gww in healthy animals vs 0.41 ± 0.04 U/gww in buffer-treated mice with pancreatitis). Therefore, a systemic gene therapy approach then was used in this model taking advantage of the natural tropism of the Ad for hepatocytes and pancreatic epithelial cells. On day −1, C57BL/6 mice underwent i.v. delivery of 1010 particles of Ad/hIL-10, Ad/vIL-10, or buffer alone, followed by induction of CDE pancreatitis on day 0. Mice were sacrificed (n = 15) 66 h after induction of CDE pancreatitis. Although a survival benefit was not observed with an i.v. approach (44%; 7 of 16 in buffer-, Ad/hIL-10-, and Ad/vIL-10-treated animals), significant reductions in the liver and pancreatic injury and liver inflammation were seen in the Ad/hIL-10 and Ad/vIL-10 treatment groups as compared with buffer alone. As shown in Fig. 4, a significant reduction in serum amylase also was seen in the Ad/hIL-10 treatment group (7,302 ± 500 U/L serum for the Ad/hIL-10 treatment group vs 10, 674 ± 857 U/L serum for the buffer only group; p = 0.03; Fig. 4,A). Serum lipase also was reduced in both the Ad/hIL-10- and Ad/vIL-10-treated animals as compared with buffer (7, 389 ± 1,058 U/L serum and 7, 137 ± 1,363 U/L serum vs 10, 414 ± 1,315 U/L serum, respectively; Fig. 4,B). Similarly, as shown in Table II, liver inflammation was markedly reduced. Liver IL-1α levels were reduced significantly in both groups of animals pretreated i.v. with Ad/hIL-10 and Ad/vIL-10. Additionally, liver IL-6 levels and myeloperoxidase activity were significantly reduced in the Ad/vIL-10 treatment group (Table II). The only cytokine that was increased in the lung after CDE pancreatitis was IL-1α (1.36 ± 0.82 ng/gww in buffer-treated mice with pancreatitis vs <0.50 in healthy murine lungs). Ad/vIL-10-treated mice with pancreatitis demonstrated baseline levels of lung IL-1α (<0.50 ng/gww) as compared with buffer-treated animals with pancreatitis; p < 0.05).

FIGURE 4.

Serum amylase and lipase concentrations after induction of CDE pancreatitis and i.v. pretreatment with Ad/IL-10. A significant reduction in serum amylase levels was seen in the mice pretreated with the Ad/hIL-10 as compared with buffer alone-treated animals. ∗, p = 0.03 (A). Reductions in serum lipase were also seen in both the mice receiving Ad/hIL-10 and Ad/vIL-10, as compared with buffer alone (B).

FIGURE 4.

Serum amylase and lipase concentrations after induction of CDE pancreatitis and i.v. pretreatment with Ad/IL-10. A significant reduction in serum amylase levels was seen in the mice pretreated with the Ad/hIL-10 as compared with buffer alone-treated animals. ∗, p = 0.03 (A). Reductions in serum lipase were also seen in both the mice receiving Ad/hIL-10 and Ad/vIL-10, as compared with buffer alone (B).

Close modal
Table II.

Liver cytokine levels following i.v. delivery of Ad/IL-10 and induction of CDE pancreatitis

Treatment groupIL-1α (ng/gww)IL-6 (ng/gww)MPO Activity (U/gww)
Healthy 2.45 ± 0.16 21.71 ± 1.49 0.17 ± 0.04 
Buffer 6.22 ± 0.31a 30.70 ± 2.47a 0.98 ± 0.25a 
Ad/empty 7.40 ± 2.18a 52.75 ± 11.47a 0.75 ± 0.17a 
Ad-hIL-10 4.32 ± 0.43b 26.03 ± 2.36 0.44 ± 0.06 
Ad/vIL-10 3.57 ± 0.30b 16.74 ± 1.97b 0.34 ± 0.04b 
Treatment groupIL-1α (ng/gww)IL-6 (ng/gww)MPO Activity (U/gww)
Healthy 2.45 ± 0.16 21.71 ± 1.49 0.17 ± 0.04 
Buffer 6.22 ± 0.31a 30.70 ± 2.47a 0.98 ± 0.25a 
Ad/empty 7.40 ± 2.18a 52.75 ± 11.47a 0.75 ± 0.17a 
Ad-hIL-10 4.32 ± 0.43b 26.03 ± 2.36 0.44 ± 0.06 
Ad/vIL-10 3.57 ± 0.30b 16.74 ± 1.97b 0.34 ± 0.04b 
a

, p < 0.05 as compared to healthy controls; determined by t test.

b

, p < 0.05 as compared to buffer-treated animals; determined by one-way ANOVA.

Serum, lung, and liver hIL-10 and vIL-10 levels at 66 h after induction of pancreatitis (3.75 days after i.v. delivery of Ad/hIL-10 and Ad/vIL-10) are shown in Table III. As seen in our previous studies (5), vIL-10 concentrations were markedly higher in the tissues as compared with hIL-10 levels, whereas hIL-10 concentrations were more readily detected in the systemic circulation, despite transduction with otherwise identical viral vectors.

Table III.

Human and viral IL-10 levels following i.v. delivery of Ad/IL-10 and induction of CDE pancreatitis

Treatment GrouphIL-10 or vIL-10 (ng/gww or ml serum)
Serum Buffer Undetectable 
 Ad/hIL-10 79.98 ± 18.43 
 Ad/vIL-10 14.22 ± 3.25 
Lung Buffer Undetectable 
 Ad/hIL-10 0.87 ± 0.25 
 Ad/vIL-10 1.91 ± 0.27 
Liver Buffer Undetectable 
 Ad/hIL-10 5.20 ± 1.3 
 Ad/vIL-10 17.09 ± 1.87 
Treatment GrouphIL-10 or vIL-10 (ng/gww or ml serum)
Serum Buffer Undetectable 
 Ad/hIL-10 79.98 ± 18.43 
 Ad/vIL-10 14.22 ± 3.25 
Lung Buffer Undetectable 
 Ad/hIL-10 0.87 ± 0.25 
 Ad/vIL-10 1.91 ± 0.27 
Liver Buffer Undetectable 
 Ad/hIL-10 5.20 ± 1.3 
 Ad/vIL-10 17.09 ± 1.87 

The question of whether these reductions in pancreatic and liver injury were attributable to the expressed IL-10 or were secondary to the adenoviral administration also were determined. Additional animals (n = 20 per group) received i.v. pretreatment with 100 μl of 1010 particles of an identical Ad/empty vector or buffer alone and were placed on the CDE diet the following day. Ten-day survival was similar in both the Ad/empty and buffer treatment groups (0% (0 of 12) vs 18% (2 of 11), respectively). Eight animals in each group also were sacrificed 66 h after induction of pancreatitis, and as shown in Table II, liver IL-1α and IL-6 levels and myeloperoxidase activity were similar or greater than the levels seen in the buffer-treated animals with pancreatitis (Table II). Serum amylase and lipase levels also were not significantly different between the two treatment groups (data not shown). This suggests the beneficial effects seen in the mice treated with Ad/hIL-10 and Ad/vIL-10 were attributable to the expression of IL-10, and not to the transduction with the Ad recombinant.

In the present report, we have explored the use of Ad-based gene therapy to deliver hIL-10 or vIL-10 either intratracheally or systemically to mice with acute necrotizing pancreatitis or zymosan-induced MSOF. It is important to note that most of these studies were performed under conditions where the Ad-delivered gene therapy was administered before the onset of the inflammatory state. Under these pretreatment conditions, the Ad gene therapy was well tolerated and did not appear to significantly exacerbate the subsequent inflammatory response. Rather, pretreatment of mice with intratracheal delivery of both Ad/hIL-10 and Ad/vIL-10 in the zymosan model improved survival, reduced weight loss, and attenuated the magnitude of the inflammatory response, as determined by plasma IL-6 concentrations. In the acute necrotizing pancreatitis model, intratracheal instillations of Ad/hIL-10 or Ad/vIL-10 did not either exacerbate or improve outcome. However, when 1010 particles of Ad were administered i.v., significant reductions in pancreatic injury and liver inflammation were observed. These findings suggest that pretreatment with Ad-based gene therapy expressing IL-10 can be safely performed during acute inflammation and can reduce the magnitude of the subsequent inflammatory process. However, what these studies did not fully address was whether administration of these adenoviral vectors would prove equally efficacious if administered after the onset of acute inflammation. In fact, in the one zymosan study performed, posttreatment benefits were not seen. It remains unresolved whether these gene therapy approaches will be applicable to clinical conditions where treatment modalities are initiated after the onset of inflammation.

Nevertheless, the use of Ad-based gene therapy offers several significant advantages over the administration of recombinant proteins. Most apparent is the observation that a single instillation of Ad results in the sustained expression of the transgene for extended periods of time. Expression with adenoviral vectors is rapid, with protein appearance usually occurring within hours. Peak expression occurs within 1–3 days after adenoviral instillation (5). For recombinant proteins like IL-10, which have a relatively short half-life that necessitates repeated or continuous administrations, adenoviral delivery offers a significant theoretical and practical advantage. We have previously shown that a single intratracheal injection of Ad/hIL-10 or Ad/vIL-10 results in expression exceeding 10 and 42 days, respectively (15).

In addition, adenoviral gene therapy offers the opportunity to target specific tissues for local expression. Because Ad has tropism for pulmonary epithelial cells (21) as well as for hepatocytes (22) and pancreatic epithelial cells (23), high local concentrations can be achieved in the absence of high systemic concentrations. In healthy mice, we observed that after intratracheal administration of Ad/hIL-10 or Ad/vIL-10, tissue levels of protein were often 100-1000 times higher than plasma levels (5, 15). vIL-10 appears to have a strong propensity for remaining in the tissues where it is expressed.

However, widespread use of Ad gene therapy has been limited by its inherent proinflammatory properties. Both intratracheal and i.v. administration of Ad induces a proinflammatory response in the lung and liver, characterized by increased TNF-α expression (4, 7). Indeed, we have shown that clearance of adenoviral vectors is a direct function of the magnitude of the innate immune response and is dependent on TNF-α expression (5). Mice lacking TNF signaling pathways often have extended adenoviral expression (4, 7).

In clinical trials with adenoviral vectors, dose-limiting inflammation has been a significant concern. The use of adenoviral vectors for cystic fibrosis has been hampered by mucosal inflammation, radiographic evidence of airway infiltration, and flu-like symptoms (24, 25). Similarly, i.v. administration of high-dose Ad to a patient with ornithine transcarbamoylase deficiency resulted in death due to fulminant hepatic and MSOF (26, 27).

However, the amount of Ad required to express a secreted protein like IL-10 in pharmacologic quantities in the target tissue is usually quite small. Theoretically, only a limited number of cells need to be transduced and secreting the protein to achieve a tissue-wide, bystander response. In fact, these preliminary studies suggest that doses as low as 107 particles in the lung produce a significant therapeutic response, and doses up to three logs higher in the lungs of healthy mice have not produced any histological evidence of inflammation despite achieving nanogram per gww concentrations of the transgene product (5).

Not surprisingly, there were significant differences in the response to adenoviral gene therapy that appeared to be dependent on the experimental injury, the timing and mode of delivery, and whether hIL-10 or vIL-10 were expressed. It is important to clarify that the use of Ad gene therapy, per se, will not obviate the difficulties associated with treating the infected or inflamed patient, which includes the need for pretreatment. Both models were chosen because of past successes with recombinant protein (8, 9, 10, 11, 28). We were initially surprised that intratracheal instillation of Ad/IL-10 would produce such significant improvements in outcome with the zymosan model, but not with acute pancreatitis. However, it became obvious that a significant component of the mortality secondary to zymosan administration was a hemorrhagic pneumonia (Fig. 3), whereas we saw no evidence of lung inflammation in mice after CDE-induced pancreatitis, as determined histologically or by lung myeloperoxidase content. In previous studies with the CDE model, we observed increased expression of the proinflammatory cytokines TNF-α and IL-1 not only in the lung, but also in the liver and pancreas (29). Because Ad has such a propensity for the liver, and to a lesser extent, the pancreas, we repeated the studies with the i.v. administration of the adenoviral vectors to target those organisms more specifically. In this case, we saw significant reductions in the magnitude of the inflammatory response in the liver, and the degree of pancreatic injury, although survival was not significantly improved.

Timing also appeared to be critical. Although pretreatment of mice with the intratracheal instillation of Ad produced significant improvements in outcome in the zymosan model, delaying treatment for five days until the second phase of the disease did not improve survival. These findings are consistent with the observations of Jansen et al. (11), who also found that although systemic pretreatment with rIL-10 was effective in improving outcome, posttreatment was not. However, other investigators have demonstrated a survival benefit when rIL-10 therapy was delivered to the mice when they developed symptoms of MSOF in the third phase of the zymosan-induced illness (30). The lack of an improvement in outcome with a posttreatment approach in the present studies as compared with the studies of Ferrer and his colleagues (30) is likely secondary to differing routes of administration, intratracheal vs systemic. The histology in Fig. 3 would strongly suggest that lung inflammation is a major component of the early response, whereas systemic inflammation not amenable to targeted lung expression may contribute significantly to the later mortality.

Finally, there were clear quantitative differences in the response to hIL-10 and vIL-10. The biological activity of vIL-10 is thought to represent a subset of activities associated with cellular IL-10. vIL-10 possesses profound anti-inflammatory and immunosuppressive activities, similar to those possessed by hIL-10. However, although hIL-10 is known to inhibit IFN-γ production, MHC class II expression, T cell proliferation, and B cell IgE production (13, 31), hIL-10 also has immunostimulatory properties that vIL-10 lacks. hIL-10 can act as a stimulatory factor for immature and mature thymocytes, mast cells, and B cells (32, 33), whereas vIL-10 lacks these properties (13, 34). These differing properties of hIL-10 and vIL-10 are thought to be attributable to a single amino acid substitution in vIL-10 (35). These contrasting immune properties may account for the differing biological responses that are seen in the Ad/hIL-10 and Ad/vIL-10 treatment groups in the zymosan and pancreatitis models.

A more simple explanation may rest in the differing pharmacokinetics and pharmacodynamics between Ad/hIL-10 and Ad/vIL-10. In a recent report, we observed that intratracheal instillation of Ad/vIL-10 resulted in peak lung concentrations that were 10–100 times greater than peak hIL-10 levels seen with Ad/hIL-10 transductions (15). vIL-10 appeared to be sequestered more effectively in tissues than hIL-10, whereas hIL-10 appeared more readily in the systemic circulation. In addition, the duration of IL-10 expression was markedly longer in the mice receiving intratracheal instillations of Ad/vIL-10 than in those receiving Ad/hIL-10. hIL-10 expression lasted only 10–14 days, whereas vIL-10 expression lasted in excess of 42 days in healthy animals (15). This latter phenomenon may explain the failure of Ad/hIL-10 to produce sustained survival through the third phase of the zymosan-induced illness (MSOF). Zymosan mice pretreated with Ad/hIL-10 started to lose weight and die 9 to 11 days after transduction, whereas mice pretreated with Ad/vIL-10 sustained their body weight and maintained survival. The increased lung vIL-10 concentrations that are typically seen after intratracheal delivery of Ad/vIL-10 as compared with Ad/hIL-10 may also explain the improvement in lung histology seen in Fig. 3.

In conclusion, the results of the present studies demonstrate that adenoviral gene therapy expressing hIL-10 and vIL-10 can be safely administered during acute inflammatory events and can improve outcome and reduce inflammation. Gene therapy offers a novel approach for the delivery of protein-based therapies in acute inflammation and can effectively target individual tissues. As gene therapy becomes a more acceptable mode of treatment, it is anticipated that adenoviral-based therapies will become available as a drug delivery system. Coupled with gene therapy, cytokine-modulating therapies like IL-10 represent an attractive therapeutic approach for the treatment of the acutely inflamed patient.

1

This work was supported in part by Grant P30 HL-59412, awarded by the National Heart, Lung and Blood Institute, Grants GM-40586 and GM-53252, awarded by the National Institute of General Medical Sciences, and a contract with Canji, Inc. R.M.M. is currently supported by a research fellowship (T32-GM-08721) in burns and trauma, awarded by the National Institute of General Medical Sciences.

3

Abbreviations used in this paper: Ad, adenovirus; CDE, choline deficient, ethionine supplemented; h, human; v, viral; MSOF, multisystem organ failure; gww, gram wet weight.

1
Anderson, W. F..
1998
. Human gene therapy.
Nature
392
:
25
2
Gallagher, W. M., R. Brown.
1999
. p53-oriented cancer therapies: current progress.
Ann. Oncol.
10
:
139
3
Crystal, R. G..
1995
. The gene as the drug.
Nat. Med.
1
:
15
4
Elkon, K. B., C. C. Liu, J. G. Gall, J. Trevejo, M. W. Marino, K. A. Abrahamsen, X. Song, J. L. Zhou, L. J. Old, R. G. Crystal, E. Falck-Pedersen.
1997
. Tumor necrosis factor α plays a central role in immune-mediated clearance of adenoviral vectors.
Proc. Natl. Acad. Sci. USA
94
:
9814
5
Minter, R. M., J. E. Rectenwald, K. Fukuzuka, C. L. Tannahill, D. La Face, V. Tsai, I. Ahmed, E. Hutchins, R. Moyer, E. M. Copeland, L. L. Moldawer.
2000
. TNF-α receptor signaling and IL-10 gene therapy regulate the innate and humoral immune responses to recombinant adenovirus in the lung.
J. Immunol.
164
:
443
6
McElvaney, N. G..
1996
. Is gene therapy in cystic fibrosis a realistic expectation?.
Curr. Opin. Pulm. Med.
2
:
466
7
Zhang, H. G., T. Zhou, P. Yang, C. K. Edwards, D. T. Curiel, J. D. Mountz.
1998
. Inhibition of tumor necrosis factor α decreases inflammation and prolongs adenovirus gene expression in lung and liver.
Hum. Gene Ther.
9
:
1875
8
Van Laethem, J. L., A. Marchant, A. Delvaux, M. Goldman, P. Robberecht, T. Velu, J. Deviere.
1995
. Interleukin 10 prevents necrosis in murine experimental acute pancreatitis.
Gastroenterology
108
:
1917
9
Van Laethem, J. L., R. Eskinazi, H. Louis, F. Rickaert, P. Robberecht, J. Deviere.
1998
. Multisystemic production of interleukin 10 limits the severity of acute pancreatitis in mice.
Gut
43
:
408
10
Deviere, J., O. Le Moine, J. L. Van Laethem, P. Eisendrath, A. Ghilain, N. Severs, M. Cohard.
2001
. Interleukin 10 reduces the incidence of pancreatitis after therapeutic endoscopic retrograde cholangiopancreatography.
Gastroenterology
120
:
498
11
Jansen, M. J., T. Hendriks, B. M. de Man, J. W. van der Meer, R. J. Goris.
1999
. Interleukin 10 mitigates the development of the zymosan-induced multiple organ dysfunction syndrome in mice.
Cytokine
11
:
713
12
Smith, R. C., K. N. Wills, D. Antelman, H. Perlman, L. N. Truong, K. Krasinski, K. Walsh.
1997
. Adenoviral constructs encoding phosphorylation-competent full-length and truncated forms of the human retinoblastoma protein inhibit myocyte proliferation and neointima formation.
Circulation
96
:
1899
13
Graham, F. L., L. Prevec.
1995
. Methods for construction of adenovirus vectors.
Mol. Biotechnol.
3
:
207
14
Vieira, P., R. de Waal Malefyt, M. N. Dang, K. E. Johnson, R. Kastelein, D. F. Fiorentino, J. E. deVries, M. G. Roncarolo, T. R. Mosmann, K. W. Moore.
1991
. Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: homology to Epstein-Barr virus open reading frame BCRFI.
Proc. Natl. Acad. Sci. USA
88
:
1172
15
Minter, R. M., M. A. Ferry, J. E. Rectenwald, F. R. Bahjat, A. Oberholzer, C. Oberholzer, D. La Face, V. Tsai, I. Ahmed, E. M. Copeland, L. L. Moldawer.
2001
. Extended lung expression and increased tissue localization of viral IL-10 with adenoviral gene therapy.
Proc. Natl Acad. Sci. USA
98
:
277
16
Goris, R. J., W. K. Boekholtz, B. I. van, J. K. Nuytinck, P. H. Schillings.
1986
. Multiple-organ failure and sepsis without bacteria: an experimental model.
Arch. Surg.
121
:
897
17
Steinberg, S., W. Flynn, K. Kelley, L. Bitzer, P. Sharma, C. Gutierrez, J. Baxter, D. Lalka, A. Sands, J. van Liew, et al
1989
. Development of a bacteria- independent model of the multiple organ failure syndrome.
Arch. Surg.
124
:
1390
18
Lombardi, B., L. W. Estes, D. S. Longnecker.
1975
. Acute hemorrhagic pancreatitis (massive necrosis) with fat necrosis induced in mice by dl-ethionine fed with a choline-deficient diet.
Am. J. Pathol.
79
:
465
19
Niederau, C., R. Luthen, M. C. Niederau, J. H. Grendell, L. D. Ferrell.
1992
. Acute experimental hemorrhagic-necrotizing pancreatitis induced by feeding a choline-deficient, ethionine-supplemented diet: methodology and standards.
Eur. Surg Res.
24
: (Suppl. 1):
40
20
Espevik, T., J. Nissen Meyer.
1986
. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes.
J. Immunol. Methods
95
:
99
21
Crystal, R. G., N. G. McElvaney, M. A. Rosenfeld, C. S. Chu, A. Mastrangeli, J. G. Hay, S. L. Brody, H. A. Jaffe, N. T. Eissa, C. Danel.
1994
. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis.
Nat. Genet.
8
:
42
22
Worgall, S., G. Wolff, E. Falck-Pedersen, R. G. Crystal.
1997
. Innate immune mechanisms dominate elimination of adenoviral vectors following in vivo administration.
Hum. Gene Ther.
8
:
37
23
Weber, M., S. Deng, T. Kucher, A. Shaked, R. J. Ketchum, K. L. Brayman.
1997
. Adenoviral transfection of isolated pancreatic islets: a study of programmed cell death (apoptosis) and islet function.
J. Surg. Res.
69
:
23
24
Zuckerman, J. B., C. B. Robinson, K. S. McCoy, R. Shell, T. J. Sferra, N. Chirmule, S. A. Magosin, K. J. Propert, E. C. Brown-Parr, J. V. Hughes, et al
1999
. A phase I study of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fibrosis.
Hum. Gene Ther.
10
:
2973
25
Knowles, M. R., K. W. Hohneker, Z. Zhou, J. C. Olsen, T. L. Noah, P. C. Hu, M. W. Leigh, J. F. Engelhardt, L. J. Edwards, K. R. Jones.
1995
. A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis.
N. Engl. J. Med.
333
:
823
26
Hollon, T..
2000
. Researchers and regulators reflect on first gene therapy death.
Nat. Med.
6
:
6
27
Marshall, E..
1999
. Gene therapy death prompts review of adenovirus vector.
Science
286
:
2244
28
Kusske, A. M., A. J. Rongione, S. W. Ashley, D. W. McFadden, H. A. Reber.
1996
. Interleukin-10 prevents death in lethal necrotizing pancreatitis in mice.
Surgery
120
:
284
29
Norman, J., W. Denham, D. Denham, J. Yang, G. Carter, A. Abouhamze, C. L. Tannahill, S. L. D. MacKay, L. L. Moldawer.
2000
. Liposome mediated nonviral gene transfer induces a systemic inflammatory response which can exacerbate pre-existing inflammation.
Gene Ther.
7
:
1425
30
Ferrer, T. J., J. W. Webb, B. H. Wallace, C. D. Bridges, H. E. Palmer, R. D. Robertson, J. B. Cone.
1998
. Interleukin-10 reduces morbidity and mortality in murine multiple organ dysfunction syndrome (MODS).
J. Surg. Res.
77
:
157
31
Vieira, P., R. de Waal-Malefyt, M. Dang, K. E. Johnson, R. Kastelein, D. F. Fiorentino, J. E. deVries, M. E. Roncarolo, T. R. Mosmann, K. W. Moore.
1991
. Isolation and expression of human cytokine synthesis inhibitory factor (CSIF/IL-10) cDNA clones: homology to Epstein-Barr virus open reading frame BCRFI.
Proc. Natl. Acad. Sci. USA
88
:
1172
32
Rousset, F., E. Garcia, T. Defrance, C. Peronne, N. Vezzio, D. H. Hsu, R. Kastelein, K. W. Moore, J. Banchereau.
1992
. Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes.
Proc. Natl. Acad. Sci. USA
89
:
1890
33
Thompson-Snipes, L., V. Dhar, M. W. Bond, T. R. Mosmann, K. W. Moore, D. M. Rennick.
1991
. Interleukin 10: a novel stimulatory factor for mast cells and their progenitors.
J. Exp. Med.
173
:
507
34
MacNeil, I. A., T. Suda, K. W. Moore, T. R. Mosmann, A. Zlotnik.
1990
. IL-10, a novel growth cofactor for mature and immature T cells.
J. Immunol.
145
:
4167
35
Ding, Y., L. Qin, S. V. Kotenko, S. Pestka, J. S. Bromberg.
2000
. A single amino acid determines the immunostimulatory activity of interleukin 10.
J. Exp. Med.
191
:
213