Association between antiphospholipid antibodies and arterial thrombosis in patients with rheumatoid arthritis.

OBJECTIVES: Antiphospholipid antibodies (aPL) are present in a proportion of patients with rheumatoid arthritis but their clinical significance remains unclear. We investigated the association between aPL and thrombotic events in rheumatoid arthritis patients. METHODS: In this cross-sectional study, aPL profiles were evaluated in 376 rheumatoid arthritis patients in accordance with the standard guidelines. Clinical and radiographic data were retrospectively collected. RESULTS: aPL were identified in 39 patients (10.4%). Lupus anticoagulant was the most common subtype (n = 25, 6.6%); anti-cardiolipin antibodies and anti-ß2 glycoprotein I antibodies were detected in six and 12 patients (1.6% and 3.2%), respectively. Compared to the aPL-negative group, aPL-positive patients included more male patients (41.0% vs. 15.4%, P < 0.001) and more smokers (41.0% vs. 16.0%, P = 0.001). There was no difference between the two groups in age, disease duration and body mass index, or the frequency of diabetes, hypertension or dyslipidaemia. Of note, arterial thromboses were more common in the aPL-positive than the aPL-negative group (12.8% vs. 2.1%, P = 0.004), whereas the frequency of venous thrombosis did not differ between the two groups (0.0% vs. 0.9%, P = 1.000). On multivariate regression analysis, aPL, age, hypertension, dyslipidaemia and baseline C-reactive protein level were independently associated with arterial thrombotic events (all P values < 0.05). CONCLUSION: aPL was found in a subset of rheumatoid arthritis patients, who were more often smokers, and aPL was independently associated with development of arterial thrombosis. This result suggests that aPL may contribute to an increased risk of arterial thrombosis in rheumatoid arthritis patients.

Metabolic Syndrome Is not an Independent Risk Factor for Hearing Impairment.

OBJECTIVE: We aimed to investigate the association between metabolic syndrome (MS) and hearing impairment (HI) using nationally representative data from Korean adults. DESIGN, SETTING AND PARTICIPANTS: A total of 16,799 subjects (≥19 years old; 7,170 men and 9,629 women) who underwent pure tone audiometry testing were included in the analysis. Data were obtained from the fifth Korea National Health and Nutrition Examination Survey (2010-2012). Subjects were divided into two groups according to the presence of MS. RESULTS: Among the subjects with MS, 47% had HI. Logistic regression analysis revealed that MS was not an independent risk factor for HI, although increased fasting plasma glucose (OR 1·4, 95% CI: 1·1-1·8) was independently associated with HI. In addition, older age, male sex, very low body mass index (≤17·5 kg/m2), lower education level, smoking history, and occupational noise exposure were independently associated with HI. For low-frequency HI, independent risk factors included older age, lower educational level, lower economic status, and very low BMI (≤17·5 kg/m2). For high-frequency HI, independent risk factors included older age, male sex, lower educational level, lower economic status, increased blood pressure, lower high-density lipoprotein cholesterol, and smoking history. CONCLUSIONS: MS itself was not an independent risk factor for HI, and, among the individual metabolic components, only increased fasting plasma glucose was independently associated with HI.

Prediction of subclinical atherosclerosis by serum osteoprotegerin in premenopausal women with systemic lupus erythematous: correlation of osteoprotegerin with monocyte chemotactic protein-1.

OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have increased risk for cardiovascular disease. Previous studies disclosed the association of serum osteoprotegerin (OPG) with the presence of symptomatic atherosclerosis in the general population and several disease conditions. We thus investigated the association between serum OPG levels and subclinical atherosclerosis in premenopausal SLE patients. METHODS: Serum OPG levels and carotid artery intima-media thickness (IMT) were measured in 181 premenopausal SLE patients and age-matched 85 control subjects. Traditional cardiovascular risk factors and SLE-related factors were analyzed. RESULTS: Patients with SLE had significantly increased serum OPG levels (1086 versus 517 pg/ml, p < 0.001) and carotid IMT (0.63 versus 0.45 mm, p < 0.001) compared with control subjects. Carotid IMT significantly increased across the quartiles of OPG. Logistic regression analysis revealed that compared to the lowest OPG quartile, the odds ratio (OR, 95% confidence interval) for increased carotid IMT in quartile 2, 3, and 4 was 1.126 (1.013-1.801), 1.562 (1.268-2.799), and 4.460 (1.126-7.128), respectively, after multiple adjustments (p for trend across quartiles < 0.001). These associations remained significant after further adjustment for inflammatory parameters. Interestingly, serum monocyte chemotactic protein-1 (MCP-1) levels were positively correlated with serum OPG levels (γ = 0.332, p < 0.001). Parallel analysis showed that serum MCP-1 was also an independent predictor of carotid IMT incrassation, but this association was lost when serum OPG was included in the model. CONCLUSION: Serum OPG levels were increased and correlated with serum MCP-1 levels in premenopausal SLE patients. Increased serum OPG was independently associated with subclinical atherosclerosis in these patients.

Adsorption behaviors of recombinant E-cadherin-IgG Fc fusion protein on polystyrene surface.

Adsorption behaviors of recombinant E-cadherin-IgG Fc (E-cad-Fc) fusion protein and mutated E-cad-Fcs on the polystyrene (PS) surface were investigated using a 27 MHz quartz-crystal microbalance (QCM) and ELISA. The amount of adsorbed E-cad-Fc on PS surface was increased with an increase of E-cad-Fc concentration as a Langmuir-type in a monolayer. Adsorbed E-cad-Fc on PS surface was stable even after washing if calcium ions are absent in the washing solution due to the calcium ion dependence in the adsorption. E-cadherin homophilic adhesion among E-cadherins during adsorption of E-cad-Fc was involved. Deglycosylation of the E-cad in the E-cad-Fc did not affect adsorption of E-cad-Fc on the PS surface although deglycosylation of the E-cad in the E-cad-Fc enhanced cell adhesion compared with E-cad-Fc.

Diffuse alveolar hemorrhage in systemic lupus erythematosus: risk factors and clinical outcome: results from affiliated hospitals of Catholic University of Korea.

This study was undertaken to investigate clinical characteristics of diffuse alveolar hemorrhage (DAH) in patients with systemic lupus erythematosus (SLE) and to determine risk factors and clinical outcomes of DAH in SLE patients. Among the 1521 patients with SLE admitted between January 1993 and June 2009 to affiliated hospitals of Catholic University of Korea, 21 SLE were admitted for DAH. The inclusion criteria for DAH was defined as new infiltrates on chest radiographs, an acute hemoglobin drop of at least 1.5 g/dl in the absence of an obvious source of bleeding, and one or more of the following signs: hemoptysis, hypoxemia, bronchoscopic or biopsy evidence of DAH. Included as disease controls were 83 SLE patients, matched for age and sex, who were admitted for other manifestations. Data based on medical records were analyzed retrospectively. There were no significantly differing demographic characteristics between SLE patients with DAH and those with other manifestations. Multivariate analysis demonstrated coexisting neuropsychiatric lupus (p = 0.002) and high SLE disease activity index scores (SLEDAI > 10) as independent risk factors in the development of DAH (p = 0.029). Among the 21 SLE patients with DAH, 13 died during the admission period (in-hospital mortality rate: 61.9%). Mortality was associated with infection and requirements of mechanical ventilation. Collectively, SLE patients who have neuropsychiatric manifestations or are in the active stage of the disease have an increased risk for developing DAH. Due to the high mortality of SLE patients with DAH, early recognition of risk factors and appropriate intervention is essential.

Effect of oestrogen on T cell apoptosis in patients with systemic lupus erythematosus.

Defective control of T cell apoptosis is considered to be one of the pathogenetic mechanisms in systemic lupus erythematosus (SLE). Oestrogen has been known to predispose women to SLE and also to exacerbate activity of SLE; however, the role of oestrogen in the apoptosis of SLE T cells has not yet been documented. In this study, we investigated the direct effect of oestrogen on the activation-induced cell death of T cells in SLE patients. The results demonstrated that oestradiol decreased the apoptosis of SLE T cells stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in a dose-dependent manner. In addition, oestradiol down-regulated the expression of Fas ligand (FasL) in activated SLE T cells at the both protein and mRNA levels. In contrast, testosterone increased FasL expression dose-dependently in SLE T cells stimulated with PMA plus ionomycin. The inhibitory effect of oestradiol on FasL expression was mediated through binding to its receptor, as co-treatment of tamoxifen, an oestrogen receptor inhibitor, completely nullified the oestradiol-induced decrease in FasL mRNA expression. Moreover, pre-treatment of FasL-transfected L5178Y cells with either oestradiol or anti-FasL antibody inhibited significantly the apoptosis of Fas-sensitive Hela cells when two types of cells were co-cultured. These data suggest that oestrogen inhibits activation-induced apoptosis of SLE T cells by down-regulating the expression of FasL. Oestrogen inhibition of T cell apoptosis may allow for the persistence of autoreactive T cells, thereby exhibiting the detrimental action of oestrogen on SLE activity.

Suppression of tumor growth in xenograft model mice by programmed cell death 4 gene delivery using folate-PEG-baculovirus.

Cancer gene therapy using tumor suppressor genes is considered to be an attractive approach for arresting cell growth and inducing apoptosis. Programmed cell death 4 (Pdcd4) is a tumor suppressor gene, which prevents tumorigenesis and tumor progression. To address the issue of whether expression of PDCD4 protein induces apoptosis in cancerous cells, the Pdcd4 gene was delivered using folate-PEG-baculovirus. Folate-PEG-baculovirus containing Pdcd4 gene (F-P-Bac-Pdcd4) was constructed by attachment of F-PEG to the baculovirus surface using chemical modification. The F-P-Bac-Pdcd4 showed enhanced transduction efficiency, efficiently expressed PDCD4 protein, and induced apoptosis in human epidermal carcinoma (KB) cells as compared with an unmodified baculovirus. In a tumor xenograft study, injection of F-P-Bac-Pdcd4 into tumors established from the KB cell line by subcutaneous implantation significantly suppressed tumor growth and induced apoptosis. Thus, this study shows a new baculovirus-mediated tumor suppressor gene delivery system for cancer therapy.

Circulating osteoprotegerin levels are elevated and correlated with antiphospholipid antibodies in patients with systemic lupus erythematosus.

Patients with antiphospholipid syndrome (APS) have an increased risk for the development of thrombotic complications. Recent studies indicate that osteoprotegerin (OPG) acts as an important molecule in the development of vascular diseases. The aim of the present study was to examine the association between serum OPG levels and APS manifestations in patients with SLE. Seventy-nine patients with SLE and ninety-two healthy controls, matched for age and sex, were included in this study. Serum levels of OPG, monocyte chemoattractant protein(MCP)-1 and soluble E-selectin were determined by ELISA. At the time of serum sampling, various clinical and laboratory parameters were assessed. We found that serum levels of OPG were significantly higher in patients with SLE than in healthy controls (1236 +/- 82 vs 967 +/- 37 pg/mL, P = 0.003). Particularly, serum OPG levels were significantly higher in SLE patients with APS than those without (1615 +/- 191 vs 1171 +/- 91 pg/mL, P = 0.006). Serum OPG levels correlated with titres of IgG anti-cardiolipin antibody (P = 0.026) and anti-beta(2)-glycoprotein I antibody (P < 0.001). Moreover, serum OPG also correlated with serum levels of sE-selectin (P = 0.002), which is an endothelial cell activation marker, and MCP-1 (P = 0.003), a well known chemokine implicated in thrombogenesis. Collectively, serum OPG levels were increased in SLE patients with APS and correlated with titres of antiphospholipid antibodies, suggesting that OPG might be linked to the development of APS.

Mesenchymal stem cells overexpressing interleukin-10 attenuate collagen-induced arthritis in mice.

Mesenchymal stem cells (MSCs) have the inherent ability to migrate to multiple organs and to exert immunosuppressive activity. The aim of this study was to investigate the anti-arthritogenic effects of interleukin (IL)-10-transduced MSCs (IL-10-MSC) on the development of inflammatory arthritis. DBA/1 mice were immunized with type II collagen (CII) to induce inflammatory arthritis and then injected weekly three times with IL-10-MSCs 21 days after primary immunization. Control mice received vehicle or MSCs alone. Serum anti-CII antibody and T cell response to CII were determined. The results showed that cultured IL-10-MSCs were able to secrete high amounts of IL-10 in vitro. Injection of IL-10-MSCs decreased the severity of arthritis significantly. However, there was no difference in arthritis severity between mice treated with MSC and vehicle alone. Anti-CII antibody titres in the sera and T cell proliferative response to CII in lymph node cells were decreased significantly in mice treated with IL-10-MSCs compared with vehicle-treated mice. Serum IL-6 level was also decreased by the administration of IL-10-MSCs. In contrast, spleen cells of IL-10-MSC-treated mice produced higher amounts of IL-4 than those of control mice. Interestingly, although not as potent as IL-10-MSCs, injection of naive MSCs alone decreased serum levels of IL-6 and anti-CII antibody, while increasing IL-4 production from cultured splenic cells. Taken together, systemic administration of genetically modified MSCs overexpressing IL-10 inhibits experimental arthritis not only by suppressing autoimmune response to CII but also by regulating cytokine production, and thus would be a new strategy for treating rheumatoid arthritis.

Up-regulated macrophage migration inhibitory factor protects apoptosis of dermal fibroblasts in patients with systemic sclerosis.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has been demonstrated to regulate the apoptosis of several cell types. Dysregulated apoptosis of fibroblasts has been implicated in a variety of fibrotic diseases, including systemic sclerosis (SSc). In this study, we investigated the role of MIF in the apoptosis of dermal fibroblasts. The concentrations of MIF were measured in sera and in culture supernatants of peripheral blood mononuclear cells (PBMCs) and dermal fibroblasts by enzyme-linked immunosorbent assay. The degree of apoptosis was determined by colorimetric assay, and signalling pathways were examined by Western blot. The results showed that serum levels of MIF were significantly higher in patients with SSc (n = 47) than in healthy controls (n = 56). Stimulation of PBMCs by anti-CD3 and anti-CD28 increased the production of MIF by fourfold over the constitutive levels. SSc dermal fibroblasts produced higher amounts of MIF than normal dermal fibroblasts. When treated with sodium nitroprusside (SNP), SSc dermal fibroblasts showed a lower degree of apoptosis compared with normal dermal fibroblasts. Exogenous MIF (1-100 ng/ml) inhibited SNP-induced apoptosis of dermal fibroblasts dose-dependently. Both extracellular regulated kinase (ERK) inhibitor (PD98059) and protein kinase B (Akt) inhibitor (LY294002) almost completely blocked the inhibitory effect of MIF on apoptosis. Furthermore, MIF increased the expression of Bcl-2, phospho-ERK and phospho-Akt activity in dermal fibroblasts. Taken together, our data suggest that MIF released by activated T cells and dermal fibroblasts decreases the apoptosis of dermal fibroblasts through activation of ERK, Akt and Bcl-2 signalling pathways, which might be associated with excessive fibrosis in SSc.

Urocanic acid-modified chitosan-mediated PTEN delivery via aerosol suppressed lung tumorigenesis in K-ras(LA1) mice.

The low efficiency of conventional therapies in achieving long-term survival of lung cancer patients calls for development of novel options. Revisiting of aerosol gene delivery may provide an alternative for safe and effective treatment for lung cancer. In this study, imidazole ring-containing urocanic acid-modified chitosan (UAC) designed in the previous study was used as a gene carrier. The potential effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) on Akt-related signals and cell cycle regulation were evaluated. Aerosols of UAC-PTEN were delivered into K-ras(LA1) lung cancer model mice through the nose-only inhalation system twice a week for total 4 weeks. Delivered PTEN suppressed lung tumor development significantly through nuclear complex formation between PTEN and p53, suppressing Akt-related signals as well as cell cycle regulation. Together, our results suggest that aerosol delivery of UAC-PTEN may be compatible with noninvasive in vivo gene therapy.

Aerosol-delivered programmed cell death 4 enhanced apoptosis, controlled cell cycle and suppressed AP-1 activity in the lungs of AP-1 luciferase reporter mice.

The long-term survival of lung cancer patients treated with conventional therapies remains poor and therefore the need for novel approaches remains high. This has led to the re-emergence of aerosol delivery as a therapeutic intervention. In this study, glucosylated polyethylenimine (GPEI) was used as carrier to investigate programmed cell death 4 (PDCD4) and PDCD4 mutant (D418A), an eIF4A-binding mutant, on PDCD4-related signaling and activator protein-1 (AP-1) activity in the lungs of AP-1 luciferase reporter mice. After confirming the efficiency of GPEI as a carrier in lungs, the effects of aerosol-delivered PDCD4 were investigated in AP-1 luciferase reporter mice. Aerosol delivery of GPEI/PDCD4 through a nose-only inhalation facilitated the apoptosis of lungs whereas aerosol PDCD4 mutant did not. Also, such aerosol delivery regulated proteins relevant to cell-cycle control and suppressed AP-1 activity. Results obtained by western blot analysis, immunohistochemistry, luciferase assay and deoxynucleotidyl-transferase-mediated nick end labeling study suggest that combined actions such as facilitating apoptosis, controlling cell cycle and suppression of AP-1 activity by PDCD4 may provide useful tool for designing lung tumor prevention and treatment by which PDCD4 functions as a transformation suppressor in the future.

Effect of interleukin-4 on vascular endothelial growth factor production in rheumatoid synovial fibroblasts.

Interleukin (IL)-4 has been demonstrated to have anti-inflammatory and anti-tumour activity. Because aberrant angiogenesis is a significant pathogenic component of tumour growth and chronic inflammation, we investigated the effect of IL-4 on the production of vascular endothelial growth factor (VEGF) by synovial fibroblasts derived from patients with rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLS) were prepared from synovial tissues of RA and incubated with different concentrations of IL-4 in the presence or absence of transforming growth factor (TGF)-beta. VEGF level was measured by enzyme-linked immunosorbent assay and semiquantitative reverse transcription--polymerase chain reaction. Treatment of FLS with IL-4 alone caused a dose-dependent increase in VEGF levels. In contrast, IL-4 exhibited the inhibitory effect on VEGF production when FLS were stimulated with TGF-beta. Combined treatment of IL-4 and IL-10 inhibited TGF-beta-induced VEGF production in an additive fashion. TGF-beta increased the induction of cyclooxygenase-2 mRNA, which was inhibited significantly by the treatment of IL-4. NS-398, a COX-2 inhibitor, inhibited TGF-beta-induced VEGF production in a dose-dependent manner. Furthermore, exogenous addition of prostaglandin E2 (PGE2) restored IL-4 inhibition on TGF-beta induced VEGF production. Collectively, our results suggest that IL-4 have an anti-angiogenic effect, especially in the inflammatory milieu of RA by inhibiting the VEGF production in synovial fibroblasts.

Aerosol delivery of Akt controls protein translation in the lungs of dual luciferase reporter mice.

Lung cancer has emerged as a leading cause of cancer death in the world; however, most of the current conventional therapies are not sufficiently effective in altering the progression of disease. Therefore, development of novel treatment approaches is needed. Although several genes and methods have been used for cancer gene therapy, a number of problems such as specificity, efficacy and toxicity reduce their application. This has led to re-emergence of aerosol gene delivery as a noninvasive method for lung cancer treatment. In this study, nano-sized glucosylated polyethyleneimine (GPEI) was used as a gene delivery carrier to investigate the effects of Akt wild type (WT) and kinase deficient (KD) on Akt-related signaling pathways and protein translation in the lungs of CMV- LucR-cMyc-IRES-LucF dual reporter mice. These mice are a powerful tool for the discrimination between cap-dependent/-independent protein translation. Aerosols containing self-assembled nano-sized GPEI/Akt WT or GPEI/Akt KD were delivered into the lungs of reporter mice through nose-only-inhalation-chamber with the aid of nebulizer. Aerosol delivery of Akt WT caused the increase of protein expression levels of Akt-related signals, whereas aerosol delivery of Akt KD did not. Furthermore, dual luciferase activity assay showed that aerosol delivery of Akt WT enhanced cap-dependent protein translation, whereas a reduction in cap-dependent protein translation by Akt KD was observed. Our results clearly showed that targeting Akt may be a good strategy for prevention as well as treatment of lung cancer. These studies suggest that our aerosol delivery is compatible for in vivo gene delivery which could be used as a noninvasive gene therapy in the future.

Vascular endothelial growth factor (VEGF) is associated with reduced severity of acute graft-versus-host disease and nonrelapse mortality after allogeneic stem cell transplantation.

This study investigated whether or not there is a correlation between the changes in the serum levels of vascular endothelial growth factor (VEGF) and the outcome of allogeneic stem cell transplantation (allo-SCT). Eighty-five patients undergoing allo-SCT were prospectively studied. The serum VEGF levels were measured on days 0, +7 and +14 after transplantation. The VEGF levels decreased significantly on day +7 and recovered on day +14. The highest levels from day +7 through day +14 were categorized by cluster analysis, which were then correlated with the nonrelapse mortality (NRM). There was a significant correlation between a low VEGF level and the occurrence of severe acute graft-versus-host disease (GVHD) including grade III-IV (P=0.029). The 1-year probability of NRM in patients with a low VEGF level was 22.5% compared with 3.5% for those with a high VEGF level (P=0.024). Multivariate analysis revealed clinically defined infections (P=0.011), advanced disease (P=0.014) and a low VEGF cluster (P=0.05) to be significantly associated with the occurrence of NRM in the cohort. In conclusion, low VEGF levels after allo-SCT are associated with NRM with an exacerbated severity of acute GVHD. VEGF monitoring after a transplant might identify those patients at risk of severe transplant-related mortality.

Hydroxychloroquine potentiates Fas-mediated apoptosis of rheumatoid synoviocytes.

Inadequate apoptosis may contribute to the synovial hyperplasia associated with rheumatoid arthritis (RA). The Fas-associated death domain protein (FADD)-like interleukin (IL)-1beta-converting enzyme (FLICE)-inhibitory protein (FLIP), which is an apoptotic inhibitor, has been implicated in the resistance to Fas-mediated apoptosis of synoviocytes. This study investigated whether hydroxychloroquine (HCQ), an anti-rheumatic drug, induces the apoptosis of rheumatoid synoviocytes, and modulates the expression of FLIP. Fibroblast-like synoviocytes (FLS) were prepared from the synovial tissues of RA patients, and were cultured with various concentrations of HCQ in the presence or absence of the IgM anti-Fas monoclonal antibodies (mAb) (CH11). Treatment with HCQ, ranging from 1 to 100 microM, induced the apoptosis of FLS in a dose- and time-dependent manner. The increase in synoviocytes apoptosis by HCQ was associated with caspase-3 activation. A combined treatment of HCQ and anti-Fas mAb increased FLS apoptosis and caspase-3 activity synergistically, compared with either anti-Fas mAb or HCQ alone. The Fas expression level in the FLS was not increased by the HCQ treatment, while the FLIP mRNA and protein levels were decreased rapidly by the HCQ treatment. Moreover, time kinetics analysis revealed that the decreased expression of FLIP by HCQ preceded the apoptotic event that was triggered by HCQ plus anti-Fas mAb. Taken together, HCQ increases the apoptosis of rheumatoid synoviocytes by activating caspase-3, and also sensitizes rheumatoid synoviocytes to Fas-mediated apoptosis. Our data suggest that HCQ may exert its anti-rheumatic effect in rheumatoid joints through these mechanisms.

Nifedipine encapsulated core-shell type nanoparticles based on poly(gamma-benzyl L-glutamate)/poly(ethylene glycol) diblock copolymers.

The diblock copolymers based on PBLG and PEO (GE) were synthesized and characterized. Nanoparticles showed spherical shape from the observations of TEM and approved core-shell structure. Drug contents were increased with use of higher initial drug concentration and higher Mw of GE. Nifedipine (NFD) release rate was slower in longer PBLG chain length and higher NFD contents than short PBLG chain length and lower drug contents of NFD due to the hydrophobic interaction between PBLG domain and NFD.

Osteonecrosis in patients with systemic lupus erythematosus: MR imaging and scintigraphic evaluation.

This study was to describe the findings of osteonecrosis in patients with SLE at MR and scintigraphic imaging. Among 415 patients with SLE, 37 patients were diagnosed to have osteonecrosis. MR images and bone scintigraphs were analyzed for sites of involvement, signal intensity, bilaterality and multiplicity. MR imaging features of osteonecrosis in patients with SLE included isointense signal intensity relative to adjacent bone marrow, hypointense rim, marginal enhancement and unusual involvement of flat bones. Bilateral and multiple involvements were common.

Visualization of transfection of hepatocytes by galactosylated chitosan-graft-poly(ethylene glycol)/DNA complexes by confocal laser scanning microscopy.

Dual-labeled galactosylated chitosan-graft-poly(ethylene glycol) (PEG) (GCP)/DNA complexes were prepared and their hepatocyte-specific delivery and cellular distribution were investigated by confocal laser scanning microscopy (CLSM). The complexes were transfected into hepatocyte through specific interaction of galactose moiety of the GCP and asialoglycoprotein receptors (ASGPR) of the hepatocytes. The GCP/DNA complexes taken up by the hepatocytes were rapidly released into the cytoplasm, but nuclear trafficking of the released complexes was slow and rate-limiting process. The more efficient transfection of the complex occurred in the human-derived HepG2 cells than in primary hepatocytes.