Cystatin C is a sensitive marker for detecting a reduced glomerular filtration rate when assessing chronic kidney disease in patients with rheumatoid arthritis and secondary amyloidosis.

OBJECTIVE: Chronic kidney disease is a predictor of end-stage renal disease, and evaluating the glomerular filtration rate (GFR) is necessary to make a definite diagnosis. We assessed the utility of serum cystatin C (cysC) for identifying a reduced GFR in patients who have rheumatoid arthritis (RA) with secondary amyloidosis. METHODS: Fifty patients with RA and secondary amyloidosis (mean age 60.9+/-11.2 years; 45 women) were evaluated. The revised 24-h creatinine clearance (r24-hC(Cr)), which was determined by multiplying the original value by 0.719, was used as a reference for the GFR. The screening potential of the serum cysC and some estimates of the GFR calculated from the serum cysC (cysC-eGFR: eGFR(Hoek) and eGFR(Rule)) for detecting a reduced GFR (r24-hC(Cr)<60 mL/min/1.73 m(2)) were analysed. RESULTS: Both cysC-eGFRs were strongly correlated with the r24-hC(Cr) (eGFR(Hoek), r=0.846, p<0.001; eGFR(Rule), r=0.820, p<0.001). The difference between the average eGFR(Rule) (37.1+/-31.2 mL/min/1.73m(2)) and average r24-hC(Cr) (35.3+/-30.9 mL/min/1.73 m(2)) was small, whereas eGFR(Hoek) and sCr-eGFR were higher than eGFR(Rule) and r24-hC(Cr). In receiver operating characteristic (ROC) curve analyses of a reduced GFR, serum cysC gave a greater area under the curve (AUC=0.958) than the sCr-eGFR (0.939-0.942). The specificity and positive predictive value (PPV) reached 100% when serum cysC >1.365 mg/L was used. CONCLUSIONS: Serum cysC can identify a reduced GFR more accurately than sCr-eGFRs. Serum cysC >1.09 mg/L (i.e. eGFR(Rule)<60 mL/min/1.73 m(2)) could be a marker of a reduced GFR, and serum cysC >1.365 mg/L would strongly suggest a reduced GFR in patients who have RA with secondary amyloidosis.

A prostacyclin agonist with thromboxane inhibitory activity for airway allergic inflammation in mice.

BACKGROUND: ONO-1301 is a novel drug that acts as a prostacyclin agonist with thromboxane A(2) (TxA(2)) synthase inhibitory activity. We investigated the effect of ONO-1301 on development of airway allergic inflammation. METHODS: Mice sensitized and challenged to ovalbumin (OVA) received ONO-1301, OKY-046 (TxA(2) synthase inhibitor), beraprost, a prostacyclin receptor (IP) agonist, ONO-1301 plus CAY10449 (selective IP antagonist) or vehicle during the challenge period. Twenty-four hours after the OVA challenge, airway hyperresponsiveness (AHR) to methacholine was assessed and bronchoalveolar lavage was performed. Lung specimens were excised for goblet cell staining and analysis of lung dendritic cells (DCs). Bone marrow-derived dendritic cells (BMDCs) were generated, in the presence or absence of drugs, for analysis of DC function. RESULTS: Mice that received ONO-1301 showed significantly lower AHR, airway eosinophilia, T-helper type 2 cytokine levels, mucus production and lung DCs numbers than vehicle-treated mice. These effects of ONO-1301 were mostly reversed by CAY10449. BMDCs treated with ONO-1301 alone showed lower DC functions, such as expression of costimulatory factors or stimulation to spleen T cells. CONCLUSIONS: These data suggest that ONO-1301 may suppress AHR and airway allergic inflammation through modulation of DCs, mainly mediated through the IP receptor. This agent may be effective as an anti-inflammatory drug in the treatment of asthma.

Patients undergoing dialysis therapy for 30 years or more survive with serious osteoarticular disorders.

AIMS: Increasing numbers of patients are undergoing long-term dialysis therapy. It is crucial for their quality of life to overcome dialysis-related complications, such as dialysis-related amyloidosis (DRA) and other osteoarticular disorder. The aim of the study was to investigate the characteristics, such as dialysis-related complications, in chronic kidney disease (CKD) Stage 5D patients undergoing dialysis therapy for more than 30 years or more. METHODS: From 2003 to 2006, 359 CKD Stage 5D patients who were admitted to a single tertiary-care center. The age and the duration of dialysis therapy, the purpose for hospital admission, and history of osteoarticular disorder, such as carpal tunnel syndrome (CTS), destructive spondyloarthropathy (DSA) and joint arthropathy, were studied. RESULTS: The proportions of the patients undergoing dialysis therapy for 20 - 24, 25 - 29 years and 30 years or more were 8.9, 5.6, and 4.5% of all admitted patients, respectively. DSA was a major cause of hospital admissions in long-term dialysis patients, especially in those treated for 30 years or more. The rate of surgery for osteoarticular disorder, such as CTS, DSA and joint arthropathy, which may show the presence of DRA, was 25.0, 66.0 and 77.8% in 20 - 24 years, 25 - 29 years and 30 years or more after the initiation of dialysis therapy, respectively. The frequency and severity of osteoarticular disorder accelerated with the duration of dialysis therapy, especially in those treated for 30 years or more. The rate of parathyroidectomy for secondary hyperparathyroidism was performed for 37.5% in 22.1 +/- 2.1 years after the initiation of dialysis treatment in the patients treated for 30 years or more. Mean age at the initiation of dialysis therapy was 27.3 +/- 8.0 years, and primary cause of CKD was mainly chronic glomerulonephritis in the patients undergoing dialysis therapy for 30 years or more. CONCLUSION: CKD stage 5D patients undergoing dialysis therapy for 30 years or more survive with characteristics of younger age at initiation of dialysis therapy, chronic glomerulonephritis as a primary cause of CKD, and serious complication of osteoarticular disorders.

Differences in lymphocyte profile between BAL fluid and human lung tissue from patients with interstitial lung disease.

Bronchoalveolar lavage (BAL) is a technique that samples the inflammatory cells from distal airways and alveoli; however, it is unclear whether or not cellular profiles in the BAL fluid reflect the cellular components of the lung parenchyma in interstitial lung disease (ILD). The aim of this study is to compare immunophenotypes of lymphocytes between BAL fluid and human lung tissue from patients with ILD. Fourteen consecutive patients with ILD who underwent BAL and surgical lung biopsy were enrolled. The diagnosis of ILD was confirmed by the presence of clinical symptoms and impaired respiratory function and on high-resolution computed tomography (CT) of the chest. Mononuclear cells in BAL were immunophenotyped for the expression of CD3, CD4, CD8, CD19, CD45, and CD103 by flow cytometry. Lung tissue obtained by surgical biopsy was digested with collagenase and then centrifuged to extract parenchymal cells. Isolated cells were also immunophenotyped for the same CD expression. Frequencies of positive cells were compared statistically between BAL and different lobes. Seven out of 14 patients were diagnosed clinically as suffering idiopathic interstitial pneumonia. Frequency of CD19+ cells from BAL was significantly lower than that from the upper/middle lobes (P < 0.05). Frequency of CD103+ cells from BAL was significantly higher than that from the upper/middle lobes and the lower lobe (P = 0.01 and P < 0.05, respectively). Comparison between different lobes demonstrated that the frequency of CD4+ cells from the upper/middle lobes was significantly lower than that from the lower lobe (P < 0.05). The results suggest that lymphocyte immunophenotype profiles from BAL may not reflect those in the inflammatory tissue of ILD.

Paraneoplastic neurological syndrome and polyglandular autoimmune syndrome type 2 in a case of small cell lung cancer.

We report a female patient with small cell lung cancer (SCLC) and clinical findings consistent with polyglandular autoimmune syndrome type 2 (PGA2) and paraneoplastic neurological syndrome (PNS). To the best of our knowledge, this is the first reported case of SCLC associated with PGA2 and PNS. All of the autoantibodies detected before anticancer treatment decreased below the upper normal limits after serial treatment, and the patient's clinical symptoms also improved. Cross reactivity of autoantibodies may have contributed to the complicated clinical picture of this patient.

Effects of edaravone, a free-radical scavenger, on bleomycin-induced lung injury in mice.

Reactive oxygen species play an important role in the pathogenesis of acute lung injury and pulmonary fibrosis. The present authors hypothesise that edaravone, a free-radical scavenger, is able to attenuate bleomycin (BLM)-induced lung injury in mice by decreasing oxidative stress. Lung injury was induced in female ICR mice by intratracheal instillation of 5 mg x kg(-1) of BLM. Edaravone (300 mg x kg(-1)) was administered by intraperitoneal administration 1 h before BLM challenge. Edaravone significantly improved the survival rate of mice treated with BLM from 25 to 90%, reduced the number of total cells and neutrophils in bronchoalveolar lavage fluid (BALF) on day 7, and attenuated the concentrations of lipid hydroperoxide in BALF and serum on day 2. The fibrotic change in the lung on day 28 was ameliorated by edaravone, as evaluated by histological examination and measurement of hydroxyproline contents. In addition, edaravone significantly increased the prostaglandin E(2) concentration in BALF on day 2. In summary, edaravone was shown to inhibit lung injury and fibrosis via the repression of lipid hydroperoxide production and the elevation of prostaglandin E(2) production in the present experimental murine system.

Appropriate timing of CD40 ligation for RNA-Pulsed DCs to induce antitumor immunity.

Dendritic cell (DC) based anti-cancer immunotherapy is believed to be a promising treatment. However, appropriate conditioning including the method of antigen loading and stimulation for DC maturation is still unclear. Total RNA pulsing is one of the most attractive methods to load antigen, since RNA is easily replicated by the PCR technique and is absolutely free of tumor cell contamination. On the other hand, CD40 ligation is capable of producing one of the most potent signals for immature DCs to start functional maturation, which is required to induce adaptive immunity, resulting in altered migration ability to secondary lymphoid organs and augmented antigen presenting activity. Here, we demonstrate that DCs pulsed with total RNA extracted from tumor cells required CD40 stimulation with an appropriate sequence to present tumor-associated antigens. RNA derived antigens were presented for both CD4+ and CD8+ T cells in an antigen-specific manner. Dendritic cells that were pulsed with RNA followed by the stimulation through CD40 successfully primed antitumor effector T cells in draining LNs and subsequently induced antitumor protective immunity.

Generation of anti-tumour effector T cells from naïve T cells by stimulation with dendritic/tumour fusion cells.

Tumour-draining lymph node T cells are an excellent source of effector T cells that can be used in adoptive tumour immunotherapy because they have already been sensitized to tumour-associated antigens in vivo. However, such tumour-specific immune cells are not readily obtained from the host due to poor immunogenicity of tumours and reduced host immune responses. One obstacle in implementation of adoptive immunotherapy has been insufficient sensitization and expansion of tumour-specific effector cells. In this study, we aim to improve adoptive immunotherapy by generating anti-tumour effector T cells from naïve T lymphocytes. We attempted to achieve this by harnessing the advantages of dendritic cell (DC)-based anti-cancer vaccine strategies. Electrofusion was routinely employed to produce fusion cells with 30-40% efficiency by using the poorly immunogenic murine B16/F10 cell line, D5 cells, and DC generated from bone marrow cells. CD62L-positive T cells from spleens of naïve mice and the fusion cells were cocultured with a low concentration of IL-2. After 9 days of culture, the antigen-specific T cells were identified with an upregulation of CD25 and CD69 expression and a downregulation of CD62L expression. These cells secreted IFN-gamma upon stimulation with irradiated tumour cells. Moreover, when transferred into mice with 3-day established pulmonary metastases, these cells with coadministration of IL-2 exhibited anti-tumour efficacy. In contrast, naïve T cells cocultured with a mixture of unfused DC and irradiated tumour cells did not exhibit anti-tumour efficacy. Our strategy provides the basis for a new approach in adoptive T cell immunotherapy for cancer.

The genetic susceptibility to IgA nephropathy: a novel functional candidate gene for incomplete O-glycosylation of IgA1.

Incompleteness of O-glycosylation in the IgA1 hinge has been implicated as a central mechanism in the development of IgA nephropathy. Although underglycosylation was reported to be an acquired abnormality, genes for enzymes of O-glycosylation, such as C1GALT1, may be responsible for susceptibility to IgA nephropathy.

Expression of allograft inflammatory factor-1 in kidneys: A novel molecular component of podocyte.

Our comprehensive gene expression profiles of the kidneys in an anti-glomerular basement membrane (GBM) nephritis model using DNA arrays revealed that allograft inflammatory factor-1 (AIF-1) was one of the highly expressed genes. Here, we explored the pathological significance of AIF-1 expression in the kidneys. The expression pattern of AIF-1 mRNA and protein in the kidneys of normal and diseased rats, such as anti-GBM nephritis and puromycin aminonucleoside nephrosis, was investigated by in situ hybridization, immunohistochemistry, and immunoelectron microscopy. Furthermore, the expression of AIF-1 in human kidneys and urinary sediments was examined. AIF-1 was expressed at both mRNA and protein levels in podocytes of normal and diseased rats, and in infiltrating cells in anti-GBM nephritis kidneys. The expression of AIF-1 in podocytes was constitutive; positive in podocytes of both normal and diseased rats. In humans, AIF-1 was expressed in podocytes and infiltrating inflammatory cells, similarly. Moreover, it was detected in urinary podocytes from patients with immunoglobulin A nephropathy. These data document for the first time that AIF-1, a constitutively expressed protein in rat and human podocytes, is a novel molecular component of podocytes, and that the upregulation of AIF-1 in an anti-GBM nephritis model may mainly be a consequence of its expression in infiltrating cells.

Etiology and prognostic significance of severe uremic pruritus in chronic hemodialysis patients.

Although uremia is well known as the most common cause of pruritus, the mechanisms of pruritus in chronic hemodialysis patients remain unclear. The purpose was to characterize uremic pruritus in more detail and to investigate whether severe pruritus is a marker for poor prognosis. A total of 1773 adult hemodialysis patients were studied. A questionnaire was given to each patient to assess the intensity and frequency, as well as pruritus-related sleep disturbance. We analyzed the relationship between clinical and laboratory data and the severity of pruritus in hemodialysis patients and followed them for 24 months prospectively. In total, 453 patients had severe pruritus with a visual analogue scale (VAS) score more than or equal to 7.0. Among them, more than 70% complained of sleep disturbance, whereas the majority of patients with a VAS score of less than 7.0 had no sleep disturbance. Male gender, high levels of blood urea nitrogen, beta2-microglobulin (beta2MG), hypercalcemia, and hyperphosphatemia were identified as independent risk factors for the development of severe pruritus, whereas a low level of calcium and intact-parathyroid hormone were associated with reduced risk. During the follow-up, 171 (9.64%) patients died. The prognosis of patients with severe pruritus was significantly worse than the others. Moreover, severe pruritus was independently associated with death even after adjusting for other clinical factors including diabetes mellitus, age, beta2MG, and albumin. Severe uremic pruritus caused by multiple factors, not only affects the quality of life but may also be associated with poor outcome in chronic hemodialysis patients.

Turbulence of glomerular hemodynamics involved in progressive glomerulosclerosis.

There is increasing evidence that changes of glomerular hemodynamics or glomerular growth responses may promote the development of glomerulosclerosis. Major problems retarding research progress include lack of suitable experimental animal models, with the exception of the ablation model, and the need for in vivo real-time analysis of glomerular hemodynamics. This study examined the sequence of pathological changes from the viewpoints of microcirculation and histopathology, from the acute stage to the chronic course and the final stage of glomerulosclerosis, using the confocal laser scanning microscope system. There is a marked difference in prognosis between sham-operated (two-kidney) and nephrectomized (one-kidney) rats after injection with anti-Thy-1 antibody. The former reversibly returns to normal and the latter irreversibly go to progressive sclerosis, respectively. The turning point determining the progression of glomerulosclerosis in both groups seemed to be the period from 7 to 14 days after disease induction, when disturbance of local intraglomerular blood flow continued in the one-kidney groups. In conclusion, this study provides the first hemodynamic-based evidence showing that disturbance of intraglomerular microcirculation is a critical marker for progressive glomerulosclerosis.

Autoantibodies against oxidized low-density lipoprotein (LDL) and carotid atherosclerosis in patients with rheumatoid arthritis.

OBJECTIVES: To examine the relationship between autoantibodies against oxidized low-density lipoprotein (oxLDL-Abs) and the progression of carotid atherosclerosis in patients with rheumatoid arthritis (RA). METHODS: Fifty RA patients without evidence of risk factors for atherosclerosis (RA group) and 30 healthy volunteers (normal group) were investigated. The mean intima-media thickness of the common carotid artery (mean CCA-IMT) was measured by high-resolution B-mode ultrasonography. The titer of IgG oxLDL-Abs was measured by enzyme-linked immunosorbent assay. The relationships among mean CCA-IMT, IgG oxLDL-Ab titer and patient factors such as body mass index, systolic blood pressure, diastolic blood pressure, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and serum lipid levels were examined. RESULTS: Mean CCA-IMT, CRP, ESR and titer of IgG oxLDL-Abs were significantly higher in the RA group than in the normal group. Although mean CCA-IMT showed a positive correlation only with age in multivariate analysis, IgG oxLDL-Ab titers in the RA group were positively associated with mean CCA-IMT and independently with age and sex by multiple regression analysis. CONCLUSIONS: IgG oxLDL-Abs appear to be associated with the degree of carotid atherosclerosis in patients with RA, and are independent of traditional risk factors for atherosclerotic diseases. These results suggest a possible link between autoimmune mechanisms and accelerated atherosclerosis in RA.

Maxacalcitol therapy decreases circulating osteoprotegerin levels in dialysis patients with secondary hyperparathyroidism.

BACKGROUND: Osteoprotegerin is a natural glycoprotein which plays a critical role in osteoclast physiology. Elevated levels of circulating osteoprotegerin may account for the development of bone and mineral metabolic abnormalities in uremia. Little is known about the effects of vitamin D therapy on the circulating osteoprotegerin levels in dialysis patients. PATIENTS AND METHODS: Fifty chronic dialysis patients whose plasma intact PTH levels were greater than 300 pg/ml were analyzed for the study. Following a four-week washout time during which all vitamin D administration was halted, 10 microg of maxacalcitol was intravenously injected thrice a week. RESULTS: The circulating intact PTH, bone-specific alkaline phosphatase and intact osteocalcin levels were significantly lowered, while the serum calcium levels were elevated after the therapy. The osteoprotegerin levels significantly decreased after the therapy (p < 0.0001). CONCLUSION: Maxacalcitol therapy reduced the circulating osteoprotegerin levels and improved secondary hyperparathyroidism. The observed effects were the opposite of those expected from previous in vitro studies. Osteoprotegerin may mediate and/or modify the effect of active vitamin D therapy in dialysis patients.

Magnetic resonance T1 gradient-echo imaging in hepatolithiasis.

BACKGROUND: We examined the role of magnetic resonance T1-weighted gradient-echo (MRT1-GE) imaging in hepatolithiasis. METHODS: MRT1-GE, precontrast computed tomography (CT), and magnetic resonance cholangiopancreatography (MRCP) of 10 patients with hepatolithiasis were compared for their diagnostic accuracies in the detection and localization of intrahepatic calculi. The diagnosis of hepatolithiasis was confirmed by surgery. For localization of the stone, we divided the bile ducts into six areas: right and left hepatic ducts and bile ducts of the lateral, medial, right anterior, and right posterior segments of the liver. Chemical analysis of the stones was performed in eight patients. RESULTS: The total number of segments proved by surgery to contain stones was 18. Although not significantly different, the sensitivity of MRT1-GE was 77.8% (14 of 18 segments), higher than that of MRCP (66.7%, 12 of 18 segments) and that of CT (50%, nine of 18 segments). The sensitivity of magnetic resonance imaging (MRCP + MRT1) was significantly higher than that of CT (p < 0.01). Multiple logistic regression analysis showed that the result of surgery was significantly affected only by the result of magnetic resonance imaging. On MRT1-GE, all the depicted stones appeared as high-intensity signal areas within the low-intensity bile duct irrespective of their chemical composition. CONCLUSION: MRT1-GE imaging provides complementary information concerning hepatolithiasis.

Hepatic portal venous gas associated with nonocclusive mesenteric ischemia in a hemodialysis patient.

Hepatic portal venous gas (HPVG) has been rarely described in chronic hemodialysis patients. We report a case of HPVG in a 59-year-old female patient with hemodialysis-dependent chronic renal failure due to diabetes who presented with acute onset of abdominal pain. Abdominal CT demonstrated the presence of gas in the portal veins. However, on laparotomy, no evidence of bowel necrosis or perforation could be found. HPVG seemed to be caused by nonocclusive mesenteric ischemia (NOMI), an increasingly recognized complication in hemodialysis patients. The patient responded favorably to intravenous hyperalimentation and antibiotics.

Local adaptation and population differentiation at the interleukin 13 and interleukin 4 loci.

A 25.6 kb region at chromosome 5q31, covering the entire human interleukin 13 (IL-13) and interleukin 4 (IL-4) genes, has been reported to be associated with bronchial asthma. We have examined nucleotide variations at this locus in African, European American, and Japanese populations, using 120 diallelic variants. A block of strong linkage disequilibrium (LD) (mid R:D'mid R:>0.7) spans a 10 kb region containing IL-4 in European American and Japanese populations, and is present but less clear in African samples. Two major haplotypes at IL-4 account for >80% of haplotypes in European Americans and Japanese. These haplotypes are common and quite diverged from each other and the ancestral haplotype, resulting in highly significant deviations from neutrality. F(ST) statistics show that European American and Japanese populations are unusually distinct at the IL-4 locus. The most common haplotype in the European American population is much less common in the Japanese population, and vice versa. This implies that natural selection has acted on IL-4 haplotypes differently in different populations. This selected variation at IL-4 may account for some genetic variance underlying susceptibility to asthma and other allergic diseases. The strong LD observed in the IL-4 region may allow more efficient disease-association studies using this locus.

Pathological study on the relationship between C4d, CD59 and C5b-9 in acute renal allograft rejection.

In order to evaluate the activation or inhibition of the later phases of classical complement cascade in renal allograft presenting with acute rejection, particularly with C4d deposition on the peritubular capillary (PTC), we observed the expression of CD59 and C5b-9 on the PTC. Subjective cases were divided into two groups, an acute rejection group, of 4 males and 6 females, and a normal donor group, of 5 males and 5 females. Renal biopsies were performed at the onset of acute rejection and at the transplant operation, before reperfusion. C4d deposition on PTC was found in three of 10 cases (30%) with biopsy proven acute rejection, whereas CD59 on PTC was positively expressed in all of the rejection cases. Although C5b-9 was not observed on PTC in the acute rejection group, it was intensively deposited on the tubular basement membrane (TBM) in five cases, including the three with positive C4d on PTC. In the normal donor group, CD59 on PTC was intensively observed, whereas C5b-9 was weakly expressed on TBM. CD59, a complement regulatory factor, works as an inhibitory factor against the formation of C5b-9, a membrane attack complex. From our data, we noted the dissociation between the depositions of C4d and C5b-9 on PTC. The substantially expressed CD59 on PTC may affect this dissociation between C4d and C5b-9 on PTC. The intensive deposition of C5b-9 on TBM in acute rejection cases may suggest an independent immunological injury attacking tubular cells.

Transforming growth factor-beta1 gene polymorphism modifies the histological and clinical manifestations in Japanese patients with IgA nephropathy.

Transforming growth factor (TGF)-beta1, a multifunctional cytokine, which regulates proliferation and differentiation of a variety of cell types, has the central role in the development and progression of renal injury in both animal models and human. Although it has been suggested that genetic variations in the TGF-beta1 gene are associated with the activity of the gene product, their clinical significance in glomerular disease is unknown. We investigated whether the polymorphisms of C-509T and T869C in TGF-beta1 account for interindividual variation in manifestations of IgA nephropathy (IgAN) using 626 Japanese subjects including 329 patients with histologically proven IgAN and 297 healthy controls with normal urinalysis. The frequencies of genotypes, alleles, and major haplotypes were similar between the patients and controls. The C-509T and T869C polymorphisms were in tight linkage disequilibrium, and the major haplotypes were C-C and T-T, which accounted for more than 95% of the total. In patients with -509CC and in those with the 869CC, urinary protein excretion was higher than in those with other genotypes, whereas no difference in other clinical manifestations was noted. Moreover, patients with -509CC and those with 869CC genotypes presented with a significant higher score of mesangial cell proliferation than in those with other genotypes. These results suggest that TGF-beta1 gene polymorphisms are specifically associated with heavy proteinuria and mesangial cell proliferation in Japanese patients with IgAN, although they do not confer susceptibility to this disease.

Post-secretion neutralization of transgene-derived effect: soluble erythropoietin receptor/IgG1Fc expressed in liver neutralizes erythropoietin produced in muscle.

BACKGROUND: The regulation of transgene expression is a key issue for the development of safe gene therapy. Various strategies have been used to regulate protein production at the levels of transgene expression, transcription, translation, and secretion. Neutralization following secretion is another important backup system to prevent super-therapeutic levels of a protein from being expressed by gene transfer. METHODS: We tested whether the soluble human erythropoietin receptor (EpoR)/IgG(1)Fc could neutralize the rat Epo at the post-secretory level and suppress erythrocytosis. RESULTS: To assess whether soluble human EpoR could bind rat Epo in vitro, we used the Epo-dependent human leukemic cell line, AS-E2. EpoR/IgG(1)Fc significantly inhibited the growth of AS-E2 cells in Epo-containing medium. To test this neutralization effect of EpoR/IgG(1)Fc in vivo, we first transferred pCAGGS-Epo into rat muscle by in vivo electroporation, confirmed erythropoiesis for 3 weeks, and then delivered EpoR/IgG(1)Fc by liver-targeted gene transfer via tail-vein injection with hydrodynamics-based transfection. Reticulocyte counts and hematocrit levels in rats that received pCAGGS-EpoR/IgG(1)Fc injections were significantly lower than in rats that received pCAGGS-EpoR, pCAGGS-IgG(1)Fc, or no injection. CONCLUSIONS: These results demonstrate that liver-targeted pCAGGS-EpoR/IgG(1)Fc transfer by tail-vein injection with hydrodynamics-based transfection is useful for neutralizing Epo delivered by in vivo electroporation. This backup strategy at the level of post-secretion could facilitate the clinical application of gene therapy in the future.