A phase I clinical study of naked DNA expressing two isoforms of hepatocyte growth factor to treat patients with critical limb ischemia.

BACKGROUND: The purpose of the present phase I clinical trial was to evaluate the safety, tolerability, and preliminary efficacy of naked DNA therapy expressing two isoforms of hepatocyte growth factor (pCK-HGF-X7) in critical limb ischemia (CLI) patients. MATERIALS AND METHODS: Twenty-one patients with CLI were consecutively assigned to receive increasing doses (cohort I: 4 mg; cohort II: 8 mg; cohort III: 12 mg; and cohort IV: 16 mg) of pCK-HGF-X7, which was administered into the ischemic calf and/or thigh muscle at days 1 and 15. A safety and tolerability evaluation and measurement of pain severity score using a visual analog scale (VAS), ulcer status, transcutaneous oxygen (TcPO(2) ) and ankle-brachial index (ABI) were performed throughout a 3-month follow-up period. RESULTS: No serious adverse events were observed in any of the 21 patients for the 3-month follow-up period. A significant reduction in pain was observed in the treated patients, with the mean VAS decreasing from 5.95-1.64 (p < 0.001). The mean ABI value increased from 0.49-0.63 (p = 0.026) at 3-month follow-up. The mean TcPO(2) value on the dorsum of the foot, the anterior calf and posterior calf significantly increased from 28.25-39.28 mmHg (p = 0.012), from 22.00-30.63 mmHg (p = 0.046) and 32.05-47.19 mmHg (p = 0.001) at 3-month follow-up, respectively. Wound healing improvement was observed in the six of nine patients that had an ulcer at baseline. CONCLUSIONS: These results support the performance of a phase II randomized controlled trial with pCK-HGF-X7.

Enhanced cardioprotective effects by coexpression of two isoforms of hepatocyte growth factor from naked plasmid DNA in a rat ischemic heart disease model.

BACKGROUND: The therapeutic potential of pCK-HGF-X7, a naked DNA designed to express two isoforms of hepatocyte growth factor (HGF(723) and HGF(728) ), was studied in the rat ischemic heart disease model. METHODS: First, the kinetics of gene expression was examined by injecting pCK-HGF-X7 DNA into the rat heart. Second, the cardioprotective effects were compared between the two naked DNA constructs, expressing a single (HGF(728) ) or both isoforms (HGF(728) and HGF(723) ) of HGF, in the rat ischemic heart disease model. The ischemic injury to the rat heart was created by ischemia-reperfusion in the anterior descending artery. The respective naked DNA constructs were injected into the anterior wall of the rat heart with the ischemia-reperfusion injury. Cardiac function, capillary density and anti-fibrotic activity were compared between the two naked DNA constructs. RESULTS: The intramyocardial administration of pCK-HGF-X7 resulted in transient and localized HGF expression for 3 weeks. At its peak, approximately 678 pg (per mg of tissue protein) of HGF was produced in the injected heart without an increase of HGF protein level in other tissues, and serum. pCK-HGF-X7 more efficiently improved the left ventricular ejection fraction and the systolic anterior wall thickness, increased the capillary density, and inhibited myocardial fibrosis, in a statistically significant manner, compared to the identical vector encoding HGF(728) only. CONCLUSIONS: These results demonstrate that transfer of the genomic-cDNA hybrid expressing both isoforms of the HGF gene might provide higher therapeutic effects than the cDNA sequence producing HGF(728) alone in the treatment of ischemic heart disease.

Human hepatocyte growth factor (VM202) gene therapy via transendocardial injection in a pig model of chronic myocardial ischemia.

BACKGROUND: Hepatocyte growth factor (HGF) may stimulate angiogenesis. We examined the safety and therapeutic potential of the HGF plasmid (VM202) in pigs with chronic myocardial ischemia. METHODS AND RESULTS: We delivered VM202 or vehicle transendocardially to 4 groups of pigs: vehicle control (n = 9); high-dose VM202 (n = 9); low-dose VM202 (n = 3); and normal control (no ischemia; n = 1). Pigs were killed 3, 30, and 60 days after injection. No adverse events were associated with VM202 treatment or delivery. Quantitative polymerase chain reaction indicated that heart injection sites had the highest levels of VM202 (day 3), which became almost undetectable by 30-60 days. Most nontarget tissues showed clearance of VM202 plasmid by day 30. Control and VM202-treated pigs did not differ in global functional data. Dobutamine-stressed myocardial-contrast echocardiogram suggested that VM202 may help preserve microvascular perfusion at 30 days; reperfusion velocity in ischemic myocardium decreased significantly in control (baseline to follow-up, 5.1 ± 1.9 to 2.7 ± 1.0; P = .031) but not in VM202 groups (high-dose: 3.1 ± 1.1 vs 3.1 ± 1.5 [P = .511]; low-dose: 3.8 ± 1.1 vs 3.9 ± 1.5 [P = .559]). Linear local shortening increased significantly from day 0 to 30 in VM202-treated versus control pigs (5.0 ± 4.7% vs 9.2 ± 7.5% vs 0.9 ± 5.8% [high-dose, low-dose, control, respectively]; P = .021). CONCLUSIONS: Transendocardial delivery of VM202 was safe and may help to preserve microcirculatory perfusion and improve wall motion.

Therapeutic angiogenesis using naked DNA expressing two isoforms of the hepatocyte growth factor in a porcine acute myocardial infarction model.

OBJECTIVE: We evaluated the potency of therapeutic angiogenesis using intramyocardial injection of naked DNA expressing two isoforms of hepatocyte growth factor (pCK-HGF-X7) in a porcine myocardial infarction model. METHODS: Four weeks after left anterior descending coronary artery ligation, 14 pigs were allocated to pCK-Null (negative control, n=7) or pCK-HGF-X7 (n=7) treatment groups. Gated myocardial single photon emission computed tomography was performed 4 and 8 weeks following coronary ligation. The effect of pCK-HGF-X7 on capillary density in the gene-injected myocardium was examined by histological analysis using alkaline phosphatase staining. RESULTS: Segmental myocardial perfusion of the underperfused area (< or =70%) from coronary ligation increased in the pCK-HGF-X7 group (p=0.051), without significant differences in changes over time between the two groups (p=0.54). Systolic wall thickening (p=0.001), left ventricular end-diastolic (p=0.045) and end-systolic volumes (p=0.009), and left ventricular ejection fraction (p=0.041) changed in both groups without significant differences in changes over time between the two groups. The increase in the left stoke volume was higher in the pCK-HGF-X7 group than in the pCK-Null group (p=0.008). Histological analysis showed that capillary density was significantly higher in the pCK-HGF-X7 group than the pCK-Null group (p<0.001). CONCLUSION: Intramyocardial injection of pCK-HGF-X7 induced significant angiogenesis at infarct-border zone, and increased the left ventricular stroke volume probably caused by reverse remodeling process.

Effects of IL-1beta on gene expression in human rheumatoid synovial fibroblasts.

IL-1 is one of the key mediators involved in the pathogenesis of rheumatoid arthritis (RA) and is known to affect the level of gene expression in various settings. We investigated the effects of IL-1beta on the expression of 240 genes in rheumatoid synovial fibroblasts (RSFs) using a cDNA microarray. Total RNAs were prepared from RSFs stimulated with IL-1beta and hybridized to the microarray. The fluorescence intensity of each gene was compared between the control and IL-1beta-treated cells. To confirm the data obtained from the microarray analysis, the level of gene expression was also examined by ELISA, Northern blot, or Western blot depending on the genes to be analyzed. The genes whose levels were significantly changed by IL-1beta in the microarray analysis could be divided into three categories; inflammatory mediators, matrix-modifying enzymes, and apoptosis-associated molecules. The increase in the mRNA levels of IL-6, IL-8, MCP-1, and GRO-1 was confirmed by determining their protein levels from the cell culture supernatant using ELISA. The increase in the level of two matrix-degrading enzymes, MMP-1 and MMP-3, was reproducibly observed by an ELISA method, while the decrease in the level of TIMP-3, an inhibitor of MMPs, was confirmed by Northern blot analysis. The fluorescence intensity of two apoptosis-related genes, caspase-3 and Bcl-xL, was significantly lowered. The decreased protein level of caspase-3 was also found. Our data suggested that IL-1beta could provoke a series of responses in RSFs leading to the pathologic status of RA, including enhancement of inflammatory cytokines, imbalanced production of MMPs and TIMPs, and dysregulation of apoptosis.

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.

Protection against collagen-induced arthritis by electrotransfer of an expression plasmid for the interleukin-4.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, leading to cartilage and bone destruction. We investigated whether the electrotransfer of IL-4 DNA could regulate the disease progress of murine collagen-induced arthritis (CIA). The maximum serum level of mIL-4 was measured by 340 pg/ml on day 1 following DNA transfer. The onset of severe CIA and the degree of synovitis and cartilage erosion were significantly reduced in mice treated with IL-4 DNA (P<0.05). The beneficial effect of IL-4 gene transfer lasted for at least 17 days subsequent to treatment. The expression of IL-1beta was considerably decreased in the paws by IL-4 DNA transfer (P<0.01). On the contrary, the ratio of TIMP2 to MMP2 significantly increased in the IL-4 DNA-treated group (P<0.01). These data demonstrated that electroporation-mediated gene transfer could provide a new approach as an IL-4 therapy for autoimmune arthritis.

Angiostatin gene transfer as an effective treatment strategy in murine collagen-induced arthritis.

OBJECTIVE: To determine the efficacy of local therapy with human angiostatin gene in murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were immunized with bovine type II collagen. Before the onset of arthritis, NIH3T3 fibroblasts, transduced with angiostatin-expressing retroviral vectors or control vectors, were transplanted into the knee cavity. The incidence of arthritis in the knee joints was evaluated histologically based on pannus formation and cartilage destruction. Paws were evaluated macroscopically for redness, swelling, and deformities and immunologically for levels of interleukin-1 beta. Angiogenesis in paws and knee joints was studied by immunohistochemistry using anti-CD31 antibody and measurement of von Willebrand factor levels. RESULTS: Pannus formation and cartilage erosion were dramatically reduced in knees transplanted with angiostatin-expressing cells. In addition, the onset of CIA in the ipsilateral paws below the knees injected with the angiostatin gene was significantly prevented. Furthermore, angiostatin gene transfer inhibited arthritis-associated angiogenesis. CONCLUSION: Local production of angiostatin in the knee was able to prevent the onset of CIA not only in the knee injected with genetically engineered cells, but also in the uninjected ipsilateral paw. This suggests that transfer of the angiostatin gene, and potentially also its protein, may provide a new, effective approach to the treatment of rheumatoid arthritis.

CNS Lesions in Tuberculous Meningitis

No abstract available.

Foreign Body in the Airway: A review of 71 patients

Seventy one children aged 3 months and 8 months with a foreign body in the airway were analysed. Most of the patients were less than 3 years of age, and male to female ratio 2.6:1. Most foreign bodies were food materials (58.2%), metallic bodies (20.9%) and plastic materials(13.4%). Of all the foreign bodies, 61 cases were lodged in the main bronchi and 3 cases were lodged in the larynx or trachea. Common symptoms and signs were cough, dyspnea, mild pyrexia, recurrent respiratory tract infection, decreased air entry, cyanosis and rales. The most common roentgenographic finding was obstructive emphysema (61.2%). A positive history of foreign body aspiration was obtained in 77.5% of the patients and it was found that carelessness of some sort is responsible in almost all the cases. Of the foreign bodies removed, 84.6% were done bronchoscopy. Direct laryngoscopy was performed in 4.6% of patients. Complications were involved in 19.7% of the cases with bronchoscopy. Three patients died of septic shock, asphyxia and hypoxic brain damage respectively.

Foreign Body in the Airway: A review of 71 patients

Seventy one children aged 3 months and 8 months with a foreign body in the airway were analysed. Most of the patients were less than 3 years of age, and male to female ratio 2.6:1. Most foreign bodies were food materials (58.2%), metallic bodies (20.9%) and plastic materials(13.4%). Of all the foreign bodies, 61 cases were lodged in the main bronchi and 3 cases were lodged in the larynx or trachea. Common symptoms and signs were cough, dyspnea, mild pyrexia, recurrent respiratory tract infection, decreased air entry, cyanosis and rales. The most common roentgenographic finding was obstructive emphysema (61.2%). A positive history of foreign body aspiration was obtained in 77.5% of the patients and it was found that carelessness of some sort is responsible in almost all the cases. Of the foreign bodies removed, 84.6% were done bronchoscopy. Direct laryngoscopy was performed in 4.6% of patients. Complications were involved in 19.7% of the cases with bronchoscopy. Three patients died of septic shock, asphyxia and hypoxic brain damage respectively.

Tubercul in Skin Test of Preschool Aged Children

The tuberculin skin test is an important aid in the detection of tuberculosis especially in child-centered approach to tuberculosis control. The Monotest and the Mantoux test were performed and B.C.G. vaccination coverage rate was assessed. A total of 139 kindergarten children in Seoul and 89 public nursery children in rural area were subjected to the tests. Each child was incoculated simultaneously to one side arm with purified protein derivative(PPD, 5u) and to another aide arm with monovacc(Institut Merieus). Results from each test were read separately and independently 72 hours after administration. At the same time B.C.G vaccination scar size was measured. B.C.G. vaccination coverage rate was 20.9% in urban area, and 15.7% in rural area. When induration of 2mm or greater was considered a positive Monotest reaction and compared to a positive Mantoux reaction of 10mm or greater, the sensitivity of Monotest to Mantoux test was 100% and the specificity was 89.29%. When induration of 5mm or greater was considered a positive Monotest reaction and compared to a positive Mantoux reaction of 10mm or greater, the sensitivity of Monotest to Mantoux test was 81.25% and the specificity was 98.9%. From these results I would assume that the Monotest can be substituted for Mantoux test as screening test, especially in preschool aged children who are apt to be fearful of needles used in the Mantoux test, and rather impatient. As screening test, 2mm or greater induration of Monotest is considered to be preferable to 5mm or greater as a positive result. All children with doubtful or positive reactions to a Monotest should be retested using the Nantoux technique because of relative highfalse positive rate.

Monotest Compared to Standard PPD, 5TU, Mantoux Test

The Monotest was compared with the Mantoux test. A total of 404 primary school children were subjected to test. Each child was innoculated simultaneously to one side arm with purified protein derivative (PPD, 5TU) and to the another side arm with Monovacc(Institut Merieux). Results from each test were read seperately 72 hours after administration. When induration of 2mm or greater was considered a positive Monotest reaction and compared to a positive Mantoux reaction of 10mm or greater, the sensitivity of Monotest to Mantoux test was 91.56% and the specificity was 71.85%. When induration of 5mm or greater was considered a positive Monotest reaction and compared to a positive Mantoux reaction of 10mm or greater, the sensitivity of Monotest to Mantoux test was 42.77% and the specificity was 94.53%. From these results I would assume that the Monotest can be substituted for Mantoux test (PPD, 5TU) as screening test, preferably 2mm or greater induration of Monotest is considered as a positive result. All children with doubtful or positive reactions to a Monotest should be retested using the Mantoux technique because of relative high false positive rate.

Ketogenic Diet Trial for The Treatment of Minor Motor Seizures

A 3-years-old girl, diagnosed as infantile spasm and 5-years-old boy, diagnosed as akinetic seizure were subjected to ketogenic diet trial because of their poor responses anticonyulsant treatment After adding ketogenic diet trial, in the former infantile spasm case, her seizures were controlled satisfactorily and in the latter akinetic seizure case, the frequency of seizures decreasd fairly. It seemed to be beneficial to add kotogenic diet the anticonvulsant therapy for the treatment of minor motor seizure children. We report these results with some discussion and a brief review of related literature