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Recent randomized controlled trials (RCTs) examined the role of adding androgen receptor signaling inhibitors (ARSIs), including abiraterone acetate (ABI), apalutamide, darolutamide (DAR), and enzalutamide (ENZ), to docetaxel (DOC) and androgen deprivation therapy (ADT) in patients with metastatic hormone-sensitive prostate cancer (mHSPC).To analyze the oncologic benefit of triplet combination therapies using ARSI + DOC + ADT, and comparing them with available treatment regimens in patients with mHSPC.Three databases and meetings abstracts were queried in April 2022 for RCTs analyzing patients treated with first-line combination systemic therapy for mHSPC. The primary interests of measure were overall survival (OS) and progression-free survival (PFS). Subgroup analyses were conducted to assess the differential outcomes in patients with low- and high-volume disease as well as de novo and metachronous metastasis.Overall, 11 RCTs were included for meta-analyses and network meta-analyses (NMAs). We found that the triplet combinations outperformed DOC + ADT in terms of OS (pooled hazard ratio [HR]: 0.74, 95% confidence interval [CI]: 0.65-0.84) and PFS (pooled HR: 0.49, 95% CI: 0.42-0.58). There was no statistically significant difference between patients with low- and high-volume disease in terms of an OS benefit from adding an ARSI to DOC +ADT (both HR: 0.79; p = 1). Based on NMAs, triplet therapy also outperformed ARSI + ADT in terms of OS (DAR + DOC + ADT: pooled HR: 0.74, 95% CI: 0.55-0.99) and PFS (ABI + DOC + ADT: HR: 0.68, 95% CI: 0.51-0.91, and ENZ + DOC + ADT: HR: 0.70, 95% CI: 0.53-0.93). An analysis of treatment ranking among de novo mHSPC patients showed that triplet therapy had the highest likelihood of improved OS in patients with high-volume disease; however, doublet therapy using ARSI + ADT had the highest likelihood of improved OS in patients with low-volume disease.We found that the triplet combination therapy improves survival endpoints in mHSPC patients compared with currently available doublet treatment regimens. Our findings need to be confirmed in further head-to-head trials with longer follow-up and among various patient populations.Our study suggests that triplet therapy with androgen receptor signaling inhibitor, docetaxel, androgen deprivation therapy prolongs survival in patients with metastatic hormone-sensitive prostate cancer compared with the current standard doublet therapy.

To assess the differential clinical outcomes of patients treated with partial nephrectomy (PN) vs. those treated with ablation therapy (AT) such as radiofrequency ablation, cryoablation and microwave ablation for cT1b compared to cT1a renal tumors.Multiple databases were searched for articles published before August 2021. Studies were deemed eligible if they compared clinical outcomes in patients who underwent PN with those who underwent AT for cT1a and/or cT1b renal tumors.Overall, 27 studies comprising 13,996 patients were eligible for this meta-analysis. In both cT1a and cT1b renal tumors, there was no significant difference in the percent decline of estimated glomerular filtration rates or in the overall/severe complication rates between PN and AT. Compared to AT, PN was associated with a lower risk of local recurrence in both patients with cT1a and cT1b tumors (cT1a: pooled risk ratio [RR]; 0.43, 95% confidence intervals [CI]; 0.28-0.66, cT1b: pooled RR; 0.41, 95%CI; 0.23-0.75). Subgroup analyses regarding the technical approach revealed no statistical difference in local recurrence rates between percutaneous AT and PN in patients with cT1a tumors (pooled RR; 0.61, 95%CI; 0.32-1.15). In cT1b, however, PN was associated with a lower risk of local recurrence (pooled RR; 0.45, 95%CI; 0.23-0.88). There was no difference in distant metastasis or cancer mortality rates between PN and AT in patients with cT1a, or cT1b tumors.AT has a substantially relevant disadvantage with regards to local recurrence compared to PN, particularly in cT1b renal tumors. Despite the limitations inherent to the nature of retrospective and unmatched primary cohorts, percutaneous AT could be used as a reasonable alternative treatment for well-selected patients with cT1a renal tumors.

Using laser powder bed fusion (L-PBF), we designed and fabricated specimens of a heat-resistant Al–4mass%Cr–1.5mass%Zr alloy (Al–4Cr–1.5Zr). The Al–4Cr–1.5Zr L-PBF specimen achieved high relative density greater than 99.9% by optimizing the laser scan conditions. The hardness and tensile and 0.2% proof stresses of the Al–4Cr–1.5Zr L-PBF specimens significantly increased after aging heat treatments, and the peak-aged specimens exhibited excellent high-temperature strength. The L-PBF specimens consisted of coarse columnar and fine equiaxed crystal grains owing to heterogeneous nucleation by the primarily crystallized Al3Zr phase. In the peak-aged L-PBF specimen, a finely dispersed Al3Zr granular phase on the order of single nanometers was precipitated in the α-Al matrix by aging. Meanwhile, the chromium was supersaturated in an α-Al solid solution even after peak aging, and partially precipitated into coarse granular Al–Cr-based compounds. These multi-scale microstructural observation results revealed that the dominant strengthening factors in the Al–4Cr–1.5Zr L-PBF specimens were precipitation hardening by the fine L12-Al3Zr phase having a strong crystallographic consistency with the α-Al matrix, solid-solution hardening with chromium, dispersion hardening due to the Al–Cr-based compounds, and hardening by the crystal grain refinement mainly with the fine equiaxed grains.

Background The purpose of this study was to investigate whether adding single immediate postoperative intravesical instillation of doxorubicin (SID) to transurethral resection of bladder tumor (TURBT) significantly reduced the risk of recurrence in patients with non–muscle-invasive bladder cancer (NMIBC). Materials and Methods We retrospectively analyzed the records of 720 patients diagnosed with primary NMIBC between 2002 through 2018 at the Kameda Medical Center. The primary outcome measure was time to recurrence. Time to progression was also compared. The cohort of SID and the cohort of TURBT alone were matched one-to-one by propensity scores. Matching was done by patient age, gender, and factors of the European Organization of Research and Treatment of Cancer recurrence risk table. The associations of adding SID and clinical outcomes were assessed with uni- and multivariate competing-risk regression models. Results After matching, a total of 364 patients, including 182 receiving SID and 182 receiving TURBT alone, were analyzed. No statistically significant differences existed among the measured baseline characteristics in propensity score-matched cohorts. In the multivariate analysis, there was a significantly longer time to recurrence in patients receiving SID (subdistribution hazard ratio, 0.68; 95% confidence interval, 0.49-0.95; P = .024) in propensity score-matched cohorts. There was no significant difference in time to progression (subdistribution hazard ratio, 0.61; 95% confidence interval, 0.11-3.49; P = .58) in univariate analysis. Conclusions Our results demonstrated that SID significantly reduced the recurrence risk of primary NMIBC. Doxorubicin could be an inexpensive alternative to other evidenced-based chemotherapeutic agents for single immediate intravesical chemotherapy.

Background Transcranial alternating current stimulation (tACS) can entrain and enhance cortical oscillatory activity in a frequency-dependent manner. In our previous study (Nakazono et al., 2016), 20 Hz (β) tACS significantly increased excitability of primary motor cortex compared with 10 Hz (α) tACS. α oscillations are a prominent feature of the primary visual cortex (V1) in a resting electroencephalogram. Hence, we investigated whether α and β tACS can differentially influence multiple visual functions. Methods Firstly, we evaluated the after-effects of α and β tACS on pattern-reversal (PR) and focal-flash (FF) visual evoked potentials (VEPs). Secondly, we determined the relationship between resting α oscillations and PR-VEPs modulated by tACS. Thirdly, the behavioral effects of tACS were assessed by contrast sensitivity. Results α tACS modulated the amplitudes of PR-VEPs, compared with β tACS, but did not modulate the FF-VEPs. Time-frequency analysis revealed that α tACS facilitated event-related α phase synchronizations without increasing power, which consequently increased the PR-VEP amplitudes. There was a significant positive correlation between PR-VEP amplitudes and resting α oscillations. These findings suggested that α tACS modulated α oscillations, and affected visual functions of contrast and spatial frequency. Indeed, α tACS also improved subjects’ contrast sensitivity at the behavioral level. Conversely, β tACS increased posterior α activity, but did not change VEP amplitudes. Conclusions α tACS can influence different neuronal populations from those influenced by β tACS. Thus, our results provide evidence that α tACS sharpens multiple visual functions by modulating α oscillations in V1.

There has been no established clinical evidence for using sequential treatment in castration-resistant prostate cancer (CRPC). Despite evident cross-resistance, androgen receptor axis-targeted agents (ARTAs), namely abiraterone (ABI) and enzalutamide (ENZ), are often used sequentially owing to less toxicity compared with chemotherapy.A multicenter retrospective review of chemotherapy-naive patients with CRPC who had received ABI followed by ENZ (ABI-to-ENZ) or ENZ followed by ABI (ENZ-to-ABI) was conducted. Combined progression-free survival (PFS), overall survival (OS), and prostate-specific antigen (PSA) response (≥ 50% PSA decline) to each drug were compared between the 2 groups at the median follow-up of 36.0 months.There were no significant differences in combined PFS (12.4 vs. 10.9 months; hazard ratio [HR], 0.94; 95% confidence interval [CI], 0.72-1.23; P = .6594) or OS (28.3 vs 29.3 months; HR, 0.96; 95% CI, 0.66-1.38; P = .8314) between the ABI-to-ENZ and ENZ-to-ABI groups. PSA response rate was not significantly different in first-line ARTAs (48.9% vs. 58.4%; P = .153) but significantly higher in ENZ as a second-line ARTA (40.4% vs. 13.7%; P .0001). Although multivariate analysis revealed that the ABI-to-ENZ sequence was associated with favorable PFS on second-line ARTA (HR, 0.65; 95% CI, 0.49-0.85; P = .0019), it was not associated with an increased combined PFS or OS.With relatively longer follow-up, ARTA sequence did not affect clinical outcomes of CRPC treatment except for PSA response and PFS on a second-line ARTA. These findings will be useful information in clinical decision-making, particularly in chemotherapy-unfit patients with CRPC.

In electrical discharge machining (EDM), it is important to evacuate EDM debris from the discharge gap for machining stability and accuracy. Especially in the processing of deep slots with high aspect ratio, EDM debris accumulated in the bottom of the groove often causes short circuit and false discharge, resulting in process instability. The tool electrode having micro holes for the machining fluid, which flush debris out of the discharge gap, enables efficient EDM machining of the deep slots. However, in conventional machining, it is difficult to drill high aspect ratio micro holes. Recently, the additive manufacturing (AM) technologies with the selective laser melting (SLM) method using metal powder attracts attention as a promising technique which can produce various complicated shapes efficiently. By using the AM method, it is possible to fabricate a high aspect ratio EDM electrode with micro holes inside. This paper describes the application of electrodes fabricated by the AM method to electrical discharge machining. Almost fully dense additive manufactured copper electrode (AM-Cu electrode) having more than 99.7% relative density was obtained by optimizing the laser scanning parameters for Cu-1.3mass%Cr (Cu-Cr) alloy powder. EDM properties of the AM-Cu tool electrode and the machinability of deep rib slots by using that electrode with micro holes inside were investigated. As a result, it was confirmed that EDM properties of the AM-Cu tool electrode were almost the same as those of conventional Cu tool electrode. Moreover, it was shown that use of the AM-Cu tool electrode with micro holes inside was found to shorten the processing time and improve machining accuracy significantly compared with the conventional solid Cu tool electrode.

We elucidated the microstructural formation and characterization mechanisms of Al–4Si–Mg selective laser melted (SLM) specimens with increasing Mg content (~2.5 mass%). First, the effect of Mg content on the processability of Al–4Si–Mg SLM specimens was systematically studied. From Al–4Si to Al–4Si–2.0Mg alloys, densified SLM specimens with a relative density of more than 99.5% were achieved by optimizing scan conditions. The relative density of the Al–4Si–2.5Mg SLM specimen decreased to approximately 99.4% due to the generation of gas pores by Mg evaporation. Then, the relation between the metal structures and mechanical properties and thermal conductivity of the Al–4Si–Mg SLM specimens was investigated. The tensile strength and 0.2% proof stress of the Al–4Si–Mg SLM specimens significantly increased with the Mg content. A multiscale metal structural analysis of the Al–4Si–Mg SLM specimens revealed that this strengthening was ascribed to multiple factors: both the crystal grain refinement and increase of solid-solute Mg in the α-Al matrix obstructed the dislocation movement. On the other hand, the elongation decreased in a stepwise manner with increasing Mg content. The SEM observations of the fracture surfaces in the Al–4Si–Mg tensile SLM specimens indicated that the decrease in elongation was attributed to the gas pores generated by Mg evaporation and shrinkage cavities. The thermal conductivity decreased slightly as the solid-solute Mg in the α-Al increased. These results can provide profound knowledge for the design of novel Al SLM alloys.

Pembrolizumab, an anti-PD-1 monoclonal antibody, revolutionized the treatment of advanced urothelial carcinoma. However, the tolerability and outcomes of pembrolizumab in patients with severe renal dysfunction [creatinine clearance (CrCl)30 ml/min] are unclear because no clinical trials included such patients. We analyzed the safety profile and outcomes of these patients in the real world.We extracted data for 739 pembrolizumab-treated patients from a Japanese nationwide cohort of platinum-refractory metastatic urothelial carcinoma. Using propensity score matching, the overall survival (OS) and adverse events (AEs) of patients with CrCl30 and ≥30 were compared.Ninety-two patients (12.4%) had CrCl30 ml/min. The median number of doses was similar between the CrCl ≥ 30 and CrCl30 groups (5 and 4, respectively), and there was no difference in the frequency of grade ≥2 treatment-related AEs between the groups (35.5% vs. 35.7%). The overall response rate was similar between the groups (27.2% vs. 29.7%, P = 0.184). Using propensity score matching, the median OS times in the CrCl ≥30 and CrCl30 groups were 10.3 (95% confidence interval [CI] = CI 7.3-13.0) and 8.1 months (95% CI = 5.4-14.6, P = 0.626), respectively. The 1-year OS rates in these groups were 41.5% and 38.2%, respectively, and the 2-year OS rates were 21.3% and 20.2%, respectively. In multivariate Cox regression analysis, performance status ≥2 (hazard ratio [HR] = 5.56, 95% CI = 2.64-11.71, P0.0001) and neutrophil-to-lymphocyte ratio ≥3 (HR = 2.20, 95% CI =1.15-4.19, P = 0.013) were independently associated with patient prognosis in the CrCl30 group.This report illustrated that pembrolizumab can be safely administered to patients with severe renal dysfunction, who had similar outcomes as patients without severe renal dysfunction.

Most patients with metastatic urothelial carcinoma experience no objective response to pembrolizumab and have poor overall survival (OS). Here, we investigated the prognostic value of fluctuation in the neutrophil-lymphocyte ratio (NLR) at 6 weeks of pembrolizumab treatment, focusing on its association with the achievement of objective response.The clinical records of 177 metastatic urothelial carcinoma patients treated with pembrolizumab were retrospectively analyzed.The median age was 72 years, and the median OS was 14 months. The objective response rate in the total cohort was 26.5% (47 of 177 patients). Multivariable analysis showed that objective response achievement (hazard ratio 0.3 [95% confidence interval 0.15-0.59], P0.001) and decline in NLR from that at baseline at 6 weeks of treatment (0.54 [0.34-0.88], P = 0.013) were independent prognostic factors for improved OS. For 47 (26.5%) patients who achieved an objective response, OS was similar regardless of NLR fluctuation at 6 weeks of treatment (P = 0.723). Intriguingly, of the 130 (73.5%) patients with no objective response, those who showed a decreased NLR at 6 weeks of pembrolizumab treatment (57 patients) from that at baseline had significantly longer OS than those with elevated NLR (73 patients) (14 vs. 6 months, P = 0.007).The fluctuation in NLR from that at baseline at 6 weeks of pembrolizumab treatment may be useful for patients without an objective response. This could potentially aid decision-making for post pembrolizumab therapies.

The association of concurrent proton pump inhibitor (PPI) use with treatment outcome of metastatic urothelial carcinoma (UC) remains controversial.We retrospectively analyzed the records of 227 patients with platinum-treated metastatic UC treated with pembrolizumab. The primary outcome was overall survival (OS). Immune progression-free survival (iPFS) and objective response per immune response evaluation criteria in solid tumors were also compared. Inverse probability of treatment weighting (IPTW)-adjusted multivariable Cox regression models and an IPTW-adjusted multivariable logistic regression model were used to evaluate the oncological outcomes. Furthermore, the heterogeneity of the treatment effect on OS was examined using interaction terms within the IPTW-adjusted univariate Cox regression models.Overall, 86 patients (37.9%) used PPIs. After weighting, no significant differences in patient characteristics were observed between PPI users and non-users. PPI use was significantly associated with a shorter OS (hazard ratio [HR]: 2.02, 95% confidence interval [CI]: 1.28-3.18, P = 0.003) and iPFS (HR: 1.70, 95% CI: 1.23-2.35, P = 0.001). Although not statistically significant, PPI use was associated with objective response as well (OR: 0.61, 95% CI: 0.36-1.02, P = 0.06). The interaction analyses showed that the effect of PPI significantly decreased with age (HR: 0.97, 95% CI: 0.93-1.00, P[interaction] = 0.048) and was increased in males (HR: 2.97, 95% CI: 1.10-8.05, P[interaction] = 0.032).PPI use was significantly associated with worse survival of patients with metastatic UC treated with pembrolizumab. Furthermore, the results suggested that its effects decreased with age and was increased in males.

Selective laser melting (SLM) has attracted considerable attention as an advanced method for the fabrication of biomedical devices. However, SLM-manufactured parts easily accumulate large amounts of residual stress due to rapid heating and cooling, which negatively affects their mechanical properties. In this study, Co–Cr–Mo alloy specimens were fabricated by SLM and then heat-treated at various temperatures (750, 900, 1050, or 1150 °C) to relieve the residual stress and improve their mechanical properties. The alloy microstructure was analyzed via confocal laser scanning microscopy, scanning electron microscopy combined with energy dispersive X-ray spectroscopy, electron backscattered diffraction, and X-ray diffraction techniques, whereas the mechanical properties of the produced specimens were evaluated by tensile and Vickers hardness tests. The results showed that increasing the heat-treatment temperature from 750 °C to 1150 °C increased the ductility of the alloy and decreased its 0.2% offset yield strength and Vickers hardness. Both γ and e phases formed in all heat-treated specimens, and the volume fraction of the e phase decreased with increasing heat-treatment temperature. After the specimens were heated to 750–1050 °C, a recovery process was initiated, which proceeded as the temperature increased; however, the residual stress in the studied specimens was not sufficiently relieved. In contrast, after heating to 1150 °C, the formation of equiaxed grains and the drastic relief of the residual stress were observed simultaneously, accompanied by an increase in the elongation of the specimen and a decrease in its strength (as compared to those of the other heat-treated specimens), indicating that the specimen completely recrystallized and that the residual stress was the driving force of this recrystallization. Thus, heat-treating at 1150 °C for 6 h is an effective method for eliminating the residual stress, leading to a homogenized microstructure and satisfactory ductility.

Purpose : The use of sentinel lymph node dissection in several cancers has been gaining attention with the emergence of indocyanine green fluorescence. We performed a meta-analysis to assess the diagnostic performance of indocyanine green fluorescence in detecting lymph node metastasis in prostate cancer patients. Methods : A literature search was conducted using PubMed, Cochrane Library, and SCOPUS on November 30, 2020, to identify eligible studies. Studies were eligible if they investigated the diagnostic performance of indocyanine green fluorescence before pelvic lymph node dissection in prostate cancer patients and reported the number of true positives, false positives, false negatives, and true negatives on lymph node–based analysis in comparison to histopathologic findings in the dissected specimen. Results : Our systematic review covered 11 studies published between 2011 and 2020, with 519 patients, and our meta-analysis included 9 studies with 479 patients. Based on lymph node analysis of indocyanine green fluorescence, the results showed pooled sensitivity and specificity at 0.75 (95% confidence interval [CI] 0.49 to 0.90) and 0.66 (95% CI 0.61 to 0.70), respectively. The diagnostic odds ratio was 6.0 (95%CI 2 to 21). Several lymphatic drainage routes also showed sentinel lymph nodes localized outside the ordinal pelvic lymph node template. Conclusions : We noted relatively low diagnostic performance for lymph node metastasis, suggesting that indocyanine fluorescence may not currently be a viable alternative to pelvic lymph node dissection in prostate cancer patients. However, this technique shows novel lymphatic drainage routes and underscores the importance of lymph nodes not removed in ordinary dissection.

Transition metals added as alloying elements to aluminum improve high-temperature strength. To design heat-resistant aluminum alloys for additive manufacturing, we have focused on the basic transition metals of iron (Fe), manganese (Mn), and chromium (Cr). The present study systematically examined the aging characteristics and the relationship between the microstructures and mechanical properties of aluminum-transition metal alloys, specifically Al-3mass%Fe, Al-3mass%Mn, and Al-3mass%Cr, fabricated by laser powder bed fusion (LPBF). All the LPBF samples were densified by optimizing laser scan parameters, achieving a relative density greater than 99.9%. The hardness values of the LPBF samples, especially Al-3mass%Fe, increased as a result of the aging heat treatment, leading to excellent high-temperature strength. From the extensive microstructural investigation, the peak aged Al-3mass%Fe and Al-3mass%Mn LPBF samples had microstructures with distinct network Al-Fe and granular/platy Al3Mn compounds with high-temperature stability, respectively. The improvement of mechanical properties in the Al-3mass%Fe and Al-3mass%Mn LPBF samples was thus attributed to the microstructural composite and dispersion hardening along with the grain boundary reinforcement. Meanwhile in the Al-3mass%Cr LPBF sample, chromium had a tendency to form a solid solution in the α-Al matrix, leading to solid-solution hardening. These results indicate that the strengthening factors significantly differed depending on the added transition metals.

The effect of the silicon content on the densification of Al-xSi binary alloys (x=0, 1, 4, 7, 10, 12, and 20 mass%) fabricated using selective laser melting (SLM) were systematically studied. By optimizing laser scanning parameters for each Al-xSi powder, almost fully dense SLM samples (more than 99.5% relative density) could be achieved for the Al-0Si (pure aluminum), and Al-4–20Si alloys. The Al-1Si SLM sample, on the other hand, contained many microcracks, considered to be solidification cracks. The Al-1Si SLM sample, in the solid-liquid coexisting state, was brittle and no healing of the cracks by the infiltration of the liquid phase occurred. Therefore, the cracks were easily generated due to thermally induced tensile stresses during solidification. The microstructures and the mechanical and thermal properties of the Al-xSi SLM samples fabricated using the optimal laser scanning parameters were examined. As the silicon content increased, the ultimate tensile strength and proof stress increased, whereas the elongation and thermal conductivity decreased. These mechanical properties were attributed to larger amounts of the crystalized phases of silicon (obstacles for the movement of dislocations) with increasing silicon content. The crystalized phases can also scatter conduction electrons, thereby decreasing the thermal conductivity.

To strengthen carbon fiber reinforced plastics (CFRPs), fiber placement along the principal stress directions have been studied as a promising approach. However, a method to sufficiently impregnate a resin between the curved carbon fibers has not established thus far, although arbitrary arrangement of carbon fibers is feasible using the automated fiber placement (AFP) machine. Previously, the authors had developed an electrodeposition resin molding method based on electrophoresis. In this method, the carbon fiber preforms sewn by the AFP machine is immersed in the electrodeposition solution containing polymers with epoxy groups, and optimal current is applied to impregnate resin between carbon fibers. In this study, we showed that a CFRP specimen could be fabricated with arranging the carbon fibers along the principal stress direction, resulting in approximately 10% higher tensile strength than that of the specimen with unidirectional carbon fibers.

We aimed to investigate the fatigue strength of Co-Cr-Mo clasps for removable partial dentures prepared by selective laser melting (SLM). The Co-Cr-Mo alloy specimens for tensile tests (dumbbell specimens) and fatigue tests (clasp specimens) were prepared by SLM with varying angles between the building and longitudinal directions (i.e., 0° (TL0, FL0), 45° (TL45, FL45), and 90° (TL90, FL90)). The clasp specimens were subjected to cyclic deformations of 0.25mm and 0.50mm for 10(6) cycles. The SLM specimens showed no obvious mechanical anisotropy in tensile tests and exhibited significantly higher yield strength and ultimate tensile strength than the cast specimens under all conditions. In contrast, a high degree of anisotropy in fatigue performance associated with the build orientation was found. For specimens under the 0.50mm deflection, FL90 exhibited significantly longer fatigue life (205,418 cycles) than the cast specimens (112,770 cycles). In contrast, the fatigue lives of FL0 (28,484 cycles) and FL45 (43,465 cycles) were significantly shorter. The surface roughnesses of FL0 and FL45 were considerably higher than those of the cast specimens, whereas there were no significant differences between FL90 and the cast specimens. Electron backscatter diffraction (EBSD) analysis indicated the grains of FL0 showed preferential close to001orientation of the γ phase along the normal direction to the fracture surface. In contrast, the FL45 and FL90 grains showed no significant preferential orientation. Fatigue strength may therefore be affected by a number of factors, including surface roughness and crystal orientation. The SLM process is a promising candidate for preparing tough removable partial denture frameworks, as long as the appropriate build direction is adopted.

We herein report the preparation of AlSi10Mg alloys with a range of porosities by selective laser melting (SLM) and subsequent hot isostatic pressing (HIP). The microstructures and mechanical properties of the resulting alloys were then investigated, and the influence of the internal porosity on the mechanical properties was investigated. Under the optimum laser irradiation conditions, the tensile strength of the alloy was very high; however, this tensile strength decreased with increasing the internal porosity. Following HIP, both the proof and tensile strengths decreased significantly due to drastic changes in the microstructures, i.e., the fine dendritic cell microstructure changed to granular precipitated Si phases. In addition, the initial porosity had a relatively small effect on the proof and tensile strengths and these strengths were comparable for all materials. In contrast, the ductility of the alloy improved significantly after HIP; however, it deteriorated as the initial porosity was increased. This was accounted for by the fact that HIP treatment was not really effective for eliminating the internal pores in the SLM AlSi10Mg alloy. Therefore, the reduction of internal pores by the optimization of laser irradiation conditions is essential in the fabrication of SLM aluminum alloys.

To investigate antimicrobial susceptibility patterns of various bacterial pathogens isolated from complicated urinary tract infection (UTI) cases, the Japanese Society of Chemotherapy, the Japanese Association of Infectious Disease, and the Japanese Society of Clinical Microbiology conducted the second nationwide surveillance from January to September 2011. With the cooperation of 42 medical institutions throughout Japan, 1036 strains belonging to 8 clinically relevant bacterial species were collected. Among methicillin-resistant Staphylococcus aureus (MRSA) strain, the vancomycin (VCM) MIC for 5.5% (3/55) of the strains was 2 μg/mL. Ampicillin, VCM, and linezolid were relatively active against 209 Enterococcus faecalis strains. The proportion of fluoroquinolone (FQ)-resistant strains was >20%. The MIC90 of FQs against the 382 Escherichia coli strains was 2-64 mg/L and the proportion resistant to FQs was approximately 30%. However, susceptibility of E. coli to sitafloxacin was still high (MIC90 = 2 mg/L). Fifty-eight (15.2%) of 382 E. coli, 6 (4.5%) of 132 Klebsiella pneumoniae, 1 (2.4%) of 41 Klebsiella oxytoca and 4 (6.8%) of 59 Proteus mirabilis strains were suspected of producing extended-spectrum beta-lactamase. Of 93 Pseudomonas aeruginosa strains, the proportions resistant to imipenem, amikacin, and ciprofloxacin were 21.5%, 4.3%, and 20.4%, respectively. Four strains (4.3%) were found to be multidrug-resistant. In complicated UTI cases, all of MRSA and E. faecalis were susceptible to all anti-MRSA agents. Sitafloxacin was active against other FQ-resistant E. coli strains. The isolation of extended-spectrum beta-lactamase-producing and multidrug-resistant strains increased.

Degarelix is a gonadotropin-releasing hormone (GnRH) antagonist that is approved for the treatment of prostate cancer. GnRH antagonists bind directly to and block GnRH receptors, without causing the initial testosterone surge associated with GnRH agonists. A pivotal phase III study indicated that degarelix induced significantly faster reduction of testosterone and prostate-specific antigen level than GnRH agonist does. In addition, its 5-year extension trial suggested that patients could be safely switched from GnRH agonist to degarelix treatment with sustained efficacy, as measured by biochemical markers. Possible benefits of GnRH antagonists over agonists were suggested especially in patients with advanced prostate cancer with metastatic and symptomatic disease. Moreover, the recent reports including pooled data analyses on degarelix suggest improved disease control, quality of life, and lower urinary tract symptoms and decreased risk of cardiovascular diseases when compared with GnRH agonists. However, interpretation of these reports should be conducted cautiously because of the potential biases involved. This article critically reviews the results of the clinical trials and subsequent analyses and evaluates the points and counterpoints of the conclusions.

We report a case of meningitis-retention syndrome followed by urodynamic tests. A 48-year-old man was admitted to the hospital for an undiagnosed fever with headache and urinary retention. Aseptic meningitis was suspected according to cerebrospinal fluid analyses, and urodynamic test showed an underactive detrusor, leading to inadequate contraction of the bladder on voiding in spite of a normal sensation during bladder filling. Clean intermittent self-catheterization was required temporarily, but normal urinary voiding without the need for medication was restored in 2 weeks after discharge from the hospital, when urodynamic tests showed normal contractility of the bladder during voiding.

Highlights • Heparin-induced thrombocytopenia is a rare but serious condition, regardless of the treatment dose or thromboprophylaxis. • A 55-year-old man, who underwent portless endoscopic radical nephrectomy, was diagnosed with non-immune mediated thrombocytopenia. • Surgeons should know that HIT is diagnosed based on clinical and serologic findings., Introduction Heparin-induced thrombocytopenia (HIT) is a rare but serious condition due to heparin use for treating thromboprophylaxis, regardless of the dosage. Here, we present a case of non-immune thrombocytopenia caused by thromboprophylaxis for urological surgery, which is sometimes difficult to discriminate from immune-mediated thrombocytopenia. Presentation of case A 55-year-old man with renal cancer underwent portless endoscopic radical nephrectomy through a single small incision and was subsequently administered unfractionated heparin as well as mechanical devices to prevent venous thromboembolism. On postoperative day 2, a subcutaneous hemorrhage developed around the surgical site and the lower abdomen, and the platelet count simultaneously decreased to 50% of the baseline value. We suspected HIT and immediately conducted the 4Ts score examination. The 4Ts score was 3 points (low probability), and the result of the platelet factor 4-heparin complex antibody assay was negative. The patient was diagnosed with non-immune mediated thrombocytopenia. We took precaution by discontinuing heparin, which fortunately did not result in any adverse effects, and this led to platelet count normalization. Discussion Due to the rarity of HIT, it is difficult to distinguish HIT from non-immune mediated thrombocytopenia. Conclusion This article emphasizes that early and accurate diagnosis of postoperative thrombocytopenia is important for accurate therapy. Hence, all surgeons should know that the HIT diagnosis is based on clinical and serologic findings.

Inflammation has been suggested to be involved in the pathogenesis of benign prostatic hyperplasia (BPH). We studied the prevalence of inflammation and BPH in Asian and Caucasian men on prostate glands (n=320) obtained during autopsy in Moscow, Russia (Caucasian men, n=220), and Tokyo, Japan (Asian men, n=100). We correlated the presence and grade of acute inflammation (AI) or chronic inflammation (CI) and BPH. AI, CI, and histologic BPH were analyzed in a blinded fashion using a grading system (0-3). We used the Cochran-Armitage test for associations between the degree of BPH and clinical variables and proportional odds logistic regression models in multivariable analysis. Histologic BPH was observed in a similar proportion of Asian and Caucasian men (p=0.94). CI was found in70% of men in both the Asian and Caucasian groups (p0.05). Higher BPH scores were associated with more CI (p0.001). In multivariate analyses, individuals with CI were 6.8 times more likely to have a higher BPH score than individuals without (p0.0001). Men included in this study presented at the hospital and their symptomatic status was not known. The prevalence of CI and BPH on autopsy is similar in Asian and Caucasian men despite very different diet and lifestyle. CI is strongly associated in both groups with BPH.In this study, we looked at the prevalence of inflammation and benign prostatic hyperplasia (BPH) on autopsy in Asian and Caucasian men. We found chronic inflammation in70% of men on autopsy. More chronic inflammation was associated with more BPH.

金沢大学医薬保健研究域 医学系, N-β-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an antibacterial substance isolated from the flesh fly, has been described as having multipotential biological activities toward various tissues. In a previous paper, we reported a novel neuroprotective action of 5-S-GAD on rat retinal ganglion cell apoptosis induced by optic nerve injury and intraocular N-methyl-d-aspartate treatment in vivo. In the present study, we further investigated the protective mechanism of this small peptide against other types of apoptosis in cultured cells of the established rat retinal ganglion cell line RGC-5. Hydrogen peroxide and serum deprivation treatments induced intracellular reactive oxygen species levels and lipid peroxidation, revealed by 4-hydroxy-2-nonenal production, in RGC-5 cells within 9-12 h. The treatments also induced cell death accompanied by nuclear condensation, DNA laddering and increases in apoptotic Bax and caspase-3 proteins in RGC-5 cells within 12-24 h. 5-S-GAD at 25-50 μM clearly suppressed the cell death and apoptotic features induced by these treatments. 5-S-GAD restored the nuclear condensation, DNA laddering and increases in apoptotic proteins. Furthermore, 5-S-GAD directly activated anti-apoptotic phospho-Akt and Bcl-2 proteins in RGC-5 cells. 5-S-GAD also quenched the reactive oxygen species production and inhibited the lipid peroxidation induced by oxidative stress. Therefore, 5-S-GAD may complementarily protect RGC-5 cells against apoptosis through dual actions as a radical scavenger and an inducer of anti-apoptotic phospho-Akt and Bcl-2. Taken together, 5-S-GAD is a high-potential tool for rescuing the retinal ganglion cell apoptosis induced by a variety of glaucomatous conditions. Crown Copyright © 2009.

Lentiviral vectors (LVs) are potential tools for genetic vaccination. To improve the safety of LV vaccines, we evaluated the selectivity, bio-distribution, persistence of expression, and immune potency of vesicular stomatitis virus G (VSV-G)-pseudotyped vectors transcriptionally targeted to antigen presenting cells (APCs) through a major histocompatibility complex class II (MHCII) promoter. Control vectors contained the ubiquitous cytomegalovirus (CMV) promoter. Bio-distribution studies after intravenous injections of LVs expressing green fluorescent protein (GFP) or luciferase were conducted by a combination of flow cytometry, immunofluorescence, real-time quantitative polymerase chain reaction (RT-Q-PCR) and whole-body bioluminescence analyses. GFP-expressing vectors showed selective expression in MHCII(+) cells of spleen and LV-CMV-GFP administration produced noticeable spleen inflammation, whereas LV-MHCII-GFP did not. Long-term optical imaging analyses of C57BL/6 mice injected with LV-CMV-LUC showed diminishing luciferase expression in the liver and spleen over time. Vaccination/boost with LV-CMV expressing the melanoma antigen tyrosinase-related protein 2 (TRP2) yielded dose-dependent antigen-specific CD8(+) T-cell reactivity and high protection against B16 melanoma challenge. Unexpectedly, administration of LVs containing the MHCII promoter resulted in persistence of luciferase expression and viral integration in MHCII(+) splenocytes and virtually no CD8(+) T-cell responses against TRP2. These studies reveal that APC transduction by LVs could lead to immune reactivity (LV-CMV) or persistence of transgene expression (LV-MHCII), providing a relevant paradigm for vaccination and gene replacement approaches.

Approaches facilitating generation of dendritic cell (DC) vaccines for clinical trials and enhancing their viability, bio-distribution, and capacity to stimulate antigen-specific immune responses are critical for immunotherapy. We programmed mouse bone marrow (BM) cells with lentiviral vectors (LV-GI4) so that they produced granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) in an autonomous manner. DC/LV-GI4 cells underwent autonomous trans-differentiation to yield typical phenotypic characteristics of DCs. DC/LV-GI4 cells that self-differentiated either ex vivo or in vivo showed persistent and robust viability and stimulated high influx of DCs into draining lymph nodes (LNs). The immunostimulatory efficacy of DC/LV-GI4 cells was evaluated using MART1 and TRP2 as co-expressed melanoma antigens. Mice vaccinated with DC/LV-GI4 cells that self-differentiated in vitro or in vivo produced potent antigen-specific responses against melanoma, which correlated with protective and long-term therapeutic anti-tumor effects. Thus, DC precursors can be genetically engineered after a single ex vivo manipulation, resulting in DC vaccines with improved activity.

There is a need to develop a computer aided detection (CAD) system for colonic polyps to assist in a physician's diagnostic load. In this paper, we propose a method for geometrically calculating curvatures based on surface-fitting. First the colonic wall region is segmented by applying a region-growing method to input images. For each point on the colonic wall (target point), we find iso-intensity points around the given processing area of interest by linear-interpolation. Surface fitting to the obtained points is performed using a least square error method. The size of the processing area is adaptively adjusted during the surface fitting process. The curvature of the target point is calculated from the first and second derivatives of the obtained surface. The shape index and curvedness defined by principal curvatures are then calculated. We extract voxels whose shapes are classified as convex and within the predefined curvedness. Colonic polyp candidates are obtained by performing connected component analysis including small component elimination. The proposed method was applied to noised artificial-figure images and abdominal CT images. Experimental results indicate that the proposed method detected less than 5 false-positives per case while maintaining 100% true positive rate, whereas the previous method generated 10 FPs under the same experimental conditions.

The characteristics of the microstructures and mechanical properties of friction stir welds of 60% Cu–40% Zn alloy (60/40 brass) were investigated. The defect-free welds were obtained in a relatively wide range of welding conditions; the tool rotation speed ranged from 1000 to 1500 rpm with a welding speed from 500 to 2000 mm/min, and 500 rpm—500 mm/min. The microstructures of the welds yielded extremely fine grains with some deformed grains in the stirred zone (SZ) and elongated grains in the thermo-mechanically affected zone. The hardness values within the SZ in all welding conditions were much higher than those of the base metal, increased with a decrease in heat input. The generation of refined grains in the SZ was a main factor which caused the hardness increase associated with decreasing heat input. The strengths of the all-SZ showed relative correspondence to the variation of the hardness values in the SZ.

Helicobacter pylori vacuolating cytotoxin, VacA, induces vacuolation, mitochondrial damage, cytochrome c release, and apoptosis of gastric epithelial cells. To detect gastric proteins that serve as VacA receptors, we used VacA co-immunoprecipitation techniques following biotinylation of the cell surface and identified p250, a receptor-like protein-tyrosine phosphatase β (RPTPβ) as a VacA-binding protein (Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999) J. Biol. Chem. 274, 36693–36699). VacA causes vacuolation of G401 cells, a human kidney tumor cell line, although they do not express RPTPβ. By co-immunoprecipitation with VacA, we identified p140 as a potential receptor in those cells. p140 purified by chromatography on a peanut agglutinin affinity matrix contained internal amino acid sequences of RGEENTDYVNASFIDGYRQK and AEGILDVFQTVK, which are identical to those in RPTPα. The peptide mass fingerprinting of p140 by time of flight-MS analysis also supported this identification. Treatment of G401 cells with RPTPα-morpholino antisense oligonucleotide before exposure to toxin inhibited vacuolation. These data suggest that RPTPα acts as a receptor for VacA in G401 cells. Thus, two receptor tyrosine phosphatases, RPTPα and RPTPβ, serve as VacA receptors.

The PC-based intelligent CAD system for cold forging process design has been developed in past research. The conventional system carries out the data input for every specific shape, and the process planning reasoning is carried out on the basis of the database made for each shape. By choosing this system constitution, it was advantageous to construct each inference system small-scale. However, the multiple of database and input/output system becomes a problem, when it is constructed by classifying system. Though system of one piece is small-scale, it becomes large as a whole system. It is necessary to integrate individual system in order to solve this problem. Construction of the input/output system that can deal with all products and the smart database become necessary for the reason. In this study, we developed the integrated process design system that constructed with classifying in order to avoid the multiple of the database. In this paper, input method of shape data that can deal with all product form is proposed. It is possible to deal with the identical format for all product forms by using this input method.

Previously, we reported a triplet repeat polymorphism in the transmembrane region within the MICA gene closely linked to HLA-B in a limited number of B27-positive Caucasian patients with ankylosing spondylitis (AS) (N = 48). In this study, we enrolled much more patients including some negative for B27, 162 AS subjects consisting of 140 B27-positive, and 22 B27-negative patients. The microsatellite allele consisting of 4 repetitions of (GCT/AGC) (A4 allele) was present at a significantly higher phenotype frequency in the patient group than in the ethnically matched control group (Pc < 0.000001). However, the frequency of the A4 allele was not significantly higher in the B27-positive and B27-negative patient groups, as compared to the B27-positive and B27-negative control groups, respectively. The higher phenotype frequency of the A4 allele in the patient group was supposed to be due to a strong linkage disequilibrium between the MICA and HLA-B genes. Thus, the possibility that the MICA gene is involved in the pathogenesis of AS can be excluded, supporting the hypothesis of a primary association of AS with HLA-B27.

We report on the first observation of surface second-harmonic generation (SHG) during the molecular beam epitaxy (MBE) growth of GaAs homoepitaxy on a GaAs(100) substrate. We used a specific optical arrangement which enabled us to observe the surface SHG and the reflection high-energy electron diffraction (RHEED) pattern simultaneously. We found that the surface SH intensity increased according to a change from an As-stabilized to a Ga-stabilized surface due to an interruption of the As flux while keeping the Ga flux constant. We show that the surface SH intensity has its maximum value when there is about 1 monolayer (ML) of excess Ga on the surface.

We have studied the atomic structure of hetero-interface and microstructural defects in Bi-Sr-Ca-Cu-O thin films by using cross-sectional high resolution transmission electron microscopy. The films were grown on (001) MgO substrates by chemical vapor deposition. From cross-sectional observation, it is proved that the interface between the thin film and the substrate is atomically very abrupt and no clear evidence of a reaction between the thin film and the substrate is found. A high degree of atomic order at the interface indicates that a Bi-O layer is initially grown on the substrates. We have also found that although multi-steps (1–2 nm) exist on the substrate surface, no anomalous growth or generation of defects have been observed at these steps. In very rare cases, facets inclined 10–20° to the substrate are observed. In this particular situation, we have discovered that the thin film is grown in such a way that the c -axis is exactly normal to one of these highly misoriented facets, indicating a rather weak epitaxial relationship in this system. In thin films, 90° twist boundaries or 90° grain boundaries are often generated. The surface region of the MgO substrate occasionally exhibits residual damage induced during the polishing process. Dislocation networks are often observed in the substrate, suggesting that it contains small angle tilt boundaries.

Bi-Sr-Ca-Cu-O thin films grown on (001) MgO substrates by chemical vapor deposition with T c at 110 K have been evaluated by transmission electron microscopy. From plan-view observations, the films are found to consist of large domains oriented along the c axis, with diameters of 15-100 μm. These domains exhibit incommensurate superstructures along the b axis. In addition, we have observed similar superstructure spots elongating in two equivalent directions normal to each other. Cross-sectional observation has indicated that the interface between the Bi-Sr-Ca-Cu-O thin film and the substrate is very abrupt. Five different perovskite-related layers along the c axis, with different thickness are successfully observed. In the thin film, 80 K phases with c = 3.0 nm are dominant and 110 K phases with c = 3.6 nm are less likely, which is very consistent with the results from X-ray diffraction. Three other phases are very rarely observed. Furthermore, boundaries where the layered structure is different on both sides are often found.

Behcet’s disease (BD) is known to be associated with HLA-B51. In order to investigate the influence of the MICB gene, located about 120 kb centromeric of the HLA-B gene, on the susceptibility to BD, (CA/TG) dinucleotide repeat microsatellite polymorphism in intron 1 of the MICB gene was investigated among 77 Japanese patients with BD, 60 randomly selected controls and 28 HLA-B51-positive unrelated healthy controls. There was no significant difference in the phenotype frequency of the microsatellite polymorphism between the BD patients and controls. This result suggests that the MICB gene itself is not responsible for the development of BD, and that the candidate gene(s) for BD is located between the MICA and HLA-C genes.

Introduction Androgen deprivation therapy (ADT) is the standard treatment of metastatic prostate cancer. Although ADT achieves disease stabilization in the beginning of the treatment, most patients eventually develop castration-resistant prostate cancer (CRPC). Docetaxel (DTX)-based chemotherapy has been proved the efficacy for CRPC, however, the prognosis of patients after the induction of chemotherapy is limited. Therefore, extending the period from the diagnosis of CRPC to the induction of chemotherapy may contribute to improvement of overall survival of those patients. Immunotherapy is being developed for several malignancies including prostate cancer. We have previously carried out phase-I/II study of personalized peptide vaccination in metastatic CRPC patients, and showed the safety and effectiveness of peptide vaccination therapy. Since cancer immunotherapy may act more effectively in early stage of the disease before systemic chemotherapy, we conducted a randomized phase II study to evaluate the efficacy of the peptide vaccination therapy for chemotherapy-naive CRPC patients. Patients and methods The patients are randomly assigned to two groups; one (vaccination group) receives the combination therapy of peptide vaccines and low-dose (1 mg/day) dexamethasone (Dx) and the other receives Dx alone. In the vaccination group, maximum of four kinds of peptide from those that have been recognized by the patient-specific immune system (serum IgG antibodies) in the peripheral blood are selected as vaccines. 3mg of each peptide is administrated separately every 2 weeks. Dexamethasone 1 mg/day p.o. is started on the first day of peptide administration. The primary end point of the study is progression-free survival and secondary end points include objective response rate and subjective effects. Results Up to now, 72 CRPC patients were enrolled in this study and 60 were evaluable and analyzed in detail. Median age was 68 years old (48–80 years) and median serum PSA was 4.8 ng/ml. The patients in vaccination group showed a significantly longer PSA progression free survival (P

In vivo administration of lentiviral vectors is a promising novel approach for genetic vaccination and for gene replacement therapy. Bio-distribution studies have shown that i.v. systemic administration of lentiviral vectors produces conspicuous gene marking of antigen presenting cells (APCs) in the liver and spleen. Expression and presentation of immunogenic antigens by APCs may explain the potential of lentiviral vectors for vaccination and the poor persistent expression in immunocompetent animals. Based on this rationale, we attempted to improve the biosafety and immunization potential of lentiviral vectors by targeting the transgene expression to APCs through the Major Histocompatibility Complex class II (MHCII) promoter. We produced third generation self-inactivating vectors with the MHCII or with the CMV promoter as ubiquitous expression control. Ex vivo transduction of MHCII+ and MHCII- cell lines and primary cells using GFP and luciferase as reporter genes confirmed the expression and selectivity of vectors with the MHCII promoter. Nu/Nu nude and C57BL/6 mice (n=3 per group) were injected i.v. with 2 mcg p24 equivalent of vectors expressing firefly luciferase (LV-CMV-LUC or LV-MHCII-LUC) and were analyzed by optical imaging on weeks 1, 2, 4, 8, 12, 16 and 24 after injections. Statistical analyses used a linear regression model comparing the overall differences (bioluminescence signal detected in liver and spleen) between the LV-CMV-LUC and LV-MHCII-LUC injected groups in early (1-8 weeks) and late (8-24 weeks) periods. Nude mice injected with LV-CMV-LUC and LV-MHCII-LUC showed increase in bioluminescence signal during the first 8 weeks, which maintained stable for the late period. Notably, a significant drop in bioluminescence signal occurred for LV-CMV-LUC expression in C57BL/6 mice throughout the observation periods (slope= -21+/-8, p=0.0112), whereas for LV-MHCII-LUC a much lower drop was observed (slope= -8+/-7.4, p=0.2827) and the signal persisted for 24 weeks. Subcutaneous administration of vectors produced similar results. Additionally, we performed immunologic studies with vectors expressing the melanoma-associated antigen TRP2. I.v. vaccination of C57BL/6 mice with LV-CMV-TRP2 produced high frequencies of TRP-2 specific CTL responses (4.5% - 12.5% of circulating CD8+ cells, measured by flow cytometry after anti-TRP-2 tetramer staining or intracellular IFN-gamma staining) and significant (p=0.0365) protection against B16 melanoma challenge. Vaccination with LV-MHCII-TRP2 yielded baseline results comparable to the placebo vaccination group. In summary, these results indicate that transgene expression regulated through the MHCII promoter may induce a tolerogenic effect that would be unsuitable for use in vaccination strategies, but might allow more persistent expression in gene replacement therapy.

Top of pageAbstract Dendritic cell (DC) vaccines are conventionally produced ex vivo by culturing DC precursors in the presence of recombinant growth factors. Methods to facilitate the generation of DCs, to prolong their viability, to facilitate their migration to lymphoid tissues and to augment their capacity to establish and maintain antigen-specific immune responses are critical for improving their effective therapeutic potential for immunotherapy. To address these issues, we have genetically engineered purified human CD14+ monocytes with lentiviral vectors (LV) for production of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), which triggered their differentiation into cells with typical characteristics of DCs. We previously demonstrated that these lentivirus programmed DCs (DC/LVs) exhibited more potent immunostimulatory function and prolonged viability in vitro than DCs generated by conventional methods. Using the same strategy, we have now generated murine DC/LVs in order to test the efficacy of this approach in vivo. Third generation self-inactivating LV (5 |[mu]|g/ml p24 equivalent) transduced non-adherent bone marrow (BM) cells harvested from C57BL/6 mice at >90% transduction efficiency. BM cells co-expressing GM-CSF and IL-4 after LV transduction produced these cytokines at concentrations similar to those commonly used for generation of conventionally cultured DCs by addition of exogenous cytokines (> 10ng/ml), resulting in the generation of DC/LVs as evidenced by their morphology and immunophenotype (CD11b+, CD11c high, CD80 high, MHCII high). DC/LVs maintained in culture were metabolically active and viable for 3 weeks, whereas conventional DCs showed a drastic decrease in viability in the first week of culture. In vivo bioluminescence imaging studies were then performed using luciferase as a marking gene to track DCs after s.c. injection into nude syngeneic mice. Persistent and robust bioluminescence signals were detectable from the injected DC/LVs for more than 45 days, whereas signals produced by conv. DCs faded away within the first two weeks. To determine whether the higher viability of DC/LVs correlated with greater potency as vaccines, we next examined their antigen presentation and immunostimulatory functions in vivo. DC/LVs and conv. DCs maintained in culture for 7 days were transduced to express the melanoma associated antigen MART-1 and injected s.c. Splenocytes harvested 2 weeks after vaccination with DC/LVs showed up to 2-fold increase in activated T cells by IFN|[gamma]|- ELISPOT assay (p

Intravenous administration of high titer VSV-G pseudotyped lentiviral vectors (LVs) leads to conspicuous gene transfer into liver, spleen and bone marrow, as previously shown by PCR, immunohistochemistry (IHC) and flow cytometry analyses. Interestingly, antigen presenting cells (APCs) corresponding to dendritic cells (DCs) and hematopoietic precursor cells homing in these tissues seem to be main target cell populations for gene delivery and expression. These results correlate with several reports showing that systemic administration of LVs expressing melanoma-associated antigens induced protective anti-tumoral immune responses. Thus, LVs are currently being explored as tools for genetic immunization, which warrants further understanding regarding the mechanisms of in vivo bio-distribution of these vectors. Furthermore, one of the logical advances toward the safety of LVs for vaccine development is the design of tissue specific promoters to restrict transgene expression APCs. In this study, we aim to characterize transcriptionally targeted LVs in vivo using bioluminescence imaging analyses, which allows to assess dynamically the spatio-temporal pattern of gene expression. Thus, nude mice were initially injected intravenously with a single dose (6 mcg of p24 equivalent) of the LV containing the CMV promoter driving transcription of firefly luciferase. After injecting mice with the luciferin substrate, in vivo whole-body optical imaging was performed using an IVIS bioluminescence optical imaging system at different time points, ranging from 3 days to 48 days post injection. Notably, during the first week the bioluminescence signal was more localized in the liver and spleen, but it gradually it dispersed towards the bones. We hypothesize that the initial gene expression pattern in the liver and spleen is due to their first-pass effect and immunologic sentinel functions, respectively, and that the subsequent shift of expression towards the bones is due to the gene expression by hematopoietic precursors present in the bone marrow that have self-renewal capacity and are able to sustain persistent LV expression. In order to explore and optimize this potential natural tropism of LVs to DCs and hematopoietic precursors for genetic immunization purposes, we are now testing LVs containing regulated promoters to restrict transgene expression in mature APCs. A LV containing the HLA-DRalpha promoter previously shown to confer high specificity of expression in MHCII+ DCs (Cui, Y., et al., Blood, 2002. 99(2): p. 399|[ndash]|408) is currently being evaluated for biodistribution through bioluminescence imaging analyses. In addition, we have generated a LV containing the human CD83 promoter (kindly provided by Dr. A. Steinkasserer), which successfully conferred up-regulation of transgene expression in DCs in vitro upon maturation with LPS or soluble CD40L. With these tools in hands and using a combination of bioluminescence imaging analyses, IHC and flow cytometry analyses, we seek to evaluate the efficacy and biosafety of these APC-transcriptionally targeted LVs for future vaccine development.