Intrasplenic electro-transfer of IL-4 encoding plasmid DNA efficiently inhibits rat experimental allergic encephalomyelitis

Most of the previous studies in which cytokine DNA plasmids were delivered by systemic administration exhibited only marginal therapeutic effects, if any, in the EAE model. One strategy to overcome this limitation would be to determine the optimal delivery route leading to significant beneficial effects both in early (prophylactic) and late (therapeutic) treatments. To address this issue, we directly compared the effects of intrasplenic (i.s.) and intramuscular (i.m.) electro-transfer of interleukin-4 (IL-4) DNA in the rat experimental allergic encephalomyelitis (EAE) model. In the preventive experiment, rats received i.m. (25 or 150 microg) or i.s. (25 microg) administration of IL-4 DNA followed by in vivo electroporation the day before MBP immunization. In the late treatment experiment, rats were treated with i.m. (150 microg) or i.s. (25 microg) administration of IL-4 DNA with electroporation 10 days after MBP immunization. As a control the same amount of vector DNA was used. Macroscopic analysis indicated that the onset of moderate to severe EAE in rats treated with i.s. transfer of 25 microg of IL-4 DNA was prevented on a significant level compared with i.m. 25 microg of the IL-4 DNA transfer group or the control group in the preventive experiments. More importantly, i.s. transfer of 25 microg of IL-4 DNA considerably suppressed the severity of EAE in late treatment experiments while i.m. transfer of 150 microg of IL-4 DNA had little effect. The MBP-specific expression of IFN-gamma from stimulated splenocytes was considerably decreased by the i.s. IL-4 DNA transfer group both in the preventive and therapeutic experiments while i.m. transfer had this effect only in the preventive protocol. Histological analysis showed that spinal cord inflammation was considerably reduced in the i.s. IL-4 DNA transfer group. These data provide the first demonstration that i.s. electro-transfer of IL-4 DNA is more effective both in the prevention and modulation of EAE than i.m. transfer and that i.s. electro-gene transfer may present a new approach to cytokine therapy in autoimmune diseases.     

Electro-transfer of small interfering RNA ameliorated arthritis in rats

RNA interference provides the powerful means of sequence-specific gene silencing. Particularly, small interfering RNA (siRNA) duplexes may be potentially useful for therapeutic molecular targeting of human diseases, although novel delivery systems should be devised to achieve efficient and organ-specific transduction of siRNA. In the present study, we demonstrated that electro-transfer of a siRNA-polyamine complex made efficient and specific gene knockdown possible in the articular synovium. Targeted suppression of the tumor necrosis factor-alpha gene through this procedure significantly ameliorated collagen-induced arthritis in rats. Our results suggest the potential feasibility of therapeutic intervention with RNA medicines for treatment of rheumatoid and other locomotor diseases.     

CTRP3 plays an important role in the development of collagen-induced arthritis in mice

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease exhibited most commonly in joints. We found that the expression of C1qtnf3, which encodes C1q/TNF-related protein 3 (CTRP3), was highly increased in two mouse RA models with different etiology. To elucidate the pathogenic roles of CTRP3 in the development of arthritis, we generated C1qtnf3^−/− mice and examined the development of collagen-induced arthritis in these mice. We found that the incidence and severity score was higher in C1qtnf3^−/− mice compared with wild-type (WT) mice. Histopathology of the joints was also more severe in C1qtnf3^−/− mice. The levels of antibodies against type II collagen and pro-inflammatory cytokine mRNAs in C1qtnf3^−/− mice were higher than WT mice. These observations indicate that CTRP3 plays an important role in the development of autoimmune arthritis, suggesting CTRP3 as a possible medicine to treat RA.     

Syngeneic fibroblasts transfected with a plasmid encoding interleukin-4 as non-viral vectors for anti-inflammatory gene therapy in collagen-induced arthritis

Background

No effective long‐term treatment is available for rheumatoid arthritis. Recent advances in gene therapy and cell therapy have demonstrated efficiency in collagen‐induced arthritis (CIA). Interleukin‐4 (IL‐4) is already known to be efficient in CIA in systemic injection or administered by gene therapy. This study was designed to evaluate the effect of a non‐viral gene therapy of CIA, involving injection of syngeneic fibroblasts transfected with a plasmid encoding for IL‐4.

Methods

Immortalised fibroblasts from DBA/1 mice (DBA/1/0 cells) were transfected with a plasmid expressing IL‐4 cDNA (DBA/1/IL‐4 cells). Xenogeneic fibroblasts from Chinese hamster ovary (CHO) transfected with a plasmid expressing IL‐4 cDNA (CHO/IL‐4) were studied also. The cells were engrafted in mice developing CIA by subcutaneous injection of 3 × 10⁶ DBA/1/0 or DBA/1/IL‐4 or CHO/IL‐4 cells.

Results

Injection of DBA/1/IL‐4 cells, on days 10 and 25 after immunisation, was associated with a significant and lasting improvement in the clinical and histological evidence of joint inflammation and destruction as compared with DBA/1/0 and CHO/IL‐4 cells. DBA/1/IL‐4 cell treatment decreased also the production of IgG2a antibody to CII and the proliferation of CIIB‐specific nodal T cells. Later treatments (engraftments on days 23 and 35 after immunisation) exerted also an anti‐inflammatory effect, as evaluated on clinical and histological signs of CIA.

Conclusions

Taken together, these findings indicate that systemic administration of syngeneic cells transfected with an anti‐inflammatory cytokine gene, namely IL‐4, with a non‐viral method is effective in CIA and may attenuate the cytokine imbalance seen in this disease. Copyright © 2002 John Wiley & Sons, Ltd.

     

Site-targeted non-viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice

The pancreas is considered an important gene therapy target because the organ is the site of several high burden diseases, including diabetes mellitus, cystic fibrosis, and pancreatic cancer. We aimed to develop an efficient in vivo gene delivery system using non-viral DNA. Direct intra-parenchymal injection of a solution containing circular plasmid pmaxGFP DNA was performed on adult anesthetized ICR female mice. The injection site was sandwiched with a pair of tweezer-type electrode disks, and electroporated using a square-pulse generator. Green fluorescent protein (GFP) expression within the injected pancreatic portion was observed one day after gene delivery. GFP expression reduced to baseline within a week of transfection. Application of voltages over 40 V resulted in tissue damage during electroporation. We demonstrate that electroporation is effective for safe and efficient transfection of pancreatic cells. This novel gene delivery method to the pancreatic parenchyma may find application in gene therapy strategies for pancreatic diseases and in investigation of specific gene function in situ.     

胶原性关节炎大鼠炎性细胞因子的动态观察

目的通过对胶原性关节炎(CIA)大鼠多个细胞因子的动态观察,了解其变化规律,为阐明RA发病机制及CIA大鼠的模型研究提供实验依据。方法建立CIA大鼠模型。分别于初次免疫后第0、10、20、30、40、50、60天记录大鼠的体重,测量大鼠后足体积和足垫厚度,计算关节炎指数;HE染色光镜下观察关节滑膜病理变化;采用ELISA方法检测大鼠血清中TNF-α、IL-1β和IL-6水平变化。结果造模成功后的CIA大鼠14 d左右出现炎症反应,30 d左右达高峰,以后炎症逐渐减轻。与同期空白对照组大鼠相比,CIA大鼠血清中TNF-α水平10 d明显升高(P<0.01),IL-1β和IL-6水平20 d明显升高(P<0.05),三者水平均30 d达高峰(P<0.01),以后持续存在,但水平有所降低。结论 CIA大鼠血清中TNF-α、IL-1β和IL-6在RA的整个病程中一直发挥着重要作用。     

In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage

BACKGROUND: Understanding the mechanisms underlying gene electrotransfer muscle damage can help to design more effective gene electrotransfer strategies for physiological and therapeutical applications. The present study investigates the factors involved in gene electrotransfer associated muscle damage. METHODS: Histochemical analyses were used to determine the extent of transfection efficiency and muscle damage in the Tibialis anterior muscles of Sprague-Dawley male rats after gene electrotransfer. RESULTS: Five days after gene electrotransfer, features of muscle degeneration and regeneration were consistently observed, thus limiting the extent of transfection efficiency. Signs of muscle degeneration/regeneration were no longer evident 21 days after gene electrotransfer except for the presence of central myonuclei. Neither the application of electrical pulses per se nor the extracellular presence of plasmid DNA per se contributed significantly to muscle damage (2.9 +/- 1.0 and 2.1 +/- 0.7% of the whole muscle cross-sectional area, respectively). Gene electrotransfer of a plasmid DNA, which does not support gene expression, increased significantly muscle damage (8.7 +/- 1.2%). When plasmid DNA expression was permitted (gene electrotransfer of pCMV-beta-galactosidase), muscle damage was further increased to 19.7 +/- 4.5%. Optimization of cumulated pulse duration and current intensity dramatically reduced gene electrotransfer associated muscle damage. Finally, mathematical modeling of gene electrotransfer associated muscle damage as a function of the number of electrons delivered to the tissue indicated that pulse length critically determined the extent of muscle damage. CONCLUSION: Our data suggest that neither the extracellular presence of plasmid DNA per se nor the application of electric pulses per se contributes significantly to muscle damage. Gene electrotransfer associated muscle damage mainly arises from the intracellular presence and expression of plasmid DNA.     

Adenovirus-mediated gene transfer of adiponectin reduces the severity of collagen-induced arthritis in mice

Adiponectin (APN) is a hormone released by adipose tissue with anti-inflammatory properties. The purpose of this study was to examine the therapeutic effects of systemic delivery of APN in murine arthritis model. Collagen-induced arthritis (CIA) was induced in male DBA1/J mice, and adenoviral vectors encoding human APN (Ad-APN) or beta-galactosidase (Ad-β-gal) as control were injected either before or during arthritis progression. Systemic APN delivery at both time points significantly decreased clinical disease activity scores of CIA. In addition, APN treatment before arthritis progression significantly decreased histological scores of inflammation and cartilage damage, bone erosion, and mRNA levels of pro-inflammatory cytokines in the joints, without altering serum anti-collagen antibodies levels. Immunohistochemical staining showed significant inhibition of complement C1q and C3 deposition in the joints of Ad-APN infected CIA mice. These results provide novel evidence that systemic APN delivery prevents inflammation and joint destruction in murine arthritis model.     

Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis

BACKGROUND: It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL-1Ra for the prevention of murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were injected in gastrocnemius muscles with plasmid DNA followed by in vivo electroporation. To uncover the optimum conditions of gene transfer, various electric field strengths and different amounts of plasmid DNA were applied. Calf muscles around the injected areas were investigated with histological methods for damage to muscle tissue. The levels of human IL-1Ra expression in the injected area and also in the serum were determined with ELISA for human IL-1Ra. Based on these data, the effects of electrotransfer of plasmid DNA were tested using the murine CIA model. DBA/1 mice were immunized with bovine collagen type II at the base of the tail. On day 21, mice were given a booster injection with the same antigen. Mice were divided into two groups on day 26. One group of mice received plasmid containing the IL-1Ra cDNA sequence, while control mice were given plasmid lacking the IL-1Ra coding sequence. The incidence of arthritis was evaluated by macroscopic analysis, histological analysis, and the levels of inflammatory cytokines. RESULTS: IL-1Ra expression increased as a function of the electrical field strength and the amount of DNA. 200 V/cm (eight pulses; 20 ms per pulse; 1 Hz) and 15 microg of plasmid DNA per mouse were found to be optimum for gene transfer. After in vivo electroporation, gene expression in both muscle and serum increased gradually, reaching a peak value on day 10. Significant levels of human IL-1Ra expression were maintained for 20 days. Macroscopic analysis showed that the onset of CIA was significantly inhibited by direct electrotransfer of plasmid DNA encoding human IL-1Ra. Histological analysis of knee joints showed that the incidence of arthritis in knee joints was also prevented. The levels of mouse IL-1beta and IL-12 in paws were significantly lower in the group treated with IL-1Ra than those in the control group. CONCLUSIONS: These results demonstrate that direct electrotransfer of plasmid containing the human IL-1Ra cDNA sequence to skeletal muscle can reduce the incidence of CIA in mice.     

TNFR-Fc fusion protein expressed by in vivo electroporation improves survival rates and myocardial injury in coxsackievirus induced murine myocarditis

Tumor necrosis factor-alpha (TNF-alpha) is one of the major cytokines that modulate the immune response in viral myocarditis, but its role has not yet been thoroughly evaluated. We antagonized TNF-alpha using the expressed soluble p75 TNF receptor linked to the Fc portion of the human IgG1 gene (sTNFR:Fc) by in vivo electroporation, and evaluated its effects on experimental coxsackieviral B3 (CVB3) myocarditis. A plasmid DNA encoding sTNFR:Fc (15microg/mouse) was injected into the gastrocnemius muscles of Balb/C male mice followed by electroporation (day -1). Control mice were injected with an empty vector. One day after electroporation, mice were infected with CVB3 (day 0). Serum levels of sTNFR:Fc increased from day 2 and peaked at day 5 following electroporation. The heart virus titers of sTNFR:Fc mice were higher than those of controls at day 3. However, subsequent to day 12, the survival rates of the sTNFR:Fc mice were significantly higher than those of the controls (36% versus 0% at day 27, P<0.01). Histopathological examination indicated that inflammation and myocardial fibrosis were significantly decreased in sTNFR:Fc mice at day 12. The expressed sTNFR:Fc could modulate the inflammatory process during the post-viremic phase of viral myocarditis.     

Protection against cartilage and bone destruction by systemic interleukin-4 treatment in established murine type II collagen-induced arthritis

Destruction of cartilage and bone are hallmarks of human rheumatoid arthritis (RA), and controlling these erosive processes is the most challenging objective in the treatment of RA. Systemic interleukin-4 treatment of established murine collagen-induced arthritis suppressed disease activity and protected against cartilage and bone destruction. Reduced cartilage pathology was confirmed by both decreased serum cartilage oligomeric matrix protein (COMP) and histological examination. In addition, radiological analysis revealed that bone destruction was also partially prevented. Improved suppression of joint swelling was achieved when interleukin-4 treatment was combined with low-dose prednisolone treatment. Interestingly, synergistic reduction of both serum COMP and inflammatory parameters was noted when low-dose interleukin-4 was combined with prednisolone. Systemic treatment with interleukin-4 appeared to be a protective therapy for cartilage and bone in arthritis, and in combination with prednisolone at low dosages may offer an alternative therapy in RA.     

Cholecystokinin octapeptide significantly suppresses collagen-induced arthritis in mice by inhibiting Th17 polarization primed by dendritic cells

Cholecystokinin octapeptide (CCK-8) is a neuropeptide, and is shown to be a potent immunomodulator with predominant anti-inflammatory effects. Although the regulatory effect of CCK-8 on macrophages and B cells has been defined, the effect of CCK-8 on dendritic cells (DCs) and T cells is not well understood. In this study, we showed that CCK-8 reduced the expression of CD80, CD86, and MHCII on DCs. Moreover, CCK-8 promoted Th1 and inhibited Th17 polarization by increasing the production of IL-12 and decreasing the production of IL-6 and IL-23 on DCs in vitro and in vivo. In addition, intraperitoneal administration of CCK-8 to mice with collagen-induced arthritis (CIA) was found to effectively reduce the incidence of arthritis, delay its onset and prevent the occurrence of joint damage. Collectively, these results suggest that CCK-8 significantly suppresses the incidence and severity of CIA in mice, through the inhibition of DC mediated Th17 polarization.     

Inhibition of established subcutaneous and metastatic murine tumors by intramuscular electroporation of the interleukin-12 gene

In vivo electroporation (EP) of the murine interleukin-12 (IL-12) gene in an expression plasmid (pIL-12) was evaluated for antitumor activity. EP transfer of pIL-12 into mouse quadriceps muscles elicited significant levels of serum IL-12 and interferon-gamma. Intramuscular EP of pIL-12 resulted in complete regression or substantial inhibition of 38C13 B-cell lymphoma, whereas pIL-12 delivered by gene gun or intramuscular injection without EP showed little therapeutic effect. Impressive antitumor activity by intramuscular EP was also demonstrated in animals with advanced malignant disease. At day 14 after 38C13 tumor inoculation, all animals were found to carry large tumors and to have metastases; without treatment, most died within a week. A single intramuscular EP of pIL-12 resulted in regression of 50% of large subcutaneous tumors and significantly prolonged the lifespan of these animals. Moreover, animals that were previously cured of 38C13 tumors by in vivo EP treatment significantly suppressed tumor growth when challenged 60 days later. In vivo EP of the IL-12 gene was also effective in suppressing subcutaneous and lung metastatic tumors of CT-26 colon adenocarcinoma and B16F1 melanoma cells. Together, these results show that intramuscular electrotransfer of the IL-12 gene may represent a simple and effective strategy for cancer treatment.     

间歇性低压低氧通过促进细胞凋亡抗大鼠胶原诱导性关节炎

本文旨在研究慢性间歇性低压低氧(chronic intermittent hypobaric hypoxia,CIHH)对大鼠胶原诱导性关节炎(collagen-induced arthritis,CIA)影响.雄性成年Sprague-Dawley大鼠50只,随机分为5组:CIHH预处理组(Pre-T)、预处理对照组(...     

Liver- and lobe-selective gene transfection following the instillation of plasmid DNA to the liver surface in mice

The present study has undertaken the liver- and lobe-selective gene transfections following the instillation of plasmid DNA (pDNA) to the liver surface in mice. The luciferase levels produced in the applied (left) liver lobe at 6 h after liver surface instillation of pDNA were significantly higher than those produced in the other tissues assayed, and ranged from 8.5-fold higher in other liver lobes to 320-fold higher in other tissues. After small intestine surface instillation of pDNA, the gene expression was a little detected in the tissues assayed. Following liver surface instillation of pDNA at a time from 2 to 48 h or at a volume from 15 to 120 microl, the gene expressions of the applied liver lobe were always significantly higher than those of other liver lobes and other tissues. We demonstrated the novel liver- and lobe-selective gene transfection utilizing the instillation to the liver surface.     

Co-expression of IL-18 binding protein and IL-4 regulates Th1/Th2 cytokine response in murine collagen-induced arthritis

We constructed a recombinant adenoviral vector containing a murine interleukin （IL）-18 binding protein （mlL-18BP） and murine IL-4 （mIL-4） fusion gene （AdmIL-18BP/mIL.4） and used a gene therapy approach to investigate the role of IL-18BP and IL-4 in modulating the T-helperl and T-helper2 （Th1/Th2） balance in mice with collagen-induced arthritis （CIA）. Mice with CIA were intra-articularly injected with 107 pfu/6 μl ofeitherAdmIL.18BP/mIL-4, or a controladenovirus, or with the control vehicle （phosphate-buffered saline）. After intra-articular gene therapy with AdmIL-18BP/mIL-4, the serum levels of tumor necrosis factor-α （TNF-α）, T-interferon （IFN-γ）, IL-4, IL-10, and IL-18 in mice with CIA were assessed by ELISA. IFN-T-expressing and IL-4-expressing CD4^＋ T cells from mice splenocytes were monitored by flow cytometry. Mice with CIA at weeks 1, 2, and 4 after intraarticular injection of AdmIL-18BP/mIL-4 showed significantly increased serum concentrations of IL-4 and IL-10 （P〈0.01 at all time points） but greatly decreased serum concentrations ofIFN-γ, TNF-α and IL-β （P〈0.01 at all time points ） compared to both the con trol adenovirus and phospha tebuffered saline control groups. The percentage of LFN-γ- producing CD4^＋ T cells was significantly decreased in response to local AdmIL-18BP/mIL-4 treatment. The percentage of IL-4-producing CD4^＋ T cells increased significantly at 1 week after local injection of AdmIL-18BP/ mIL-4 then returned to normal by week 4. These data indicated the significant modifying effects on the Th1/Th2 imbalance in murine CIA produced by local overexpression of IL-18BP and IL-4. Combination treatment with IL-18BP and IL-4 is a promising potential therapy for rheumatoid arthritis.     

The production of two Th2 cytokines, interleukin-4 and interleukin-10, is controlled independently by locus Cypr1 and by loci Cypr2 and Cypr3, respectively

 The strains BALB/cHeA (BALB/c) and STS/A (STS) differ in production of IL-4 and IL-10, two Th2 cytokines, after stimulation of spleen cells with Concanavalin A, STS being a low and BALB/c a high producer. We analyzed the genetic basis of this strain difference using the recombinant congenic (RC) strains of the BALB/c-c-STS/Dem (CcS/Dem) series. This series comprises 20 homozygous strains. Each CcS/Dem strain contains a different, random set of approximately 12.5% genes of the "donor" strain STS and approximately 87.5% of the "background" strain BALB/c. We selected for further analysis the RC strain production intermediate between BALB/c and STS. In (CcS-20×BALB/c)F₂ hybrids we found that different loci control expression of IL-4 and IL-10. Cypr1 (cytokine production 1) on chromosome 16 near D16Mit15 controls IL-4 production, whereas the production of IL-10 is influenced by loci Cypr2 near D1Mit14 and D1Mit227 on chromosome 1 and Cypr3 marked by D5Mit20 on chromosome 5. In addition, the relationship between the level of these two cytokines depends on the genotype of the F₂ hybrids at a locus cora1 (correlation 1) on chromosome 5. This differential genetic regulation may be relevant for the understanding of biological effects of T-helper cells in mice of different genotypes. Received: 2 March 1998 / Revised: 8 June 1998