Hyaluronic acid pretreatment for Sendai virus-mediated cochlear gene transfer.

Gene therapy with viral vectors is one of the most promising strategies for sensorineural hearing loss. However, safe and effective administration of the viral vector into cochlear tissue is difficult because of the anatomical isolation of the cochlea. We investigated the efficiency and safety of round window membrane (RWM) application of Sendai virus, one of the most promising non-genotoxic vectors, after pretreatment with hyaluronic acid (HA) on the RWM to promote efficient viral translocation into the cochlea. Sendai virus expressing the green fluorescent protein reporter gene was detected throughout cochlear tissues following application combined with HA pretreatment. Quantitative analysis revealed that maximum expression was reached 3 days after treatment. The efficiency of transgene expression was several 100-fold greater with HA pretreatment than that without. Furthermore, unlike the conventional intracochlear delivery methods, this approach did not cause hearing loss. These findings reveal the potential utility of gene therapy with Sendai virus and HA for treatment of sensorineural hearing loss.

Pneumonia due to varicella-zoster virus reinfection in a renal transplant recipient.

Varicella pneumonia is one of the serious complications of primary varicella zoster virus (VZV) infection in adults. A 36-year-old woman with end-stage renal disease underwent renal transplantation from a living donor in 1998, receiving immunosuppressive treatment with cyclosporine, mycophenolate mofetil, and methylprednisolone. She had a history of VZV infection during childhood. The patient developed an intractable cough on December 10, 2006, but there were no abnormalities in the laboratory data or chest radiograph for several weeks. On January 1, 2007, she was admitted to our hospital with cutaneous vesicles covering the entire body. We learnt that when her symptoms developed, her son was diagnosed with varicella. The chest radiograph at this stage showed a diffuse miliary pattern in the entire lung field. We started intravenous administration of acyclovir. VZV antigen was detected in the cutaneous lesions and VZV antibody in the serum after the start of these treatments, so we continued to administer acyclovir for 18 days. The cutaneous lesions healed and the pneumonia improved based on the chest radiograph. She was discharged from the hospital on January 19, 2007. In conclusion, this report documents VZV reinfection in a transplant patient.

Successful steroid withdrawal after long-term administration in renal transplant patients.

OBJECTIVE: This study evaluated the effect of steroid withdrawal after long-term administration on stably functioning renal transplant recipients. METHODS: Between April 2000 and October 2006, steroid administration was safely withdrawn in 47 patients with stable graft function for >1 year after renal transplantation. The period between renal transplantation and steroid withdrawal varied from 12 to 234 months. We also investigated the current steroid doses of all 274 outpatients who had undergone renal transplantation at our hospital between July 1977 and October 2006. RESULTS: Twelve patients out of 47 had to resume steroid administration, 10 (21%) owing to acute rejection with/without recurrent glomerulonephritis, 1 owing to treatment of subacute thyroiditis, and the other owing to accompanying cessation of azathioprine for ovarian cancer. Thirty-five patients have maintained stable graft function for 12 to 90 months (median, 49) after steroid withdrawal as confirmed by follow-up. At present, only 1 of the 47 patients had to resume hemodialysis owing to chronic deterioration of renal graft function. The current steroid doses (prednisolone equivalent) of the 274 outpatients at our hospital are as follows: The number of patients for withdrawn, <5 mg, 5 mg, >5 to 10 mg, and >10 mg/d is 38, 20, 155, 57, and 4, respectively. Of 294 patients, 213 (77.7%) are maintaining stable renal graft functions on

A free radical scavenger, edaravone, prevents ischemia-reperfusion injury in liver grafts from non-heart-beating donors.

BACKGROUND: Due to the increase in liver transplantation, the donor shortage has become a serious problem, requiring marginal, non-heart-beating donors (NHBDs). The aims of this study were to evaluate the cytoprotective effect of edaravone, a free radical scavenger, on warm ischemia-reperfusion (I/R) injury of liver grafts from NHBDs. METHODS: Rat livers were harvested from heart-beating donors (HB group) or from NHBDs undergoing cardiac arrest for 30 minutes led by thoracotomy (NHB group), and reperfused for 60 minutes with Krebs-Henseleit bicarbonate buffer after cold preservation for 6 hours. In another group (ED group), warm ischemic livers from NHBDs were reperfused with buffer containing edaravone (1 mg/L) after cold preservation. RESULTS: In the ED group, portal flow volume, bile production, and energy charge were significantly ameliorated. Lipid peroxidation, elevation of hepatic enzymes, and release of tumor necrosis factor-alpha and interleukin-1 beta were significantly alleviated, compared with the NHB group. CONCLUSIONS: These results suggested that edaravone has suppressive effects on warm I/R injury in liver grafts from NHBDs.

New strategy for ABO-incompatible living donor liver transplantation with anti-CD20 antibody (rituximab) and plasma exchange.

It is more difficult to control humoral rejection in living donor liver transplantations (LDLT) across the ABO blood group barrier than in matched or compatible combinations. We achieved excellent results in ABO-incompatible transplantation with novel immunosuppressive regimens and plasma exchange (PE). Among 82 LDLT were 10 cases of ABO-incompatible recipients, including three who were administered rituximab for rescue or prophylactic therapy. Pretransplantation PE was performed as necessary to maintain hemagglutinin titers below 1:16 and posttransplantation PE was performed when there were signs of hyperacute rejection associated with high titers. Induction immunosuppression consisted of FK506, steroid, mycophenolate mofetil (MMF), and rituximab. The first patient was administered rituximab with deoxyspergualin (DSG), steroid pulse therapy, and PE on postoperative day (POD) 7, because of biopsy-proven humoral acute rejection. The titers and LFTs improved drastically. The second and third patients were administered rituximab just after the operation with other routine immunosuppressants for prophylaxis of hyperacute rejection. The second patient showed a slight deterioration in LFTs with an elevated titer, which normalized after steroid pulse therapy and PE. The third patient had no episodes of rejection. At present, that is 27, 17, and 6 months after the operations respectively, the 3 transplant recipients are in stable condition.

Experience with recipient's superficial femoral vein as conduit for middle hepatic vein reconstruction in a right-lobe living donor liver transplant procedure.

Middle hepatic vein reconstruction during the right-lobe living donor liver transplant procedure has been recognized to be a significant factor. We initially reconstructed only a single middle hepatic vein orifice draining into segment 8. In cases where the right-lobe liver graft has several major middle hepatic vein tributaries, including veins draining segment 5 that are remote from the right hepatic vein orifice, a long and thick interposition conduit is necessary for reconstruction. Among 11 consecutive adult patients who received a right-lobe liver graft without a middle hepatic vein at our institution, 8 underwent reconstruction of all major middle hepatic vein tributaries using a vein graft from the recipient's superficial femoral vein. The remaining 3 patients had no major middle hepatic vein tributaries. Posttransplant-computed tomography imagings showed increased liver mass with a patent superficial femoral vein graft in 8 patients. In the absence of a venous system from a deceased donor, a recipient superficial femoral vein offers an excellent size match to maintain the venous outflow of middle hepatic vein tributaries. Reconstruction with recipient superficial femoral vein plays an important role in maximizing liver function and minimizing morbidity in the early posttransplant period.

Analysis of the gene expression of SPARC and its prognostic value for bladder cancer.

PURPOSE: We analyzed the gene expression of the glycoprotein termed secreted protein, acidic and rich in cysteine (SPARC), also called osteonectin and BM40, in bladder cancer and its relationship with conventional clinical-histopathological manifestations, evaluated its prognostic value for patient outcome and determined the possible mechanism underlying the effect of SPARC on bladder cancer progression. MATERIALS AND METHODS: Tissue samples from 63 patients with bladder cancer were used for analysis. Gene expression levels of SPARC and matrix metalloproteinase-2 were analyzed using reverse transcription-polymerase chain reaction. Correlations of the expression of SPARC with histopathological findings or patient outcome and with matrix metalloproteinase-2 were evaluated. RESULTS: Significantly higher expression of SPARC was observed in grades 3 and 2 than in grade 1 tumors (p <0.001 and <0.05, respectively). Stage T2 or greater invasive tumors expressed a significantly higher level of SPARC than stages T1 or less superficial tumors (p <0.0001). Patients in whom the lesions showed high SPARC expression had a significantly worse prognosis than those with low SPARC expression disease (p <0.0001). Even in those with invasive bladder cancer high SPARC expression was associated with significantly worse survival than low expression (p <0.01). Moreover, gene expression of SPARC significantly correlated with matrix metalloproteinase-2 gene expression (p <0.0001), implying that regulation of matrix metalloproteinase-2 expression may be a possible mechanism underlying the effect of SPARC on bladder cancer progression. CONCLUSIONS: A significant correlation was detected of the gene expression level of SPARC with histological grade, pathological stage and bladder cancer prognosis. SPARC may have an important role in bladder cancer progression and provide some additional information in patients with bladder cancer.

Secondary amyloidosis of the bladder causing macroscopic hematuria.

Bladder involvement in amyloidosis is unusual. The case of an 80-year-old man with macroscopic hematuria caused by secondary amyloidosis of the bladder is described. Cystoscopic examination revealed only a diffuse edematous area and bleeding. No tumor-like lesions were identified. Transurethral biopsy revealed amyloid deposits. Macroscopic hematuria disappeared spontaneously after cystoscopy and bladder biopsy. The patient has been followed up without treatment and is currently free of symptoms.

A centrifuge-less plasma separation method from whole blood anticoagulated with EDTA-2K for the use of clinical laboratory tests.

In the modern medical laboratory system, simple and rapid processing of specimens are required. In the current system with the transportation line, its centrifugation part would disturb smooth flow of the testing because it needs much time for the centrifugation. To solve the problems, a serum separation method was tried for the whole blood specimen using poly-L-lysine, concanavalin A and phyto-hemoagglutinin. Ploy-L-lysine with molecular weight 130,000 to 210,000 in a final concentration of 0.1% could accelerate blood sedimentation, although its supernatant contaminated platelets. Concanavalin and phytohemoagulutinin could accelerate the sedimentation and obtained plasma, but the method could yield enough amount of supernatant by 1 hour standing. As the purpose of this study is to develop a centrifugeless method, a sieve method using a steel mesh and a magnet was applied to the mixture of EDTA blood, red-cell adhesives and thrombin. The method was unique to separate plasma, but the yield was not so high and chemistry data were not fitted with serum data in some of tests. Thus, the trial would be a new technology, but it was judged that some further improvement will be needed technically.

Expression of vascular endothelial growth factor isoforms and platelet-derived endothelial cell growth factor in bladder cancer.

We analyzed the expression of vascular endothelial growth factor (VEGF) messenger ribonucleic acid (mRNA) isoforms and platelet-derived endothelial cell growth factor (PDECGF) mRNA in bladder cancer. We also attempted to determine if correlation exists between their expression level and conventional clinical variables in patients with bladder cancer. Tissues obtained from 60 patients with bladder carcinoma were used for analysis. Expression levels of VEGF isoforms and PDECGF were examined using reverse transcription-polymerase chain reaction (RT-PCR). Correlations between the expression levels of each VEGF isoform and PDECGF and histopathologic findings were evaluated. Four VEGF isoforms corresponding to VEGF121, 165, 189, and 206 were detected in bladder cancer tissue by RT-PCR. Gene expression of all VEGF isoforms as a ratio of the target to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) showed no correlation with pathologic stage of bladder cancer. However, with regard to relative expression levels of VEGF isoform, which is the ratio to the sum of total VEGF isoforms, the levels of VEGF206 and VEGF189 in tumor samples of grade pT2 or higher were significantly lower than those in tumors of grade pT1 or lower (P<.05). In contrast, the levels of VEGF121 in >/=pT2 tumors tended to be higher than those in

Mechanism of primary graft non-function in a rat model for fatty liver transplantation.

We established a fatty liver model in rat suitable for the model of human liver with steatosis by cholesterol enriched chow, and investigated the mechanism of primary graft non-function in fatty liver transplantation (LTx) using this model. Grafts with steatosis caused primary graft dysfunction after LTx following even short cold preservation; however, no significant difference was recognized in mitochondrial function of the graft during preservation. Morphological findings were not different at 1 h after reperfusion between non-steatotic and steatotic livers. Focal necrosis of hepatocytes was seen and the sinusoidal endothelial cells were injured 24 h after reperfusion. In addition, the fluidity of the plasma membrane decreased in fatty liver. Our results indicate that deterioration of sinusoidal endothelial cells after reperfusion causes graft dysfunction in LTx of steatotic liver.

Regulation of cell cycle and apoptosis by human immunodeficiency virus type 1 Vpr.

Biological effects of HIV-1 Vpr on CD4(+) cells were studied by an infection system. High-titered HIV-1 stocks pseudotyped with vesicular stomatitis virus G protein were prepared and used to inoculate into CD4(+ )T cells at high multiplicity of infection. Both cell- and virion-associated Vpr were demonstrated to arrest the cell cycle at the G2/M phase, and to induce cell apoptosis. Of note, morphologically apoptotic cells were shown to be arrested at the G2/M stage. No appreciable effect of Vpr on the anti-Fas antibody-mediated apoptosis was observed in this system.

Physiological significance of apoptosis in animal virus infection.

In contrast to insect viruses, animal viruses can produce considerable amounts of progeny virus in cells undergoing apoptosis. Nevertheless, viruses in general have acquired the ability to escape apoptosis of infected cells. These facts indicate that the role of apoptosis in virus infection is different in insect virus and animal virus, although both viruses need to avoid apoptosis of the infected cells for a viral life cycle in nature. In animal virus infection, the primary role of apoptosis is considered not to be a premature lysis of the infected cells (and the following abortion of virus multiplication) but to allow the dying cells to be phagocytosed by macrophages. This phagocytosis is able to prevent dysregulated inflammatory reactions at the site of virus infection and to initiate a specific immune response against the infected virus.

A chemiluminescent assay for hydroperoxide level of phosphatidylcholine hydroperoxide fraction purified by two Sep-Pak cartridges in biological samples.

A chemiluminescent assay for hydroperoxide level of phosphatidylcholine hydroperoxide (PCOOH) fraction purified from biological samples was presented. This method utilized of two Sep-Pak cartridges. A lipid soluble fraction was isolated from each homogenized tissue or blood by Folch's method. The mixture of phosphatidylcholine (PC) and PCOOH was separated from the lipid soluble fraction by a Sep-Pak silica cartridge. A Sep-Pak tC18 cartridge made complete separation of both PCOOH and PC possible. The hydroperoxide level of PCOOH fraction was quantified by the reaction with ferrous ion using 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin++ +-3-one as a chemiluminescent dye. The mixture of positional isomers, 1-hexadecanoyl-2-[9, or 10-hydroperoxyl octadecanoyl]-sn-glycero-3-phosphocholine was used as an authentic standard. The good recovery rate for authentic PCOOH of 87.1 +/- 11.6% (mean +/- S.E., n = 4) was obtained by using two Sep-Pak cartridges. Linear calibration curve was obtained in the range from 2.5 to 20 nmol, and the detection limit of the standard was 10 pmol (signal-to-noise ratio > 3). This method was applied to the investigation of the lipid peroxidation induced by reperfusion of the liver with cold preservation, mimicking liver transplantation in rats. The effect of liposome-encapsulated dichloromethylene diphosphonate (LEDD), which eliminate of Kupffer cells to prevent the generation of oxygen radicals on the lipid peroxidation, was compared with the untreated group as a control. After 1 h reperfusion at 37 degrees C the hydroperoxide level obtained the liver without preservation in the untreated group was 12.4 +/- 2.4 nmol/100 mg lipid (n = 4) and levels increased significantly by prolongation of the preservation time. On the other hand, the hydroperoxide level in the LEDD treated group did not change up to 24 h preservation. These results suggest that this improved assay for hydroperoxide level of PCOOH fraction in biological samples can be applied to investigations involving lipid peroxidation because of its simplicity and accuracy.

MHC-I expression in HTLV-1-positive and -negative cells.

The expression level of major histocompatibility class I (MHC-I) and the extent of down-regulation of MHC-I after an anti-MHC-I antibody treatment in numerous human T-cell leukemia virus type 1 (HTLV-1)-positive and -negative lymphocytic cell lines were examined. While there was no clear correlation between the expression level of MHC-I and the presence of HTLV-1 genome, a relatively low level of MHC-I down-regulation was generally induced in HTLV-1-positive cells by the antibody. The results may suggest the potential involvement of MHC-I in HTLV-1 leukemogenesis.

Cell-dependent requirement of human immunodeficiency virus type 1 gp41 cytoplasmic tail for Env incorporation into virions.

Growth kinetics in lymphocytic H9 and M8166 cells of two mutants of human immunodeficiency virus type 1 (HIV-1) with deleted gp41 cytoplasmic tails were examined. While the mutant viruses designated CTdel-44 and CTdel-144 were able to grow in M8166 cells, they were unable to grow in H9 cells. Transfection and single-round infectivity assays demonstrated that they are defective in the early phase of viral replication in H9 cells. Analysis of the mutant virions revealed drastically reduced incorporation of Env gp120 (compared with the incorporation of wild-type virions) in H9 cells but normal incorporation in M8166 cells. These results indicate that the HIV-1 cytoplasmic tail of gp41 determines virus infectivity in a cell-dependent manner by affecting incorporation of Env into virions and suggest the involvement of a host cell factor(s) in the Env incorporation.

Nef-induced major histocompatibility complex class I down-regulation is functionally dissociated from its virion incorporation, enhancement of viral infectivity, and CD4 down-regulation.

The N-terminal alpha-helix domain of the human immunodeficiency virus type 1 (HIV-1) Nef protein plays important roles in enhancement of viral infectivity, virion incorporation of Nef, and the down-regulation of major histocompatibility complex class I (MHC-I) expression on cell surfaces. In this study, we demonstrated that Met 20 in the alpha-helix domain was indispensable for the ability of Nef to modulate MHC-I expression but not for other events. We also showed that Met 20 was unnecessary for the down-regulation of CD4. These findings indicate that the region governing MHC-I down-regulation is proximate in the alpha-helix domain but is dissociated functionally from that determining enhancement of viral infectivity, virion incorporation of Nef, and CD4 down-regulation.

Induction of apoptosis in Herpesvirus saimiri-immortalized T lymphocytes by blocking interaction of CD28 with CD80/CD86.

We have previously shown that Herpesvirus saimiri (HVS) immortalizes primary macaque monkey T lymphocytes. In this study, we examined the characteristics of the immortalized T cells. The cells showed the phenotype of activated T lymphoblasts (CD3(+) CD25(+) CD69(+) MHC-IIDR(+)) and produced no infectious virus while viral DNA was detected in the Hirt DNA. Interestingly, both a major costimulatory molecule, CD28, and its ligands, CD80/CD86, were coexpressed on the immortalized T cells. The treatment of the cells with a neutralizing monoclonal antibody against CD28, which blocks interaction of CD28 with CD80/CD86, resulted in retarded cell growth and in induction of apoptosis. The effect of the antibody treatment was not overcome by exogenous interleukin-2 treatment. These findings demonstrate the requirement of interaction of CD28 with CD80/CD86 for the optimal growth of HVS-immortalized T cells.