Homoharringtonine is a transdermal granular permeation enhancer.

Although modulation of claudin-1-based tight junction (TJ) in stratum granulosum is an option for transdermal absorption of drugs, granular permeation enhancers have never been developed. We previously found that homoharringtonine (HHT), a natural alkanoid, weakened intestinal epithelial barrier with changing expression and cellular localization of TJ components such as claudin-1 and claudin-4. In the present study, we investigated whether HHT is an epidermal granular permeation enhancer. Treatment of normal human epidermal keratinocytes (NHEK) cells with HHT decreased claudin-1 and claudin-4 but not zonula occludens-1 and E-cadherin. HHT lowered TJ-integrity in NHEK cells, accompanied by permeation-enhancement of dextran (4 kDa) in a dose-dependent manner. Transdermal treatment of mice with HHT weakened epidermal barrier. HHT treatment enhanced transdermal absorption of dextran with a molecular mass of up to 10 kDa. Together, HHT may be a transdermal absorption enhancer.

A phase I dose-escalation, safety/tolerability, and preliminary efficacy study of the intratumoral administration of GEN0101 in patients with advanced melanoma.

Despite recent advance in immunotherapy agents, safe new therapies that enhance the effects of immune checkpoint inhibitors are still required to develop. We previously demonstrated that hemagglutinating virus of Japan-envelope (HVJ-E) induced not only direct tumor cell death but also antitumor immunity through the activation of T and natural killer (NK) cells, thereafter, developed a manufacturing process of HVJ-E (GEN0101) for clinical use. We here performed a phase Ia clinical trial of intratumoral GEN0101 administration in six patients with stage IIIC or IV malignant melanoma. The primary aim was to evaluate the safety and tolerability of GEN0101, and the secondary aim was to examine the objective tumor response. Patients were separated into two groups (n = 3 each) and received a low dose of 30,000 and high dose of 60,000 mNAU of GEN0101. All patients completed a two-week follow-up evaluation without severe adverse events. The overall response rate was 33% (2 of 6), with 2 partial responses in the high-dose group and 2 with stable disease, and 2 with progressive disease in the low-dose group. Local complete or partial responses were observed in 11 of 18 (61%) target lesions. One patient demonstrated shrinkage of lung metastases after the treatment. The activity of NK cells and interferon-γ levels were increased in the circulation, indicating augmentation of antitumor immunity by GEN0101. This trial showed not only the safety and tolerability but also the significant antitumor effect of GEN0101, suggesting that GEN0101 might be a promising new drug for patients with advanced melanoma.

Impact of surgical resection on survival in patients with metastatic soft tissue sarcoma and comparison between synchronous and metachronous metastases.

BACKGROUND: The role of local surgery in patients with metastatic soft tissue sarcoma (STS) remains unknown. The study aims to assess the clinical outcomes and impact of surgical resection on survival in patients with metastatic STS and elucidate the survival differences between synchronous and metachronous metastatic groups. METHODS: Among the 272 patients with STS treated between 2000 and 2018, 84 with synchronous or metachronous metastasis were included. Associations between overall survival and primary tumor resection and metastasectomy were assessed using multivariate Cox regression analyses to adjust for baseline differences between surgically and non-surgically treated patients. Propensity score matching was applied to compare synchronous and metachronous metastasis. RESULTS: Among the 84 patients included, 69 (82%) and 41 (49%) underwent primary tumor resection and metastasectomy, respectively. The 2- and 5-year overall survivals of all patients after first detection of metastasis were 51.1% and 24.4%, respectively. Multivariate analysis showed that size <8 cm, grade <3, and number of metastases <4 were associated with longer overall survival. After adjusting for baseline demographic and tumor characteristics, primary tumor resection and metastasectomy still had favorable effects on survival. Tumor subtypes, grade, and number of metastases differed significantly between synchronous and metachronous groups. However, after adjusting for these valuables, both groups exhibited comparable survival. CONCLUSIONS: Approximately one fourth of the patients with metastatic STS survived for >5 years. Our results showed that surgical resection of primary tumors or metastatic lesions had favorable impact on survival even after adjusting for patient backgrounds, with comparable survival observed between those with synchronous and metachronous metastases.

Intratumoral injection of hemagglutinating virus of Japan-envelope vector yielded an antitumor effect for advanced melanoma: a phase I/IIa clinical study.

Hemagglutinating virus of Japan (HVJ; Sendai virus) is an RNA virus that has cell fusion activity. HVJ-envelope (HVJ-E) is a UV-irradiated HVJ particle that loses viral replication and protein synthesis activity but retains cell fusion activity. We recently reported that HVJ-E has antitumor effects on several types of tumors. Here, we describe the results of a first-in-human phase I/IIa study in patients with advanced melanoma, receiving intratumoral administration of HVJ-E. The primary aim was to evaluate the safety and tolerability of HVJ-E, and the secondary aim was to examine the objective tumor response and antitumor immunity. Six patients with stage IIIC or IV progressive malignant melanoma with skin or lymph metastasis were enrolled. Patients were separated into two groups (n = 3 each) and received low and high doses of HVJ-E. Five of the six patients completed 4 weeks of follow-up evaluation; one patient discontinued treatment owing to progressive disease. Complete or partial responses were observed in 3 of 6 (50%) injected target lesions, 7 of 15 (47%) noninjected target lesions, and 10 of 21 (48%) target lesions. Induction of antitumor immunity was observed: activation of natural killer cells, a marked increase in interferon-γ levels in the peripheral blood, and infiltration of cytotoxic T cells into both injected and noninjected tumor lesions. Thus, intratumoral injection of HVJ-E in advanced melanoma patients showed safety and tolerability with local regression of the tumor mediated by antitumor immunity. The results suggest that HVJ-E might be a new treatment approach in patients with advanced melanoma.

Therapeutic potential of TAS-115 via c-MET and PDGFRα signal inhibition for synovial sarcoma.

BACKGROUND: The prognosis of synovial sarcoma (SS), an aggressive soft tissue sarcoma, remains poor. We previously reported that c-MET or platelet-derived growth factor receptor α (PDGFRα) signalling pathway is related to SS progression based upon the findings of phospho-receptor tyrosine kinase (RTK) arrays. TAS-115 is a novel c-MET/ vascular endothelial growth factor receptor-targeting tyrosine kinase inhibitor that has been shown to inhibit multiple RTKs. Here we aimed to investigate the therapeutic potential of TAS-115 against SS. METHODS: We first evaluated which signalling pathway was relevant to the viability of three human SS cell lines: Yamato-SS, SYO-1 and HS-SY-II. Next, we assessed the anticancer activity and mechanism of action of TAS-115 in these SS cell lines. Finally, we compared the ability of TAS-115 to inhibit c-MET and PDGFRα phosphorylation with that of pazopanib. RESULTS: We classified the SS cell lines as c-MET-dependent or PDGFRα-dependent based upon the differences in the signalling pathway relevant for growth and/or survival. We also found that c-MET and PDGFRα were the primary activators of both phosphatidylinositol 3-kinase/AKT and mitogen-activated protein kinase pathways in c-MET-dependent and PDGFRα-dependent SS cells, respectively. TAS-115 treatment blocked the phosphorylation of PDGFRα as well as that of c-MET and their downstream effectors, leading to marked growth inhibition in both types of SS cell lines in in vitro and in vivo studies. Furthermore, PDGFRα phosphorylation, on at least four representative autophosphorylation sites, was impeded by TAS-115 equivalently to pazopanib. CONCLUSIONS: These experimental results have demonstrated the significance of c-MET and PDGFRα signalling for growth and/or survival of SS tumours. TAS-115 monotherapy may benefit SS patients whose tumours are dependent upon either c-MET or PDGFRα signalling by functioning as a multiple tyrosine kinase inhibitor to suppress c-MET as well as PDGFRα pathways.

Radiofrequency ablation of osteoid osteoma using a three-dimensional navigation system.

OBJECTIVE: This study aimed to report the clinical outcomes for patients with osteoid osteoma (OO) treated by radiofrequency ablation (RFA) using a three-dimensional (3D) navigation system. METHODS: We performed RFA using a 3D navigation system on 32 patients with clinically and radiologically diagnosed OO. This study included 25 males and 7 females with a median age of 20 years (range, 10-39 years). The median duration of follow-up was 18 months (range, 1-65 months). We investigated technical specifications, tumor localization, technical success, clinical success, biopsy success, complications, incomplete treatment, and recurrences. RESULTS: Eighteen tumors were located in the femur, seven in the tibia, two in the humerus, and one each in the fibula, scapula, patella, lumbar vertebra, and acetabula. All procedures were technically successful, and pain relief was achieved in all patients. However, local recurrence developed in one patient, needing additional RFA. The clinical success rate was 96.8%. Biopsy showed OO in 12 patients (37%). Complications occurred in three patients (9%), two cases of fractures and one of osteomyelitis. CONCLUSIONS: A 3D navigation provides real-time imaging and enables us to set the RFA needle in the correct position, particularly in case of OO-aroused complex anatomical structures. Our initial results indicated that radiofrequency ablation using a 3D navigation system is feasible and safe for patients with OO.

Combined targeting of mTOR and c-MET signaling pathways for effective management of epithelioid sarcoma.

BACKGROUND: Epithelioid sarcoma (EpS) is a high-grade malignant soft-tissue sarcoma characterized by local recurrences and distant metastases. Effective treatments for EpS have not been established and thus novel therapeutic approaches against EpS are urgently required. mTOR inhibitors exert antitumor effects on several malignancies but AKT reactivation by mTOR inhibition attenuates the antitumor effects of mTOR inhibitors. This reactivation is receptor tyrosine kinase (RTK)-dependent due to a release of negative feedback inhibition. We found that c-MET was the most highly activated RTK in two human EpS cell lines, Asra-EPS and VAESBJ. Here we investigated the functional and therapeutic relevance of mTOR and/or c-MET signaling pathways in EpS both in vitro and in vivo. METHODS: We first examined the effects of an mTOR inhibitor, RAD001 (everolimus), on cell proliferation, cell cycle, AKT/mTOR signaling, and xenograft tumor growth in EpS cell lines. Next, we determined whether RAD001-induced AKT reactivation was blocked by silencing of c-MET or treatment with a selective c-MET inhibitor, INC280. Finally, we evaluated the antitumor effects of RAD001 combined with INC280 on EpS cell lines compared with either single agent or control in vitro and in vivo. RESULTS: Constitutive AKT phosphorylation was observed in Asra-EPS and VAESBJ cells. RAD001 suppressed EpS cell growth by inducing cell cycle arrest but enhanced AKT phosphorylation, which resulted in intrinsic resistance to mTOR inhibitors. In both EpS cell lines, RAD001-induced AKT phosphorylation was dependent on c-MET signaling. INC280 inhibited phosphorylation of c-MET and its downstream molecules, and decreased RAD001-induced phosphorylation of both AKT and ERK in EpS. Compared with a single agent or control, the combination of RAD001 and INC280 exerted superior antitumor effects on the growth of EpS cell lines in vitro and in vivo. CONCLUSIONS: Targeting of mTOR and c-MET signaling pathways significantly abrogates the growth of EpS in preclinical models and may be a promising therapeutic approach for patients with EpS.

A giant periosteal chondroma of the distal femur successfully reconstructed with synthetic bone grafts and a bioresorbable plate: a case report.

Periosteal chondromas are rare benign cartilaginous tumors that arise adjacent to the cortex beneath the periosteum. These lesions are usually slow-growing and rarely exceed 3 cm in the greatest dimension. Here, we describe a 17-year-old boy who had a giant periosteal chondroma of the right distal femur, which was treated with intralesional resection and intensive curettage. In addition, we report a novel application of a bioresorbable plate in the management of the large bone defect after resection of a benign bone tumor.

Radiofrequency ablation for treatment for osteoid osteoma of the scapula using a new three-dimensional fluoroscopic navigation system.

Osteoid osteoma is a relatively common benign skeletal tumor. The traditional standard treatment has been surgical resection of the nidus. Recently, computed tomography (CT)-guided radiofrequency ablation (RFA) has gained favor as a more precise alternative due to potentially less bone destruction. However, CT-guided RFA is limited in treatment for osteoid osteoma involving complex anatomic structures such as cervical spine, pelvis, or scapula because of difficulty in approach and proximity to neurovascular structures. To solve this problem, we investigated RFA using a new real-time three-dimensional fluoroscopic navigation system. We report its technical procedure and use in a rare case of osteoid osteoma of the scapula.

Remote audit practice for inspection of structural and equipment standards for cell processing facilities under the act on the safety of regenerative medicine in Japan.

Introduction: The Act on the Safety of Regenerative Medicine enforced in Japan in 2014, regulates the manufacture of cellular processed products. However, with regards to the manufacturing facilities at medical institutions, only the submission of necessary documents is required for a license, and the need for third-party inspection has been highlighted. Remote activities are becoming more prominent with the spread of the Severe Acute Respiratory Syndrome Coronavirus 2 infection; therefore, the current assessment of compliance with structural facility standards was conducted remotely. Methods: The entire process, including start-up meetings, preparation of the survey schedule, submission and review of preliminary materials, audits, and reporting of results, was conducted via e-mail and web conferencing systems. The survey was conducted remotely, to minimize the risk of contamination of the cell processing facility (CPF) and reduce the burden on surveyors, while contributing to the establishment of suitable structural facilities by identifying and highlighting the areas or items that were considered to be non-compliant with the regulations. The series of audits were completed in ten weeks, with a period of six weeks between the start-up meeting and the audit implementation. The audit was completed in approximately 3 h on the day of the inspection. Results: The audit results were delivered in the report, with four items requiring improvement and several other recommended items listed as non-conformities. Conclusions: We believe that this remote method allows the effective inspection of regenerative medicine manufacturing facilities and assessment of more cell culture processing facilities than the current in-person audit method, with limited human resources.

Identification of unapproved orthopedic regenerative medicine: Usefulness of the Act on Safety of Regenerative Medicine.

In Japan, the Act on Safety of Regenerative Medicine regulates unapproved regenerative medicine. Other nations market regenerative medicine products, bypassing regulatory approval. To identify unapproved orthopedic regenerative medicine, we have used data based on the Act. Platelet-rich plasma was often used. The common target was the knee. Prices averaged $2,490.

Bone tissue engineering with bone marrow-derived stromal cells integrated with concentrated growth factor in Rattus norvegicus calvaria defect model.

Concentrated growth factor (CGF) is an autologous leukocyte-rich and platelet-rich fibrin (L-PRF) biomaterial termed "second-generation platelet concentrate". CGF contains autologous osteoinductive platelet growth factors and an osteoconductive fibrin matrix. The purpose of this study was to assess the ability of CGF combined with bone marrow stromal cells (BMSCs) to heal critical-size rat calvaria defects in vivo and to modulate the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. In the in-vivo study, the CGF group regenerated bone better than the control group, and combined therapy with CGF and BMSCs almost completely repaired critical-size bone defects within 12 weeks after surgery. In the in-vitro study, the CGF extract, at concentrations between 1 and 10%, promoted proliferation, osteogenic maturation, and mineralization of hTERT-E6/E7 human MSCs in a dose-dependent manner but had an inhibitory effect at higher concentrations. In conclusion, a CGF extract promoted the proliferation, osteogenic maturation, and mineralization of mesenchymal stem cells in vitro, and combination therapy with CGF and BMSCs resulted in excellent healing of critical-size bone defects in vivo.

Safety and immunogenicity of BK-SE36/CpG malaria vaccine in healthy Burkinabe adults and children: a phase 1b randomised, controlled, double-blinded, age de-escalation trial.

Background: BK-SE36/CpG is a recombinant blood-stage malaria vaccine candidate based on the N-terminal Plasmodium falciparum serine repeat antigen5 (SE36), adsorbed to aluminium hydroxide gel and reconstituted, prior to administration, with synthetic oligodeoxynucleotides bearing CpG motifs. In healthy Japanese adult males, BK-SE36/CpG was well tolerated. This study assessed its safety and immunogenicity in healthy malaria-exposed African adults and children. Methods: A double-blind, randomised, controlled, age de-escalating clinical trial was conducted in an urban area of Ouagadougou, Burkina Faso. Healthy participants (n=135) aged 21-45 years (Cohort 1), 5-10 years (Cohort 2) and 12-24 months (Cohort 3) were randomised to receive three vaccine doses (Day 0, 28 and 112) of BK-SE36/CpG or rabies vaccine by intramuscular injection. Results: One hundred thirty-four of 135 (99.2%) subjects received all three scheduled vaccine doses. Vaccinations were well tolerated with no related Grade 3 (severe) adverse events (AEs). Pain/limitation of limb movement, headache in adults and fever in younger children (all mild to moderate in intensity) were the most frequently observed local and systemic AEs. Eighty-three of BK-SE36/CpG (91%) recipients and 37 of control subjects (84%) had Grade 1/2 events within 28 days post vaccination. Events considered by the investigator to be vaccine related were experienced by 38% and 14% of subjects in BK-SE36/CpG and control arms, respectively. Throughout the trial, six Grade 3 events (in 4 subjects), not related to vaccination, were recorded in the BK-SE36/CpG arm: 5 events (in 3 subjects) within 28 days of vaccination. All serious adverse events (SAEs) (n=5) were due to severe malaria (52-226 days post vaccination) and not related to vaccination. In all cohorts, BK-SE36/CpG arm had higher antibody titres after Dose 3 than after Dose 2. Younger cohorts had stronger immune responses (12-24-month-old > 5-10 years-old > 21-45 years-old). Sera predominantly reacted to peptides that lie in intrinsically unstructured regions of SE36. In the control arm, there were no marked fold changes in antibody titres and participants' sera reacted poorly to all peptides spanning SE36. Conclusion: BK-SE36/CpG was well-tolerated and immunogenic. These results pave the way for further proof-of-concept studies to demonstrate vaccine efficacy. Clinical trial registration: https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=1921, PACTR201701001921166.

Biased usage of T cell receptor ß-chain variable region genes of Wilms' tumor gene (WT1)-specific CD8+ T cells in patients with solid tumors and healthy donors.

Wilms' tumor gene 1 (WT1) protein is a promising tumor-associated antigen. In patients with WT1-expressing malignancies, WT1-specific CTLs are spontaneously induced as a result of an immune response to the WT1 protein. In the present study, we performed single cell-level comparative analysis of T cell receptor ß-chain variable region (TCR-BV) gene families of a total of 750 spontaneously induced WT1(126) peptide (amino acids 126-134, WT1(126))-specific CTLs in both HLA-A*0201(+) patients with solid tumors and healthy donors (HDs). This is the first report of direct usage analysis of 24 kinds of TCR-BV gene families of WT1(126)-specific CTLs at the single cell level. Usage analysis with single-cell RT-PCR of TCR-BV gene families of individual FACS-sorted WT1(126) tetramer(+) CD8(+) T cells showed, for the first time, that: (i) BVs 3, 6, 7, 20, 27, and 28 were commonly biased in patients and HDs; (ii) BVs 2, 11, and 15 were biased only in patients; and (iii) BVs 4, 5, 9, and 19 were biased only in HDs. However, statistical analysis of similarity of individual usage frequencies of 24 kinds of TCR-BV gene families between patients and HDs indicated that the usage frequencies of TCR-BV gene families in patients reflected those in HDs. These results should provide us with a novel insight for a better understanding of WT1-specific immune responses.

Biomechanical evaluation of porous bioactive ceramics after implantation: micro CT-based three-dimensional finite element analysis.

Hydroxyapatite ceramics have been widely investigated for bone regeneration due to their high biocompatibility. However, few studies focus on their mechanical characteristics after implantation. In this study, the finite element (FE) method was used to evaluate the mechanical properties of a fully interconnected porous hydroxyapatite (IPHA) over time of implantation. Based on the micro-CT images obtained from the experiments dealing with IPHA implanted into rabbit femoral condyles, three-dimensional FE models of IPHA (1, 5, 12, 24, and 48 weeks after implantation) were developed. FE analysis indicated that the elastic modulus gradually increased from 1 week and reached the peak value at 24 weeks, and then it kept at high level until 48 weeks postoperatively. In addition, as a local biomechanical response, strain energy density became to distribute evenly over time after the implantation. Results confirmed that the mechanical properties of IPHA are strongly correlated to bone ingrowth. The efficiency of the proposed numerical approach was validated in combination with experimental studies, and the feasibility of applying this approach to study such implanted porous bioceramics was proved.

Nkx3.2-induced suppression of Runx2 is a crucial mediator of hypoxia-dependent maintenance of chondrocyte phenotypes.

Hypoxia is a key factor in the maintenance of chondrocyte identity. However, crucial chondrogenic transcription factors in the Sox families are not activated in this phenomenon, indicating that other pathways are involved. Nkx3.2 is a well-known chondrogenic transcription factor induced by Sonic hedgehog (Shh); it suppresses a key osteogenic transcriptional factor, Runt-related transcription factor 2 (Runx2), to maintain the chondrogenic phenotype in mesenchymal lineages. The purpose of this study was to examine the function of Nkx3.2 in hypoxia-dependent maintenance of chondrocyte identity. C3H10T1/2 pluripotent mesenchymal cells were cultured with rh-BMP2 (300 ng/ml) to induce chondrogenesis under normoxic (20% O(2)) or hypoxic (5% O(2)) conditions. Immunohistological detection of Nkx3.2 in a micromass cell culture system revealed that hypoxia promoted expression of the Nkx3.2 protein. Real-time RT-PCR analysis revealed that hypoxia promoted Nkx3.2 mRNA expression and suppressed Runx2 mRNA expression; however, Sox9 mRNA expression was not altered by oxygen conditions, as previously described. Over-expression of exogenous Nkx3.2 promoted glycosaminoglycan (GAG) production and inhibited Runx2 mRNA expression and, based on a dual luciferase assay, Runx2 promoter activity. Interestingly, downregulation of Nkx3.2 using RNAi abolished hypoxia-dependent GAG production and restored Runx2 mRNA expression and promoter activity. These results demonstrated that Nkx3.2-dependent suppression of Runx2 was a crucial factor in hypoxia-dependent maintenance of chondrocyte identity.

Eleven cases of cardiac metastases from soft-tissue sarcomas.

OBJECTIVE: Cardiac metastasis is a highly life-threatening condition because it leads to cardiac failure. However, it is difficult to diagnose because its precise clinical features are unknown. Here, we report 11 cases of cardiac metastasis from soft-tissue sarcoma, and discuss its diagnosis and treatment. METHODS: Of 641 patients with soft-tissue sarcoma treated in our institute between 1996 and 2009, we retrospectively reviewed the medical records of 11 patients whose cardiac metastases were diagnosed while they were alive. RESULTS: The most common primary tumor was leiomyosarcoma (n= 5), followed by clear cell sarcoma (n= 2). In all cases, metastases to other organs, including lungs (n= 10), soft tissues (n= 5) and bones (n= 4) were found along with cardiac metastases. Cardiac metastasis was diagnosed by echocardiography in six cases and by computed tomography in four cases. In four patients, cardiac metastasis was not detected by chest computed tomography as follow-up to lung metastases and echocardiography was required to make the diagnosis. Although five patients complained of exertional dyspnea, four were asymptomatic. Seven cases were treated with radiotherapy. No patient had surgery for their cardiac metastasis. The median survival of patients who received radiation therapy was 10.5 months; that of those who did not was 3.5 months. CONCLUSIONS: Cardiac metastasis is often asymptomatic. Echocardiography is better than computed tomography for diagnosing cardiac metastasis, and should be considered in all patients presenting with soft-tissue metastases. Owing to the highly life-threatening nature of cardiac metastases and the possibility of soft-tissue dissemination, treatment with radiation therapy is recommended immediately on diagnosis.

High frequencies of less differentiated and more proliferative WT1-specific CD8+ T cells in bone marrow in tumor-bearing patients: an important role of bone marrow as a secondary lymphoid organ.

In tumor-bearing patients, tumor-associated antigen (TAA)-specific CTLs are spontaneously induced as a result of immune response to TAAs and play an important role in anti-tumor immunity. Wilms' tumor gene 1 (WT1) is overexpressed in various types of tumor and WT1 protein is a promising pan-TAA because of its high immunogenicity. In this study, to clarify the immune response to the WT1 antigen, WT1-specific CD8(+) T cells that were spontaneously induced in patients with solid tumor were comparatively analyzed in both bone marrow (BM) and peripheral blood (PB). WT1-specific CD8(+) T cells more frequently existed in BM than in PB, whereas frequencies of naïve (CCR7(+) CD45RA(+)), central memory (CCR7(+) CD45RA-), effector-memory (CCR7- CD45RA(-)), and effector (CCR7- CD45RA(+)) subsets were not significantly different between BM and PB. However, analysis of these subsets for the expression of CD57 and CD28, which were associated with differentiation, revealed that effector-memory and effector subsets of the WT1-specific CD8(+) T cells in BM had less differentiated phenotypes and more proliferative potential than those in PB. Furthermore, CD107a/b functional assay for WT1 peptide-specific cytotoxic potential and carboxyfluorescein diacetate succinimidyl ester dilution assay for WT1 peptide-specific proliferation also showed that WT1-specific CD8(+) T cells in BM were less cytotoxic and more proliferative in response to WT1 peptide than those in PB. These results implied that BM played an important role as a secondary lymphoid organ in tumor-bearing patients. Preferential residence of WT1-specific CD8(+) T cells in BM could be, at least in part, explained by higher expression of chemokine receptor CCR5, whose ligand was expressed on BM fibroblasts on the WT1-specific CD8(+) T cells in BM, compared to those in PB. These results should provide us with an insight into WT1-specific immune response in tumor-bearing patients and give us an idea of enhancement of clinical response in WT1 protein-targeted immunotherapy.

Influence of knee positions on T2, T*2, and dGEMRIC mapping in porcine knee cartilage.

We examined the influence of flexed knee positions on cartilage MR assessments. Sagittal T(2), T*(2), and delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) maps of the femoral cartilage were obtained in eight 6-month-old porcine femorotibial joints in the extended knee position (position A: flexion 0 degrees and femoral shaft in parallel with the amplitude of static field), flexed knee position (position B: flexion 40 degrees and femoral shaft oriented at 40 degrees to the amplitude of static field), and oblique-placed knee-extended position (position C: flexion 0 degrees and femoral shaft oriented at 40 degrees to the amplitude of static field). Comparison of the MR parameters between positions A and C showed isolated influence of the magic-angle effect, and comparison between positions A and B showed effects of knee flexion. Proteoglycan and hydroxyproline content in cartilage specimen at each region of interest had no significant correlation with T(2), T*(2), and dGEMRIC values. At the central zone, located on a weight-bearing area and parallel to the amplitude of static field, T(2)/T*(2)/dGEMRIC values increased by 6.8/11/0.8% at position C and by 24/44/31% at position B compared with position A. There was a significant increase in T(2) and T*(2) values at position B compared with those at position A. The substantial changes in T(2), T*(2), and dGEMRIC were shown in response to knee flexion, presumably due to the compounding influence of the magic-angle effect and change in the intra-articular biomechanical condition.