Clinical-scale expansion of human cytomegalovirus-specific cytotoxic T lymphocytes from peripheral blood mononuclear cells requiring single-peptide stimulation and feeder cells but not additional antigen-presenting cells

BACKGROUND: The aim of this study was to find a simple and feasible method for ex vivo expansion of human cytomegalovirus (HCMV)-specific cytotoxic T cells from peripheral blood mononuclear cells (PBMNCs) without the aid of exogenous antigen-presenting cells (APCs) such as cultured dendritic cells. STUDY DESIGN AND METHODS: PBMNCs from three HLA-A*2402-seropositive donors were stimulated with HCMV pp65(341-350) peptide on Day 1 and then cultured with interleukin-2 and allogeneic feeder cells for 3 to 4 weeks. HCMV peptide-specific T cells were purified with HLA-A*2402/pp65(341-350) tetramer on Days 12 to 13 and harvested on Days 23 to 27. RESULTS: The initial numbers of PBMNCs were 2 x 10(7), 1.5 x 10(7), and 2.5 x 10(7) and the increases in HCMV peptide-specific T cells were 3.5 x 10(4)-, 2.0 x 10(3)-, and 1.1 x 10(3)-fold, respectively. The estimated final numbers of tetramer-positive cells were 9.1 x 10(7), 9.0 x 10(6), and 5.3 x 10(6), respectively. The purities of the tetramer-positive cell population in culture were 72.6, 75.0, and 80.9 percent, respectively. The cells killed peptide-pulsed B-lympoblastoid cell lines and secreted interferon-gamma in a HLA-restricted manner. They did not have natural killer cell activity or lymphokine activated killer cell activity. Most of them had an effector-memory phenotype. They did not express killer inhibitory receptors. CONCLUSION: This method makes it possible to obtain more than 1 x 10(7) HCMV-specific T cells from approximately 2 x 10(7) to 5 x 10(7) PBMNCs without exogenous APCs such as cultured dendritic cells.     

In vitro inhibitory effects of Kampo medicines on metabolic reactions catalyzed by human liver microsomes

BACKGROUND: Although it is well known that drug-drug interactions may lead to toxicity and therapeutic failure, little is known about the incidence and consequences of herb-drug interactions in patients receiving Kampo medicines. METHODS: We evaluated the frequency of the combined use of Kampo medicines and Western drugs at Osaka University Hospital, and investigated the effects of these formulae on the metabolic activity of different cytochrome P450 (CYP) isoforms using pooled microsomes obtained from human liver. RESULTS: Twenty-two Kampo formulae were used together with 40 Western drugs catalyzed by the CYP isoforms CYP3A4, CYP2C9, CYP2D6 and CYP1A2. Among the Kampo medicines, HOCHUEKKI-TO, SHOSAIKO-TO, NINJINYOUEI-TO, SAIREI-TO and KAKKON-TO were most frequently used during the study period (1996-2000). These were co-administered with 11 categories of drugs, which are substrates for CYP3A4. HOCHUEKKI-TO and SAIREI-TO were competitive inhibitors of CYP3A4 with Ki values of 0.65 and 0.1 mg/mL, respectively. HOCHUEKKI-TO, SHOSAIKO-TO and SAIREI-TO inhibited the metabolic activities of CYP2C9, but had no effect on CYP2D6. HOCHUEKKI-TO and SAIREI-TO exhibited non-competitive inhibition of the metabolic activity of CYP2C9 with a similar Ki value (0.7-0.8 mg/mL). SAIRE-TO (0.25 mg/mL) was a potent inhibitor of CYP1A2 (inhibition > 68%). CONCLUSIONS: Frequently used Kampo medicines may interact with Western drugs, which are substrates for CYP3A4, CYP2C9 and CYP1A2. Their co-administration should be undertaken with care.     

The agreement of left ventricular function parameters between 99mTc-tetrofosmin gated myocardial SPECT and gated myocardial MRI

Objective

The aim is to compare and evaluate the agreement of quantification of left ventricular functional parameters obtained by two different methods, ^99mTc-tetrofosmin gated myocardial perfusion SPECT (MPS) and cardiac magnetic resonance imaging (CMR).

Methods

Ten healthy male volunteers participated. Gated MPS data were acquired using 32 frames, which were also combined into 16- and 8-frame data set for the investigation. Gated CMR data were acquired using 8, 16 and 32-frame for the different sets. All examinations were conducted in resting and at exercise conditions. Quantitative measurements of end-diastolic volume (EDV), end-systolic volume (ESV), left ventricular ejection fraction (LVEF), peak ejection rate (PER), peak filling rate (PFR) and time to peak filling (TTPF) were done for each study, respectively. Finally, we evaluated the concordance of parameters between gated MPS and gated CMR by % difference and Bland?CAltman plot analysis.

Results

LVEF showed favorable concordance in both rest and exercise conditions (% differences were around 10%). PER, PFR and TTPF also showed good concordances in rest conditions, under 32-frame gated collections particularly (% differences were around 10%). In exercise conditions, although the concordances were relatively good, certain variances were noted (% differences were around 20?C25%). Regarding left ventricular volumes, the concordance were worse in both conditions (% differences were around 30?C40%).

Conclusions

In quantifying of left ventricular function parameter, gated CMR provides similar quantitative values comparing with gated MPS except for ventricular volumes in rest conditions. In contrast, there were certain variations except for LVEF in exercised examinations. When we follow patients by the same cardiac parameters with CMR and MPS, using parameters across the two modalities proved to be possible under rest condition. However, it is limited at exercise condition.     

Effects of separate application of three growth factors (TGF-beta1, EGF, and PDGF-BB) on mechanical properties of the in situ frozen-thawed anterior cruciate ligament

OBJECTIVE: To clarify effects of a separate application of TGF-beta1, EGF, and PDGF-BB on the material properties of the in situ frozen-thawed anterior cruciate ligament. DESIGN: Twenty-eight rabbits were divided into four groups after undergoing the in situ freeze-thaw treatment in the right anterior cruciate ligament. In 3 of the 4 groups, 4 ng TGF-beta1, 20 ng EGF, and 4 microg PDGF-BB was applied to the frozen anterior cruciate ligament, respectively. In the remaining sham treatment group, only fibrin sealant as a vehicle was applied. Each animal was sacrificed at 12 weeks after surgery. BACKGROUND: If the role of growth factors in ligament healing and remodeling is understood, better therapies can be designed for ligament trauma. METHODS: The freeze-thaw treatment was performed three times using the originally developed cryo-probe. The cross-sectional area of the anterior cruciate ligament was measured by the optical non-contact method. After preconditioning, each specimen was stretched to failure. The ligament strain was determined with a video dimension analyzer. RESULTS: The tensile strength and the tangent modulus of the anterior cruciate ligament in the TGF-beta1 group was significantly higher than in the sham group, but significantly lower than in the normal control group. There were no significant differences in the strength and the modulus between the EGF group, the PDGF-BB group, and the sham group. CONCLUSIONS: In this model, an application of 4 ng TGF-beta1 significantly inhibited some of the material deterioration that occurs in the in situ frozen-thawed anterior cruciate ligament, while an application of 20 ng EGF or 4 microg PDGF-BB did not significantly affect the deterioration. RELEVANCE: This information will be useful in the future to develop a new biological therapy for ligament reconstruction to prevent the graft deterioration after transplantation.     

Intravesical administration of exogenous microRNA-145 as a therapy for mouse orthotopic human bladder cancer xenograft

We previously reported that the level of microRNA (miR)-145 is attenuated in human bladder cancer cells. In this current study, we investigated whether intravesical administration of miR-145 could be a potential therapeutic strategy for controlling bladder cancer by using an orthotopic human bladder cancer xenograft model. Following transfection of 253J B-V cells with miR-145, the effects of the ectopic expression of miR-145 were examined by performing MTT, Western blotting analysis, Hoechst33342 staining, and wound healing assay in vitro. Also, a mouse orthotopic human bladder cancer model was established by inoculating 253J B-V cells into the bladder wall of mice. The anti-cancer effects of intravesical injections of miR-145 into these mice were then assessed. Transfection of 253J B-V cells with miR-145 induced apoptosis and suppression of cell migration in vitro. Western blotting showed that the levels of c-Myc, socs7, FSCN1, E-cadherin, β-catenin, and catenin δ-1 were decreased and that the PI3K/Akt and Erk1/2 signaling pathways were increased in compensatory fashion. In vivo, mice treated with miR-145 showed 76% inhibition of tumor growth, with a significant prolongation of animal survival (p = 0.0183 vs. control). Western blotting showed that both apoptosis and cell motility-related genes were significantly decreased as seen in vitro. Furthermore, PI3k/Akt and Erk1/2 signaling pathways, which were activated in a compensatory manner in vitro, were decreased in vivo. Intravesical administration of exogenous miR-145 was thus concluded to be a valid therapy for bladder cancer in this human bladder cancer xenograft model.     

Binding of plasma fibronectin to human polymorphonuclear leukocytes in normal subjects and patients with aplastic anemia and thyroid dysfunction

The binding of iodine 125-labeled fibronectin to polymorphonuclear leukocytes (PMNs) from human peripheral blood was examined. The optimum temperature and time for the binding were 37 degrees C and 30 minutes, respectively. On increase in the amount of 125I-labeled fibronectin, its binding to PMNs became saturated. Scatchard analysis of data on binding indicated the presence of a single class of binding sites. PMNs from 15 normal subjects had approximately 6.3 +/- 1.6 x 10(3) sites per cell and a dissociation constant of 10.2 +/- 2.4 x 10(-9) mol/L, indicating that they had high affinity for soluble fibronectin. Arg-Gly-Asp-Ser inhibited the binding of fibronectin to PMNs, strongly suggesting that the fibronectin receptor is one of the Arg-Gly-Asp receptor family. The plasma level of fibronectin was higher in patients with hyperthyroidism and lower in patients with hypothyroidism than in normal subjects, without any significant change in the number of fibronectin binding sites of the PMNs. However, the number of binding sites of fibronectin on PMNs of patients with aplastic anemia was increased, probably because of sensitization of the PMNs with immune complex and other factors.     

Molecular targeted therapy in lung cancer

Lung cancer continues to be the leading cause of cancer death worldwide, and nonsmall cell lung cancer(NSCLC) is the most common type of lung cancer. Despite many clinical trials of platinum-based chemotherapy in combination with various drugs, the median survival time of NSCLC patients remains poor. The overall 5-year survival rate is approximately 15%, and has improved only marginally over the last few decades despite the introduction of new therapeutic agents. A recent milestone in this field has been the development of molecular-targeting drugs, among which gefitinib and erlotinib targeting the epidermal growth factor receptor (EGFR) have improved the efficacy of therapy for NSCLC. Anti-angiogenetic drug, such as bevacizumab, had become clinical use in the treatment for NSCLC. Moreover, discovery of EML4-ALK made the marvelous progress in cancer research in NSCLC. In this review, we discuss about the development of molecular-targeting drugs, such as EGFR-TKI, anti-angiogenetic drug, and EMLA-ALK inhibitors.