Transforming growth factor ß1 T868C gene polymorphism is associated with cerebral infarction in Japanese patients with type 2 diabetes.

It is likely that the C allele of the polymorphism at position 29 of the translated sequence of transforming growth factor (TGF)-ß1 gene, which codes a pleiotropic cytokine expressed in a variety of cells, is a susceptibility allele for cerebral infarction in Japanese type 2 diabetic patients.

Aldose reductase C-106T gene polymorphism is associated with diabetic retinopathy in Japanese patients with type 2 diabetes.

It is likely that the C allele of the polymorphism at position -106 in the promoter of aldose reductase gene, which codes a rate-limiting enzyme of the polyol pathway, is a susceptibility allele for diabetic retinopathy in Japanese type 2 diabetic patients.

Combined effect of oxidative stress-related gene polymorphisms on atherosclerosis.

It is well known that oxidative stress plays critical roles in the pathogenesis of atherosclerosis. In this study, we enrolled 1746 type 2 diabetic subjects, determined 4 common genetic variants related to oxidative stress (glutamate-cysteine ligase modifier subunit (GCLM) C-588T, myeloperoxidase G-463A, human paraoxonase 1 Gln192Arg and NAD(P)H oxidase p22phox C242T polymorphisms), and measured carotid intima-media thickness (IMT) as a surrogate marker for atherosclerosis. GCLM C-588T polymorphism was associated with average IMT (AveIMT) (r=0.090, p=0.0008), but the association between the other 3 polymorphisms and AveIMT did not reach the statistical significance. However, AveIMT was significantly greater as the total number of 4 concomitant "pro-oxidant alleles" in each subject was increased (r=0.108, p<0.0001). Furthermore, the number of "pro-oxidant alleles" was a risk factor for a high AveIMT independently of conventional risk factors (p=0.0003). In conclusion, accumulation of oxidative stress-associated alleles was associated with carotid atherosclerosis in type 2 diabetic patients.

Metabolism and interaction of bisphenol A in human hepatic cytochrome P450 and steroidogenic CYP17.

The metabolism of bisphenol A (BPA) was determined for 11 forms of human hepatic cytochromes P450 (CYPs) expressed in the yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli. Additionally, the effect of BPA on the progesterone 17alpha-chydroxylase activity of CYP17 was investigated. CYP2C18 catalyzed BPA metabolism most efficiently, followed by CYP2C19 and CYP2C9. CYP2C9 and CYP2C18 exhibited the highest affinity (Km=3.9 microM) for BPA metabolism. The Vmax of CYP2C18 (8.10 nmol x min(-1) x nmol CYP(-1)) was 5 times higher than that of CYP2C9. Although the Vmax of CYP2C19 was 1.5 times higher than that of CYP2C18, the affinity of CYP2C19 was 12 times lower than that of CYP2C9 and CYP2C18. Therefore the intrinsic clearance (Vmax/Km) of CYP2C18 was more than 5 times higher than that of CYP2C9 and CYP2C19. On the other hand, BPA exhibited a competitive-type inhibition of the progesterone 17alpha-hydroxylase activity of CYP17 with a Ki value of 71 microM, whereas no metabolism of BPA by CYP17 was detected. These results suggest that BPA is mainly metabolized by the CYP2C subfamily in human liver, and that BPA inhibits human steroidogenic CYP17 activities.

Improved superoxide-generating ability by interferon gamma due to splicing pattern change of transcripts in neutrophils from patients with a splice site mutation in CYBB gene.

Chronic granulomatous disease (CGD) is an inherited disorder of host defense against microbial infections caused by defective activity of the phagocyte NADPH oxidase. Based on an increase of neutrophil superoxide-generating ability in response to interferon gamma (IFN-gamma) in a single patient with CGD, multicentered group studies demonstrated a beneficial effect of prophylactic IFN-gamma. However, no apparent increase of the phagocyte superoxide generation was found in patients enrolled in these studies. The present report offers an additional kindred in whom an IFN-gamma-dependent increase in neutrophil superoxide production was observed in 3 affected patients. The defect in the CYBB gene for gp91-phox was identified as an otherwise silent mutation adjacent to the third intron of the CYBB gene that alters messenger RNA splicing. By molecular analysis, significant differences were found in the splicing pattern of CYBB gene transcripts in patient neutrophils between 1 and 25 days after administration of IFN-gamma. Furthermore, a complete transcript containing the missing exons could be detected in all specimens after the treatment. The changes in the splicing pattern of the transcripts and the prolonged effect on superoxide-generating ability of patient neutrophils indicate that IFN-gamma induced a partial correction of the abnormal splicing of CYBB gene transcripts in myeloid progenitor cells.

Combined revascularization strategy for acute myocardial infarction in patients with intracoronary thrombus: preceding intracoronary thrombolysis and subsequent mechanical angioplasty.

Thrombus in the infarct-related artery is one of the limitations for flow restoration in primary percutaneous transluminal coronary angioplasty (PTCA) treatment for acute myocardial infarction (AMI). The present study investigated the benefit of preceding intracoronary thrombolysis (ICT) by retrospectively analyzing acute phase flow restoration in 80 AMI patients with intracoronary thrombus: 40 undergoing primary PTCA alone (primary PTCA group) and 40 treated with preceding ICT plus PTCA (combined group). Acute phase Thrombolysis in Myocardial Infarction (TIMI) grade flow was as follows: TIMI 0/1: 35.0% vs 12.5% for the primary PTCA group and the combined group, p=0.06; TIMI 2: 7.5% vs 15.0%, p=NS; TIMI 3: 57.5% vs 72.5%, p=NS). In the subgroup analysis, it was also less in the combined group among 33 patients with a left anterior descending coronary artery (LAD) lesion (42.1 % vs 7.1%, p=0.08), but not among the remaining 47 with either a right coronary artery or left circumflex artery lesion. The combined therapy may potentially provide better acute phase flow restoration in AMI patients with an intracoronary thrombus in a LAD lesion.

Plasma monocyte chemoattractant protein-1 antigen levels and the risk of restenosis after coronary stent implantation.

Monocyte chemoattractant protein-1 (MCP-1) plays a fundamental role in monocyte recruitment and has been implicated in atherosclerosis. The present study tested the hypothesis that increased levels of MCP-1 are associated with an increased risk for restenosis post stent implantation. The plasma MCP-1 antigen levels were measured pre-stenting, and at 24 and 48 h and 6 months post stenting in 41 patients with stable exertional angina (SEA) who had undergone successful stent implantation. Nineteen patients with chest pain syndrome were selected as a control group. Initial plasma MCP-1 antigen levels (mean +/- SE, pg/ml) in the patients with SEA were significantly higher than those in the control group (852.3+/-51.4 vs 418.2+/-26.7, p<0.001). The patients with SEA were divided into 2 groups based on follow-up angiographic findings: 17 patients with restenosis (R group); 24 patients without restenosis (N group). The lesion was significantly longer in the R group than in the N group (p<0.03). Plasma MCP-1 antigen levels at pre-stenting were not significantly different between the 2 groups (820.6+/-69.1 in the R group vs 874.7+/-73.8 in the N group). Serial changes of plasma MCP-1 levels were plotted as percent changes from the initial levels (mean +/- SE, %) and were significantly higher in the R group than in the N group at 48 h and at 6 months post stent implantation (104.6+/-4.8 vs 89.2+/-3.4, p<0.01, 109.6+/-11.2 vs 98.5+/-5.0, p<0.05). The study concludes that MCP-1 production at stented coronary arterial sites is associated with an increased risk for restenosis post stent implantation.

Improved procedure for the enantiometric synthesis of 1-hydroxy/acetoxy-2,6-diaryl-3,7-dioxabicycl.

Short enantiomeric syntheses of the 1-hydroxy/acetoxy-3,7-dioxabicyclo[3.3.0]octane lignans, paulownin, and (+)-phrymarin I and II, were accomplished by starting from the chiral synthon, (R)-(+)-3-hydroxybutanolide, and employing photocyclization as the key step.

Pulse infusion thrombolysis (PIT) for large intracoronary thrombus: preventive effect against the 'no flow' phenomenon in revascularization therapy for acute myocardial infarction.

Because large thrombus is a limitation for revascularization in acute myocardial infarction (AMI), the present study evaluated the effectiveness of pulse infusion thrombolysis (PIT) in patients with an AMI with a large (>15 mm) coronary thrombus, focusing on the occurrence of the 'no flow' phenomenon. The retrospective study compared patients treated before (1988-95; Group A, n=74) and after (1996-99; Group B, n=40) the use of PIT, using the following parameters: lesion success (<50% stenosis during 30-min observation), procedural success (lesion success plus TIMI grade 3 flow), procedural no flow (TIMI grade 0 flow during the procedure with 'back and forth movement' of contrast dye after lesion success), persistent no flow (consistent no flow without any flow improvement at the final visualization despite intensive treatment), reocclusion rate and in-hospital death. Group B was significantly better than Group A in procedural success (90% vs 66%; p=0.005), procedural 'no flow' (51% vs 15%; p<0.001), and persistent 'no flow' (34% vs 10%; p<0.05). Subgroup comparison was performed among the following groups: Direct-BA group (n=44): treated with mechanical angioplasty alone; ICT-BA group (n=40): treated with prior intracoronary thrombolysis and angioplasty; and PIT-BA group (n=30): treated with PIT and angioplasty. There were no differences in thrombus length and lesion success among these 3 groups. Procedural success was best achieved in PIT-BA: 97% vs 52% for Direct-BA (p=0.003) and 68% for ICT-BA (p=0.009). Procedural 'no flow' was least in PIT-BA: 50% vs 3.3% for Direct-BA (p=0.003) and 25% vs 3.3% for ICT-BA (p=0.042). Persistent 'no flow' was less frequent in PIT-BA than Direct-BA: 32% vs 3.3% (p=0.009). However, the difference between ICT-BA and Direct-BA was insignificant: 13% vs 3.3% (p=0.53). There were no differences in reocclusion rate and in-hospital death among the 3 subgroups. And there were no differences between Direct-BA and ICT-BA in any parameters. PIT was effective in preventing 'no flow' in the mechanical revasculalization for AMI especially those cases with a large thrombus.

Increased plasma antigen levels of monocyte chemoattractant protein-1 in patients with restenosis after percutaneous transluminal coronary angioplasty.

Monocyte chemoattractant protein-1 (MCP-1) plays an important role in the progression of atherosclerosis in coronary arteries. To examine whether or not plasma antigen levels of MCP-1 are related to restenosis after percutaneous transluminal coronary angioplasty (PTCA), the plasma antigen levels of MCP-1 were measured by enzyme-linked immunosorbent assay (pg/ml) before, 24 and 48 h, and 3 months after elective PTCA for stable exertional angina performed between June 1997 and March 1998. Restenosis was defined as recurrence of stenosis greater than 50% of the diameter in the dilated segment at 3-month follow-up angiography. There were no differences in plasma MCP-1 antigen levels before and at 24 h after PTCA between restenosis (R; n=27) and no-restenosis (N; n=43) groups (R vs N: 633+/-35 vs 589+/-34, and 669+/-41 vs 575+/-36 pg/ml before and at 24 h after PTCA, respectively), but plasma MCP-1 antigen levels were higher at 48 h and 3 months after PTCA in the R than in N group (R vs N: 678+/-41 vs 558+/-35, and 735+/-35 vs 571+/-32 pg/ml at 48 h and 3 months after PTCA, respectively). These data suggest that the MCP-1 production and macrophage accumulation in the balloon-injured site is partially associated with restenosis after PTCA.

[Genetic analysis of a patient with dyskeratosis congenita].

Dyskeratosis congenita (DKC) is a rare inherited disease characterized by reticulated pigmentation of the skin, nail dystrophy and oral leukoplakia. More than 90% of DKC cases are inherited as an X-linked recessive trait. Half the patients develop progressive pancytopenia by the age of 11 yr, and this is the leading cause of death. We experienced a 11-year-old boy with the above symptomatic triad of DKC, complicated by progressive pancytopenia as well as cerebellar ataxia. Genetic analysis of mRNA from his cultured peripheral lymphocytes revealed a missense mutation resulting in substitution of 1,150 C with T in the DKC1 gene. This is identical to the mutation reported by Knight et al. to be prevalent in X-linked cases of DKC (11 out of 21 patients). Existence of the identical mutation in Japan suggests that this mutation has been selected on the basis of not only the DNA structural sequence of dyskerin, but also its biological function. We report the detailed clinical course of this Japanese DKC patient with a mutation in the DKC1 gene, and describe the results of genetic analysis.

Statistical and mutational analysis of chronic granulomatous disease in Japan with special reference to gp91-phox and p22-phox deficiency.

Chronic granulomatous disease (CGD) is a group of inherited disorders of host defense caused by a mutation in any of the four components of phagocyte NADPH oxidase, namely gp91-, p22-, p47-, and p67-phox. We have made a precise statistical analysis of 229 registered patients from 195 families in Japan and mutation analysis of 28 and 5 independent patients, respectively, with gp91- and p22-phox deficiency. The gp91- and p22-phox proteins form the membrane cytochrome b558, which plays important roles in the assembly of the active oxidase and electron-transfer reaction, and the lesions in either subunit account for more than 80% of cases. The ratio of male to female patients was 6.6/1, the incidence was calculated to be about 1 out of 220,000 birth, and the life expectancy of the patients born in the 1970s was estimated to be 25-30 years old. For the X-linked gp91-phox deficiency, we found five missense and nine nonsense mutations, seven deletions, three insertions, and four splice site mutations, which included the following novel mutations: four missense, five nonsense, six deletions, one insertion, and two splice site abnormalities. With regard to p22-phox deficiency, two homozygous nonsense mutations and one homozygous deletion, a missense mutation together with a splice site mutation, and two different missense mutations were found. These mutations have not been reported before. Based on the present and reported data from Japan, we discuss the molecular defects of the disease and the difference in statistics between western countries and Japan.

Inhibition of drug-metabolizing enzyme activity in human hepatic cytochrome P450s by bisphenol A.

Effect of bisphenol A on drug-metabolizing enzyme activities by human hepatic cytochrome P450s (CYP) was investigated. We measured aminopyrine N-demethylation by eleven kinds of cDNA-expressed CYPs. CYP2C19 and CYP2B6 catalyzed most efficiently the aminopyrine N-demethylation, followed by CYP2C8 and CYP2D6. Bisphenol A (1 mM) most efficiently inhibited aminopyrine N-demethylation by CYP2C8 and CYP2C19 by 82% and 85%, respectively, whereas inhibition of the activities by CYP 2B6 and 2D6 was less than 40%. Bisphenol A exhibited a noncompetitive-type inhibition of aminopyrine N-demethylase activity by CYP2C8 with Ki value of 97 microM. Additionally, we investigated the inhibitory effect of bisphenol A on CYP2C19-mediated S-mephenytoin 4-hydroxylation. Bisphenol A exhibited a mixed-type inhibition with Ki value of 113 microM. These results suggest that bisphenol A inhibits human hepatic CYP activities, especially CYP2C8 and CYP2C19.

Growth regulation of bovine retinal pericytes by transforming growth factor-beta2 and plasmin.

PURPOSE: Transforming growth factor -beta2 (TGF-beta2) is a predominant isoform of TGF-betas in the eye and plasmin is a peptidase with many functions. To better understand the pathogenesis of retinal microcirculation disorders, the effects of TGF-beta2 and plasmin on cultured bovine retinal pericytes were investigated. METHODS: Exogenous TGF-beta2 or plasmin was added to some cultures, DNA synthesis during cell cycle progression was investigated using [(3)H]thymidine incorporation. Anti-TGF-beta2 antibody was added to neutralize the effects of TGF-beta2. TGF-beta2 in the culture medium was measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: Exogenous TGF-beta2 (10 pg to 100 ng/mL) suppressed DNA synthesis. Pericytes produced TGF-beta2. Anti-TGF-beta2 antibody neutralized TGF-beta2 and accelerated DNA synthesis, which shows that pericytes regulate their own cell cycle by action of the autocrine and/or paracrine system of TGF-beta2. Plasmin (0.2 to 0.5 U/mL) accelerated DNA synthesis in a dose-dependent manner, while addition of aprotinin, a protease inhibitor, counteracted this effect of plasmin. The concentration of TGF-beta2 in the culture medium decreased with the addition of plasmin. Simultaneous addition of both plasmin and anti-TGF-beta2 antibody accelerated DNA synthesis. High and low glucose concentrations of the culture medium did not affect DNA synthesis. CONCLUSIONS: Our results suggest that TGF-beta2 and plasmin respectively decrease and increase DNA synthesis. In a retinal microcirculation disorder, they may play competitive roles in the cell cycle of pericytes.

Relation between coronary thrombus and angiographic no-flow during primary angioplasty in patients with acute myocardial infarction.

No flow is an unsolved issue in primary percutaneous transluminal coronary angioplasty (PTCA) for patients with acute myocardial infarction (AMI), and the pathophysiology of no-flow is undetermined. To evaluate the potential participation of coronary thromboembolism in no-flow during primary PTCA, the present study reviewed cinefilms of 256 consecutive patients who underwent primary PTCA for AMI within 24h after the onset of chest pain between January 1992 and June 1998, focusing on the thrombus size. Angiographic no-flow was defined as the cessation of flow into the distal coronary circulation of the treated vessel with a to-and-fro contrast movement, not attributable to high-grade stenosis or spasm of the original target lesion. The coronary thrombus size was determined by using the 2-cm balloon catheter as a reference after crossing the infarct-related occluded artery with a guide wire. Angiographic no-flow was observed in 37 patients (37/256, 14%): 14 of 29 cases (48%) with a large thrombus (> or =2cm) versus 23 of 227 cases (9%) with a small thrombus (<2cm, 14/29 vs 23/227, p<0.01). Among 37 patients who experienced angiographic no-flow, overt distal emboli were observed in 14 patients. A thrombolytic agent was used through a guiding catheter in 102 cases prior to or after balloon dilatation to prevent or attenuate distal embolism, particularly in all those cases with a large thrombus (29/29 100%), and angiographic no-flow was seen in 27 cases of this subgroup (27/102, 26%). It is suggested that distal thromboembolism plays an important role in the mechanism of angiographic no-flow during primary PTCA performed for AMI.

[Statistical evaluation of chronic granulomatous disease in Japan and basic studies for gene therapy for CGD patients].

Chronic granulomatous disease (CGD) is an inherited immune deficiency caused by mutations in any of the following four phox genes encoding subunits of the superoxide generating phagocyte NADPH oxidase. It consists of membranous cytochrome b558 composed of gp91-phox and p22-phox, and four cytosolic components, p47-phox, p67-phox, rac p21 and p40-phox, which translocate to the membrane upon activation. In our group study, more than 220 CGD patients has been enrolled. The incidence of CGD patients was estimated as 1 out of 250,000 births. The expected life span of the CGD patients is 25 to 30 years old by the Kaplan Meier analysis. Comparing with the ratio of CGD subtype in US and Europe, that with p47-phox deficiency is lower (less than 10% vs. 23%) and that of gp91-phox deficiency is higher (more than 75% vs. 60%). Prophylactic administration of ST antibiotics and IFN-gamma and bone marrow transplantation have been successfully employed in our therapeutic strategy. However, it is necessary to develop the gene therapy technology for CGD patients as more promising treatment. In the current study we constructed two retrovirus vectors; MFGS-gp91/293 SPA which contains only the therapeutic gp91-phox gene, a bicistronic retrovims pHa-MDR-IRES-gp91/PA317 which carries a multi drug resistant gene (MDR1) and the gp91-phox gene connected with an internal ribosome entry site (IRES). We demonstrate high efficiency transduction of gp91-phox to CGD EB virus established cell line with high levels of functional correction of the oxidase by MFGS-gp91 and by pHa-MDR-IRES-gp91, respectively. We also demonstrate sufficient transduction of gp91-phox to CD34+ haematopoietic stem cell from the patients with gp91-phox deficiency by MFGS-gp91/293 SPA. Our current studies suggest that the combination of the 293-SPA packaging system and the bicistronic retrovirus system inserted MDR1 gene make our CGD gene therapy more feasible for clinical application.

Contribution of human hepatic cytochrome P450s and steroidogenic CYP17 to the N-demethylation of aminopyrine.

1. Aminopyrine N-demethylase activity was determined for 11 forms of human hepatic cytochrome P450s (P450s) expressed in yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli. 2. Among the hepatic P450s, the N-demethylation of aminopyrine was catalysed most efficiently by CYP2C19, followed by CYP2C8, 2D6, 2C18 and 1A2, whereas the activity with CYP2E1 was negligible. The kinetics of the N-demethylation process by CYP1A2, 2C8, 2C19 and 2D6 were studied by fitting to Michaelis-Menten kinetics by Lineweaver-Burk plots. CYP2C19 exhibited the highest affinity and a high capacity for the aminopyrine N-demethylation. CYP2C8 showed the highest Vmax, followed by CYP2C19, 2D6 and 1A2, whereas the Km for CYP2C8, 2D6 and 1A2 were 10-17 times higher than that for CYP2C19. Accordingly, the Vmax/Km for CYP2C19 was more than nine times higher than that of other P450s. 3. Human steroidogenic CYP17 also catalysed aminopyrine N-demethylation and the activity was comparable with that for CYP3A4 which is a dominant P450 in human liver. The activity was increased 1.5-fold by the addition of cytochrome b5, whereas the activity was not affected by the addition of Mg2+. 4. These results suggest that several human hepatic P450s, especially CYP2C19, and steroidogenic CYP17 exhibit aminopyrine N-demethylase activity.

Outcome of target sites escaping high-grade (>70%) restenosis after percutaneous transluminal coronary angioplasty.

This study examined the fate of target sites that escaped high-grade restenosis (> or = 70% diameter narrowing) after percutaneous transluminal coronary angioplasty. Although favorable long-term prognosis after successful percutaneous transluminal coronary angioplasty is well documented, little is known about the stability of target sites. Long-term follow-up (mean 6.5 years, range 1.0 to 12.0) was performed in 693 patients with 948 narrowings (stenosis <70% in diameter at follow-up coronary angiography). Among them, 249 patients (36%) with 303 target sites received late follow-up coronary angiography. The relation of target sites to the culprit lesions for coronary events or newly developed angina was angiographically reviewed and progression/regression was also examined, focusing on the target sites. Regression was observed in 16 of 255 target sites in subjects with <50% stenosis and in 21 of 48 sites in the group with midgrade stenosis of 50% to 69% luminal narrowing (16 of 255, 6.3% vs 21 of 48, 43.8%, p <0.001). Progression was observed in 33 and 4 sites (33 of 255, 12.9% vs 4 of 48, 8.3%; p = NS) in each group, respectively. The rest remained within the same range of stenosis. Culprit lesions for 2 acute myocardial infarctions, 7 unstable anginas, and 17 newly developed anginas were related to the original target sites. Three lesions developed in the midgrade stenosis group. Those 26 lesions were a component of 8.6% of 303 angiographically confirmed sites and 2.7% of total target sites. Target sites that escape high-grade restenosis frequently regress and become stable plaques and rarely trigger coronary events.

Effects of methanol and temperature on enzyme immunoassay with monoclonal antibodies specific to the insecticide etofenprox.

We examined the effects of methanol and temperature on the reactivity of monoclonal antibodies specific to the insecticide etofenprox. When the antigen-antibody reaction was done at 4 degrees C in 10% methanol, the sensitivity in the enzyme immunoassay with each antibody was more than 10-fold higher than that measured at 37 degrees C. Although in 10% methanol one of the antibodies reacted equally with both etofenprox and the carbonate-derivative of etofenprox, in 50% methanol the antibody reacted with etofenprox, but not with the derivative.

[Gene therapy for inherited diseases using heamatopoietic stem cells--gene therapy for patients with chronic granulomatous disease].

The possibility of gene therapy for inherited diseases with a single gene mutation in Figure 1 had been verified by the successful treatment with bone marrow transplantation. As the gene therapy method and theory has been progressing rapidly, it is expected that gene therapy will overcome the complications of bone marrow transplantation. Of these inherited diseases, chronic granulomatous disease (CGD) is the one of the most expected disease for gene therapy. CGD is an inherited immune deficiency caused by mutations in any of the following four phox genes encoding subunits of the superoxide generating phagocyte NADPH oxidase. It consists of membranous cytochrom b558 composed of gp91 phox and p22 phox, and four cytosolic components, p47 phox, p67 phox, rac p21 and p40 phox, which translocate to the membrane upon activation. In our group study, more than 220 CGD patients has been enrolled. The incidence of CGD patients was estimated as 1 out of 250,000 births. The expected life span of the CGD patients is 25 to 30 years old by the Kaplan Meier analysis. Comparing with the ratio of CGD subtype in US and Europe, that with p47phox deficiency is lower (less than 10%/o vs. 23%) and that of gp91 phox deficiency is higher (more than 75% vs. 60%). Prophylactic administration of ST antibiotics and IFN-gamma and bone marrow transplantation have been successfully employed in our therapeutic strategy. However, it is necessary to develop the gene therapy technology for CGD patients as more promising treatment. In the current study we constructed two retrovirus vectors; MFGS-gp91/293 SPA which contains only the therapeutic gp91 phox gene, a bicistronic retrovirus pHa-MDR-IRES-gp91/PA317 which carries a multi drug resistant gene (MDR1) and the gp91phox gene connected with an internal ribosome entry site (IRES). We demonstrate high efficiency transduction of gp 91 phox to CGD EB virus established cell line with high levels of functional correction of the oxidase by MFGS-gp91 and by pHa-MDR-IRES-gp91, respectively. We also demonstrate sufficient transduction of gp91 phox to CD34+ hematopoietic stem cell from the patients with gp91 phox deficiency by MFGS-gp91/293 SPA. Our current studies suggest that the combination of the 293-SPA packaging system and the bicistronic retrovirus system inserted MDR1 gene make our CGD gene therapy more feasible for clinical application.