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1.  Kinesin-5: cross-bridging mechanism to targeted clinical therapy 
Gene  2013;531(2):133-149.
Kinesin motor proteins comprise an ATPase superfamily that goes hand in hand with microtubules in every eukaryote. The mitotic kinesins, by virtue of their potential therapeutic role in cancerous cells, have been a major focus of research for the past 28 years since the discovery of the canonical Kinesin-1 heavy chain. Perhaps the simplest player in mitotic spindle assembly, Kinesin-5 (also known as Kif11, Eg5, or kinesin spindle protein, KSP) is a plus-end-directed motor localized to interpolar spindle microtubules and to the spindle poles. Comprised of a homotetramer complex, its function primarily is to slide anti-parallel microtubules apart from one another. Based on a multi-faceted analysis of this motor from numerous laboratories over the years, we have learned a great deal about the function of this motor at the atomic level for catalysis and as an integrated element of the cytoskeleton. These data have, in turn, informed the function of motile kinesins on the whole, as well as spearheaded integrative models of the mitotic apparatus in particular and regulation of the microtubule cytoskeleton in general. We review what is known about how this nanomotor works, its place inside the cytoskeleton of cells, and its small-molecule inhibitors that provide a toolbox for understanding motor function and for anticancer treatment in the clinic.
doi:10.1016/j.gene.2013.08.004
PMCID: PMC3801170  PMID: 23954229
kinesin; motor protein; phylogeny; evolution; cytoskeletal motor; catalysis; ATP hydrolysis; mitosis; mitotic spindle; microtubule; mitotic motor protein; targeted inhibitor; Eg5; KSP; Kif11; structural biology; ispinesib; monastrol; allosteric inhibition; L5 loop; review
2.  A Rapid and Efficient Method for Construction of an Infectious Clone of Tomato yellow leaf curl virus 
The Plant Pathology Journal  2014;30(3):310-315.
Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, is responsible for one of the most devastating viral diseases in tomato-growing countries and is becoming a serious problem in many subtropical and tropical countries. The climate in Korea is getting warmer and developing subtropical features in response to global warming. These changes are being accompanied by TYLCV, which is now becoming a large problem in the Korean tomato industry. The most effective way to reduce damage caused by TYLCV is to breed resistant varieties of tomatoes. To accomplish this, it is necessary to establish a simple inoculation technique for the efficient evaluation of resistance to TYLCV. Here, we present the rolling circle amplification (RCA) method, which employs a bacteriophage using phi-29 DNA polymerase for construction of infectious TYLCV clones. The RCA method is simple, does not require sequence information for cloning, and is less expensive and time consuming than conventional PCR based-methods. Furthermore, RCA-based construction of an infectious clone can be very useful to other emerging and unknown geminiviruses in Korea.
doi:10.5423/PPJ.NT.03.2014.0025
PMCID: PMC4181116  PMID: 25289018
infectious clone; rolling circle amplification; tomato; Tomato yellow leaf curl virus
3.  Optimizing conditions for E-and Z-ajoene formation from garlic juice using response surface methodology 
Food Science & Nutrition  2014;2(5):605-611.
The optimum conditions for the formation of E- and Z-ajoene from garlic juice mixed with soybean oil were determined using response surface methodology. A central composite design was used to investigate the effects of three independent variables temperature (°C, X1), reaction time (hours, X2), and oil volume (multiplied by weight, X3). The dependent variables were Z-ajoene (Y1) and E-ajoene (Y2) in oil-macerated garlic. The optimal conditions for E- and Z-ajoene using ridge analysis were 98.80°C, 6.87 h, and weight multiplied by weight 2.57, and 42.24°C, 9.71 h, and weight multiplied by weight 3.08, respectively. These conditions resulted in E- and Z-ajoene compound predicted values of 234.17 and 752.62 μg/g from garlic juice, respectively. The experimental values of E- and Z-ajoene were 222.75 and 833.59 μg/g, respectively. The estimated maximum values at the predicted optimum conditions were in good agreement with experimental values.
doi:10.1002/fsn3.148
PMCID: PMC4237492  PMID: 25473520
Ajoene; High-performance liquid chromatography; oil-macerated garlic; response surface methodology
4.  The Association between the Low Muscle Mass and Osteoporosis in Elderly Korean People 
Journal of Korean Medical Science  2014;29(7):995-1000.
The purpose of this study was to predict osteoporosis risk as decreasing muscle mass and to declare the cut-off value of low muscle mass in an elderly Korean population. This study was based on data from the 2008-2010 Korea National Health and Nutritional Examination Surveys (KNHANES). The subjects included 1,308 men and 1,171 women over 65 yr. Bone mineral density (BMD) and appendicular skeletal muscle (ASM) were measured by dual energy X-ray absorptiometry (DXA), and appendicular skeletal muscle was adjusted by height as a marker of sarcopenia. After confirming the correlation between low muscle mass and BMD, the best cut-off value of muscle mass to estimate osteoporosis was suggested through the receiver operating characteristic (ROC) curve. For both men and women, BMD correlated positively with low muscle mass when ASM/Ht2 was used as a marker for sarcopenia. The ROC curve showed that ASM/Ht2 was the best marker for osteoporosis at a cut-off value of 6.85 kg/m2 for men and 5.96 kg/m2 for women. When these cut-off values were used to determine sarcopenia, the risk of osteoporosis increased 4.14 times in men and 1.88 times in women. In particular, men (OR 2.12) with sarcopenia were more greatly affected than women (OR 1.15), even after adjusting for osteoporosis risk factors. In elderly Korean people, sarcopenia is positively correlated with BMD and there is a strong correlation between sarcopenia and osteoporosis with risk of bone fracture.
doi:10.3346/jkms.2014.29.7.995
PMCID: PMC4101790  PMID: 25045234
Osteoporosis; Sarcopenia; Korea; KNHANES; Elderly Population
5.  RNAseq-based Transcriptome Analysis of Burkholderia glumae Quorum Sensing 
The Plant Pathology Journal  2013;29(3):249-259.
Burkholderia glumae causes rice grain rot and sheath rot by producing toxoflavin, the expression of which is regulated by quorum sensing (QS). The QS systems of B. glumae rely on N-octanoyl homoserine lactone, synthesized by TofI and its cognate receptor TofR, to activate the genes for toxoflavin biosynthesis and an IclR-type transcriptional regulator gene, qsmR. To understand genome-wide transcriptional profiling of QS signaling, we employed RNAseq of the wild-type B. glumae BGR1 with QS-defective mutant, BGS2 (BGR1 tofI::Ω) and QS-dependent transcriptional regulator mutant, BGS9 (BGR1 qsmR::Ω). A comparison of gene expression profiling among the wild-type BGR1 and the two mutants before and after QS onset as well as gene ontology (GO) enrichment analysis from differential expressed genes (DEGs) revealed that genes involved in motility were highly enriched in TofI-dependent DEGs, whereas genes for transport and DNA polymerase were highly enriched in QsmR-dependent DEGs. Further, a combination of pathways with these DEGs and phenotype analysis of mutants pointed to a couple of metabolic processes, which are dependent on QS in B. glumae, that were directly or indirectly related with bacterial motility. The consistency of observed bacterial phenotypes with GOs or metabolic pathways in QS-regulated genes implied that integration RNAseq with GO enrichment or pathways would be useful to study bacterial physiology and phenotypes.
doi:10.5423/PPJ.OA.04.2013.0044
PMCID: PMC4174805  PMID: 25288952
Burkholderia glumae; gene ontology enrichment; pathway; quorum sensing; RNAseq
6.  The improvement of quality of life in patients treated with bariatric surgery in Korea 
Purpose
Bariatric surgery is considered an efficient treatment for severe obesity, but postoperative complications and psychosocial problems may impact quality of life (QoL). Although QoL is an important aspect of bariatric surgery, few studies have evaluated the changes in QoL. We examined whether severely obese patients who had undergone bariatric surgery had better QoL compared with severely obese adults who had not undergone bariatric surgery in Korea.
Methods
Data were obtained from 78 participants in two groups; bariatric surgery group (n = 53) and nonsurgery group (n = 25). EuroQoL-5D (EQ-5D), the impact of weight on quality of life-lite (IWQoL-lite) and the obesity-related psychosocial problem scale (OP-scale) were used to assess the improvement of QoL.
Results
A total of 78 patients completed the QoL forms as part of their surgical consultation. In the EQ-5D, the changes of EQ-5D 3 level and EQ-5D visual analogue scale in the surgery group was 0.174 and 24.6 versus 0.017 and 17.8 in the nonsurgery group (P = 0.197 and P = 0.179). The changes of IWQoL-lite and OP-scale were significantly improved after bariatric surgery. In the IWQoL-lite, the mean changes in the surgery group was 33.4 versus 14.3 points in the nonsurgery group (P = 0.000). In the OP-scale, the mean changes in the surgery group patients scored 39.3 versus 9.0 points in the nonsurgery group (P = 0.000).
Conclusion
We demonstrated significant improvement of QoL observed after bariatric surgery compared to nonsurgical procedure. The results of this comparative study favor bariatric surgery for the treatment of severe obesity.
doi:10.4174/jkss.2013.84.3.131
PMCID: PMC3594640  PMID: 23486845
Bariatric surgery; Obesity; Quality of life
7.  Methodological Considerations in Ambulatory Skin Conductance Monitoring 
Little is known how much skin conductance (SC) recordings from the fingers are affected by factors such as electrode site deterioration, ambient temperature (TMP), or physical activity (ACT), or by age, sex, race, or body mass index.
We recorded SC, TMP, and ACT in 48 healthy control subjects for a 24-hour period, and calculated SC level (SCL), its standard deviation, the coefficient of SC variation, and frequency and amplitude of non-specific SC fluctuations. One method of assessing electrode site deterioration showed an average decline of 20 %, while a second method found no significant change. All SC measures were higher during waking than sleep. Other factors influenced different measures in different ways. Thus, 24-hour SC recording outside the laboratory is feasible, but some measures need to be corrected for the influence of confounding variables.
doi:10.1016/j.ijpsycho.2011.02.002
PMCID: PMC3075336  PMID: 21320551
skin conductance; non-specific fluctuations; ambulatory recording; arousal
8.  NSC 622124 Inhibits Human Eg5 and Other Kinesins Via Interaction with the Conserved Microtubule-Binding Site 
Biochemistry  2009;48(8):1754-1762.
Kinesin-5 proteins are essential for formation of a bipolar mitotic spindle in most, and perhaps all, eukaryotic cells. Several Kinesin-5 proteins, notably the human version, HsEg5, are targets of a constantly expanding group of small-molecule inhibitors, which hold promise both as tools to probe mechanochemical transduction and as anti-cancer agents. Although most such compounds are selective for HsEg5 and closely related Kinesin-5 proteins, some, such as NSC 622124, exhibit activity against at least one kinesin from outside the Kinesin-5 family. Here we show NSC 622124, despite identification in a screen that yielded inhibitors now known to target the HsEg5 monastrol-binding site, does not compete with 14C-monastrol for binding to HsEg5, and is able to inhibit the basal and microtubule-stimulated ATPase activity of the monastrol-insensitive Kinesin-5, KLP61F. NSC 622124 competes with microtubules, but not ATP, for interaction with HsEg5, and disrupts the microtubule binding of HsEg5, KLP61F and Kinesin-1. Proteolytic degradation of an HsEg5•NSC622124 complex revealed that segments of the α3 and α5 helices map to the inhibitor-binding site. Overall, our results demonstrate that NSC 622124 targets the conserved microtubule-binding site of kinesin proteins. Further, unlike compounds previously reported to target the kinesin microtubule-binding site, NSC 622124 does not produce any enhancement of basal ATPase activity, and thus acts solely as a negative regulator through interaction with a site traditionally viewed as a binding region for positive regulators (i.e., microtubules). Our work emphasizes the concept that microtubule-dependent motor proteins may be controlled at multiple sites by both positive and negative effectors.
doi:10.1021/bi801291q
PMCID: PMC3244877  PMID: 19236100
9.  Twenty-four hour Skin Conductance in Panic Disorder 
Journal of psychiatric research  2010;44(16):1137-1147.
Skin conductance, physical activity, ambient temperature, and mood were recorded for 24 hours in 22 panic disorder (PD) patients and 29 healthy controls. During the day, subjects performed standardized relaxation tests (ARTs). We hypothesized that tonically elevated anticipatory anxiety in PD during waking and sleeping would appear as elevated skin conductance level (SCL) and greater skin conductance (SC) variability. Mean SCL was higher during both usual waking activities and sleeping in PD, but not during the ARTs. Group SC variability differences did not reach significance, perhaps because of variance unrelated to anxiety. Analyses indicated that in the PD group, antidepressant medication reduced mean SCL whereas state anxiety had the opposite effect during the day. Depressive symptoms reported during the day were related to elevated mean SCL on the night of the recording. The rate and extent of SCL deactivation over the night was equal in the two groups. However, PD patients had more frequent interruptions of deactivation that could have arisen from conditioned arousal in response to threat cues during sleep.
doi:10.1016/j.jpsychires.2010.04.012
PMCID: PMC2937198  PMID: 20537349
Panic Disorder; Anxiety; Skin Conductance; Ambulatory Monitoring; Sleep
10.  p53 Interacts with RNA Polymerase II through Its Core Domain and Impairs Pol II Processivity In Vivo 
PLoS ONE  2011;6(8):e22183.
The tumor suppressor p53 principally functions as a gene-specific transcription factor. p53 triggers a variety of anti-proliferative programs by activating or repressing the transcription of effector genes in response to genotoxic stress. To date, much effort has been placed on understanding p53's ability to affect transcription in the context of its DNA-binding activity. How p53 regulates transcriptional output independent of DNA binding is less well understood. Here we provide evidence that human p53 can physically interact with the large subunit of RNA polymerase II (Pol II) both in in vitro interaction assays and in whole cell extracts, and that this interaction is mediated (at least in part) through p53's core DNA-binding domain and the Ser5-phosphorylated CTD of Pol II. Ectopic expression of p53, combined with mutations in transcription elongation factors or exposure to drugs that inhibit Pol II elongation, elicit sickness or lethality in yeast cells. These phenotypes are suppressed by oncogenic point mutations within p53's core domain. The growth phenotypes raise the possibility that p53 impairs Pol II elongation. Consistent with this, a p53-dependent increase in Pol II density is seen at constitutively expressed genes without a concomitant increase in transcript accumulation. Additionally, p53-expressing yeast strains exhibit reduced transcriptional processivity at an episomal reporter gene; this inhibitory activity is abolished by a core domain point mutation. Our results suggest a novel mechanism by which p53 can regulate gene transcription, and a new biological function for its core domain that is susceptible to inactivation by oncogenic point mutations.
doi:10.1371/journal.pone.0022183
PMCID: PMC3150338  PMID: 21829606
11.  A Comparison of Comorbidity and Psychological Outcomes in Children and Adolescents with Attention-Deficit/Hyperactivity Disorder 
Psychiatry Investigation  2011;8(2):95-101.
Objective
The purpose of this study was to compare psychiatric comorbid disorders and psychological outcomes in children and adolescents with Attention-deficit/hyperactivity disorder (ADHD).
Methods
Subjects were divided into a child group (aged under 12 years) and an adolescent group (aged 12 years and above). All subjects were diagnosed with ADHD based on the DSM IV diagnostic criteria using the Kiddie-Schedule for Affective Disorders and Schizophrenia-Present and Lifetime Korean Version (K-SADS-PL-K). The K-SADS-PL-K was also used to evaluate those psychiatric disorders comorbid with ADHD. And the Korean version of the Child Behavior Checklist (K-CBCL) was used to examine the subjects' psychological outcomes.
Results
The rate of comorbidity in adolescent group was significantly higher than that in the child group. In particular, the adolescent group had a significantly higher ratio of comorbid conduct disorder and mood disorder than the child group. With respect to the predominantly inattentive type and Not Otherwise Specified, the school subscale scores on the K-CBCL for the children were significantly higher than those for the adolescents.
Conclusion
These results suggest that the psychiatric comorbidity may differ between adolescents and children with ADHD. Therefore when treating adolescents with ADHD, more careful assessment and treatment targeting a range of comorbidities are needed.
doi:10.4306/pi.2011.8.2.95
PMCID: PMC3149117  PMID: 21852984
Children; Adolescent; Comorbidity; ADHD
12.  SYMPATHETIC ACTIVATION IN BROADLY DEFINED GENERALIZED ANXIETY DISORDER 
Journal of psychiatric research  2007;42(3):205-212.
The definition of Generalized Anxiety Disorder (GAD) has been narrowed in successive editions of DSM by emphasizing intrusive worry and deemphasizing somatic symptoms of hyperarousal. We tried to determine the clinical characteristics of more broadly defined chronically anxious patients, and whether they would show physiological signs of sympathetic activation. A group whose chief complaint was frequent, unpleasant tension over at least the last six weeks for which they desired treatment, was compared with a group who described themselves as calm. Participants were assessed with structured interviews and questionnaires. Finger skin conductance, motor activity, and ambient temperature were measured for 24 hours. Results show that during waking and in bed at night, runs of continuous minute-by-minute skin conductance level (SCL) declines were skewed towards being shorter in the tense group than in the calm group. In addition, during waking, distributions of minute SCLs were skewed towards higher levels in the tense group, although overall mean SCL did not differ. Thus, the tense group showed a failure to periodically reduce sympathetic tone, presumably a corollary of failure to relax. We conclude that broader GAD criteria include a substantial number of chronically anxious and hyperaroused patients who do not fall within standard criteria. Such patients deserve attention by clinicians and researchers.
doi:10.1016/j.jpsychires.2006.12.003
PMCID: PMC2262283  PMID: 17250853
tension; anxiety; Generalized Anxiety Disorder; sympathetic activation; skin conductance; autonomic nervous system; sleep
13.  Central Pontine Myelinolysis in a Patient with Acute Lymphoblastic Leukemia after Hematopoietic Stem Cell Transplantation: A Case Report 
Journal of Korean Medical Science  2008;23(2):324-327.
We describe a 37-yr-old man who developed central pontine myelinolysis (CPM) after allogeneic hematopoietic stem cell transplantation (HSCT) for acute lymphoblastic leukemia. After HSCT, desquamation developed on the whole body accompanied by hyperbilirubinemia. The liver biopsy of the patient indicated graft-versus-host disease-related liver disease, and the dose of methylprednisolone was increased. Then, the patient developed altered mentality with eye ball deviation to the left, for which electroencephalogram and magnetic resonance imaging (MRI) scans were done. Brain MRI scan demonstrated the imaging findings consistent with central pontine myelinolysis and extrapontine myelinolysis. He did not have any hyponatremia episode during hospitalization prior to the MRI scan. To the best of our knowledge, presentation of CPM after allogeneic HSCT is extremely rare in cases where patients have not exhibited any episodes of significant hyponatremia. We report a rare case in which hepatic dysfunction due to graft-versus-host disease has a strong association with CPM after HSCT.
doi:10.3346/jkms.2008.23.2.324
PMCID: PMC2526450  PMID: 18437020
Myelinolysis, Central Pontine; Hematopoietic Stem Cell Transplantation; Graft versus Host Disease
14.  The Gene yggE Functions in Restoring Physiological Defects of Escherichia coli Cultivated under Oxidative Stress Conditions† 
DNA microarray analysis showed that yfiD, yggB, and yggE genes were up-regulated when superoxide dismutase (SOD)-deficient Escherichia coli IM303 (I4) was cultivated under the oxidative stress generated by photoexcited TiO2, and pYFD, pYGB, and pYGE were constructed by inserting the respective genes into a pUC 19 vector. The content of reactive oxygen species (ROS) in IM303 (I4) cells carrying pYGE was reduced to 31% of ROS content in the control cells with pUC 19. In the culture of wild-type strain, E. coli MM294, in the medium with paraquat (10 μmol/l), maximum specific growth rate of the cells with pYGE was about five times higher than that of the control cells, with a decreased ROS content in the former cells. The introduction of pYGE also suppressed the occurrence of the cells with altered amino acid requirement in the culture of MM294 cells with paraquat.
doi:10.1128/AEM.71.5.2762-2765.2005
PMCID: PMC1087592  PMID: 15870370
15.  A Case of Hypertrophic Osteoarthropathy Associated with Epithelioid Hemangioendothelioma 
Journal of Korean Medical Science  2004;19(3):484-486.
Epithelioid hemangioendothelioma is a rare vascular tumor, which occurs in the lung, liver, bone, and soft tissue. Hypertrophic osteoarthropathy is a syndrome characterized by subperiosteal new bone formation, joint effusion and clubbing, and may be associated with cyanotic heart disease, chronic pulmonary disease, liver disease, and other miscellaneous diseases. The activation of endothelium and platelets has been suggested to be involved in the development of hypertrophic osteoarthropathy. We report a rare case of hypertrophic osteoarthropathy, which developed in association with hepatic epithelioid hemangioendothelioma with pulmonary metastasis. We also discuss the role of vascular endothelial growth factor in its pathogenesis.
doi:10.3346/jkms.2004.19.3.484
PMCID: PMC2816858  PMID: 15201523
Hemangioendothelioma, Epithelioid; Osteoarthropathy, Secondary Hypertrophic; Vascular Endothelial Growth Factor
16.  The 17 Nucleotides Downstream from the env Gene Stop Codon Are Important for Murine Leukemia Virus Packaging 
Journal of Virology  2000;74(18):8775-8780.
We have identified a previously unknown nucleotide sequence important for the packaging of murine leukemia virus. This nucleotide sequence is located downstream from the stop codon of the env gene but does not overlap the polypurine tract. Deletion of 17 bp from this region resulted in a more than 10-fold decrease in viral titer. Consistent with this result, the deletion mutant showed a 20- to 30-fold drop in the amount of virion RNA in the culture supernatant. The total amount of virion protein in the culture supernatant was comparable for the deletion mutant and the parental virus, suggesting that the mutant construct could release the empty viral particles. These results suggested that the packaging signal sequence might be present at the two extreme sites of the viral genome, one in the region around the splice donor sequence downstream from the 5′ long terminal repeat (LTR) and the other immediately upstream from the 3′ LTR. Implications for gene therapy, especially in regard to construction of retroviral vectors and packaging constructs, are discussed.
PMCID: PMC116393  PMID: 10954583
17.  Gene therapy for established murine collagen-induced arthritis by local and systemic adenovirus-mediated delivery of interleukin-4 
Arthritis Research  2000;2(4):293-302.
To determine whether IL-4 is therapeutic in treating established experimental arthritis, a recombinant adenovirus carrying the gene that encodes murine IL-4 (Ad-mIL-4) was used for periarticular injection into the ankle joints into mice with established collagen-induced arthritis (CIA). Periarticular injection of Ad-mIL-4 resulted in a reduction in the severity of arthritis and joint swelling compared with saline- and adenoviral control groups. Local expression of IL-4 also reduced macroscopic signs of joint inflammation and bone erosion. Moreover, injection of Ad-mIL-4 into the hind ankle joints resulted in a decrease in disease severity in the untreated front paws. Systemic delivery of murine IL-4 by intravenous injection of Ad-mIL-4 resulted in a significant reduction in the severity of early-stage arthritis.
Introduction:
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that is characterized by joint inflammation, and progressive cartilage and bone erosion. Recent research has identified certain biologic agents that appear more able than conventional therapies to halt effectively the progression of disease, as well as ameliorate disease symptoms. One potential problem with the use of biologic agents for arthritis therapy is the need for daily or weekly repeat dosing. The transfer of genes directly to the synovial lining can theoretically circumvent the need for repeat dosing and reduce potential systemic side effects [1,2]. However, although many genes have been effective in treating murine CIA if administrated at a time before disease onset, local intra-articular or periarticular gene transfer has not been highly effective in halting the progression of established disease. IL-4, similar to tumor necrosis factor (TNF)-α and IL-1 inhibitors, has been shown be therapeutic for the treatment of murine CIA when administered intravenously as a recombinant protein, either alone or in combination with IL-10. IL-4 can downregulate the production of proinflammatory and T-helper (Th)1-type cytokines by inducing mRNA degradation and upregulating the expression of inhibitors of proinflammatory cytokines such as IL-1 receptor antagonist (IL-1Ra) [3,4]. IL-4 is able to inhibit IL-2 and IFN-γ production by Th1 cells, resulting in suppression of macrophage activation and the production of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNF-α by monocytes and macrophages [4,5,6,7,8,9].
Objective:
In order to examine the therapeutic effects of local and systemic IL-4 expression in established CIA, an adenoviral vector carrying the gene for murine IL-4 (Ad-mIL-4) was generated. The ability of Ad-mIL-4 to treat established CIA was evaluated by local periarticular and systemic intravenous injection of Ad-mIL-4 into mice at various times after disease onset.
Materials and methods:
Male DBA/1 lacJ (H-2q) mice, aged 7-8 weeks, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were immunized intradermally at the base of tail with 100 μ g bovine type II collagen. On day 21 after priming, mice received a boost injection (intradermally) with 100 μ g type II collagen in incomplete adjuvant. For the synchronous onset of arthritis, 40 μ g lipopolysaccharide (Sigma, St Louis, MO, USA) was injected intraperitoneally on day 28. Ad-mIL-4 was injected periarticularly into the hind ankle joints of mice on day 32 or intravenously by tail vein injection on day 29. Disease severity was monitored every other day using an established macroscopic scoring system ranging from 0 to 4: 0, normal; 1, detectable arthritis with erythma; 2, significant swelling and redness; 3, severe swelling and redness from joint to digit; and 4, maximal swelling with ankylosis. The average of macroscopic score was expressed as a cumulative value for all paws, with a maximum possible score of 16 per mouse. Cytokine production by joint tissue or serum were assessed using enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA).
Results:
To examine the therapeutic effects of IL-4 gene transfer in a murine model of arthritis, 5×108 particles of Ad-mIL-4 and enhanced green fluorescent protein (Ad-eGFP) were administered by periarticular injection into the ankle joints of mice with established disease 4 days after lipopolysaccharide injection. All mice had established disease at time of injection. As shown in Figure 1, the severity of arthritis (Fig. 1a), paw thickness (Fig. 1b), and the number of arthritic paws (Fig. 1c) were all significantly reduced in the Ad-mIL-4 group, compared with the saline- and Ad-eGFP-treated groups. Analysis of the bones in the ankle joints of control arthritic mice showed evidence of erosion with an associated monocytic infiltrate around the joint space compared with the Ad-mIL-4-treated and nonarthritic control joints. In addition, injection of the ankle joints in the hind legs resulted in a therapeutic effect in the front paws. A similar contralateral effect has been observed with adenoviral-mediated delivery of viral (v)-IL-10. Interestingly, a high level of murine IL-10 also was detected from the joint lysates of Ad-mIL-4-treated naïve and arthritic mice, with the production of endogenous IL-10 correlating with the dose of Ad-mIL-4. The administration of recombinant IL-4 protein systemically has been shown to be therapeutic in murine CIA models if given before disease onset. To examine the effect of systemic IL-4 delivered by gene transfer, 1×109 particles of Ad-mIL-4 were injected via the tail vein of collagen-immunized mice the day after lipopolysaccharide injection. Whereas the immunized control mice, injected with Ad-eGFP, showed disease onset on day 3 after lipopolysaccharide injection, Ad-mIL-4-treated mice showed a delay in disease onset and as a reduction in the total number of arthritic paws. Also, systemic injection of Ad-mIL-4 suppressed the severity of arthritis in CIA mice according to arthritis index.
Discussion:
Gene therapy represents a novel approach for delivery of therapeutic agents to joints in order to treat the pathologies associated with RA and osteoarthritis, as well as other disorders of the joints. In the present study we examined the ability of local periarticular and systemic gene transfer of IL-4 to treat established and early-stage murine CIA, respectively. We have demonstrated that both local and systemic administration of Ad-mIL-4 resulted in a reduction in the severity of arthritis, as well as in the number of arthritic paws. In addition, the local gene transfer of IL-4 reduced histologic signs of inflammation and of bone erosion. Interestingly, local delivery of Ad-mIL-4 was able to confer a therapeutic effect to the untreated, front paws through a currently unknown mechanism. In addition, both local and systemic expression of IL-4 resulted in an increase in the level of endogenous IL-10, as well as of IL-1Ra (data not shown). Previous experiments have shown that gene transfer of IL-10 and IL-1 and TNF inhibitors at the time of disease initiation (day 28) is therapeutic. However, delivery of these agents after disease onset appeared to have only limited therapeutic effect. In contrast, the present results demonstrate that IL-4, resulting from local periarticular and systemic injection of Ad-mIL-4, was able partially to reverse progression of established and early-stage disease, respectively. These results, as well as those of others, support the potential application of IL-4 gene therapy for the clinical treatment of RA.
PMCID: PMC17812  PMID: 11056670
adenoviral vectors; collagen-induced arthritis; gene therapy; IL-4; IL-10; rheumatoid arthritis
18.  Construction of Retroviral Vectors with Improved Safety, Gene Expression, and Versatility 
Journal of Virology  1998;72(2):994-1004.
Murine leukemia virus (MLV)-based retroviral vectors are the most frequently used gene delivery vehicles. However, the current vectors are still not fully optimized for gene expression and viral titer, and many genetic and biochemical features of MLV-based vectors are poorly understood. We have previously reported that the retroviral vector MFG, where the gene of interest is expressed as a spliced mRNA, is superior in the level of gene expression with respect to other vectors compared in the study. As one approach to developing improved retroviral vectors, we have systematically performed mutational analysis of the MFG retroviral vector. We demonstrated that the entire gag coding sequence, together with the immediate upstream region, could be deleted without significantly affecting viral packaging or gene expression. To our knowledge, this region is included in all currently available retroviral vectors. In addition, almost the entire U3 region could be replaced with the heterologous human cytomegalovirus immediately-early promoter without deleterious effects. We could also insert internal ribosome entry sites (IRES) and multicloning sites into MFG without adverse effects. Based on these observations, we have constructed a series of new, improved retroviral constructs. These vectors produced viral titers comparable to MFG, expressed high levels of gene expression, and stably transferred genes to the target cells. Our vectors are more convenient to use because of the presence of multicloning sites and IRESs, and they are also more versatile because they can be readily converted to various applications. Our results have general implications regarding the design and development of improved retroviral vectors for gene therapy.
PMCID: PMC124570  PMID: 9444992

Results 1-18 (18)