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Collection of CD3+ lymphocytes via lymphapheresis is essential for manufacturing autologous chimeric antigen receptor (CAR) T cells. Optimization of timing and procedures for lymphapheresis for each patient is critical because patients often have progressive diseases and receive medications that could reduce T cell counts. We conducted a retrospective study of clinical data from 28 patients who underwent lymphapheresis for CD19-directed CAR-T therapy with tisagenlecleucel to identify factors that could affect CD3+ lymphocyte yields. The numbers of CD3+ cells in peripheral blood were significantly correlated with CD3+ cell yields (correlation coefficient r = 0.84), which enabled us to estimate the volume of blood to process before apheresis. We also found that small cell ratio (SCR) at the apheresis site precisely reflected the proportion of lymphocytes, especially in patients without circulating blasts (coefficient of determination: r2 = 0.9). We were able to predict the CD3+ cell yield and prevent excessive apheresis by measuring pre-apheresis circulating CD3+ cell counts and monitoring SCR. Collectively, these results will help us to establish a strategy for optimization of lymphapheresis procedures for CAR-T cell production on a patient-by-patient basis.

Background De novo complement-binding donor-specific anti-human leukocyte antigen antibodies (DSAs) are reportedly associated with an increased risk of kidney graft failure, but there is little information on preformed complement-binding DSAs. This study investigated the correlation between preformed C1q-binding DSAs and medium-term outcomes in kidney transplantation (KT). Methods We retrospectively studied 44 pretransplant DSA-positive patients, including 36 patients who underwent KT between April 2010 and October 2016. There were 17 patients with C1q-binding DSAs and 27 patients without C1q-binding DSAs. Clinical variables were examined in the 2 groups. Results Patients with C1q-binding DSAs had significantly higher blood transfusion history (53.0% vs 18.6%; P = .0174), complement-dependent cytotoxicity crossmatch (CDC-XM)-positivity (29.4% vs 0%; P = .0012), and DSA median fluorescence intensity (MFI) (10,974 vs 2764; P = .0009). Among patients who were not excluded for CDC-XM-positivity and underwent KT, there was no significant difference in cumulative biopsy-proven acute rejection rate (32.5% vs 33.5%; P = .8354), cumulative graft survival, and 3-month and 12-month protocol biopsy results between patients with and without C1q-binding DSAs. Although patients with C1q-binding DSAs showed a higher incidence of delayed graft function (54.6% vs 20.0%; P = .0419), multivariate logistic regression showed that DSA MFI (P = .0124), but not C1q-binding DSAs (P = .2377), was an independent risk factor for delayed graft function. Conclusions In patients with CDC-XM-negativity, preformed C1q-binding DSAs were not associated with incidence of antibody-mediated rejection and medium-term graft survival after KT. C1q-binding DSAs were highly correlated with DSA MFI and CDC-XM-positivity.

The BRAF inhibitors dabrafenib and vemurafenib induce remarkable clinical responses in patients with BRAF-mutated melanomas. However, adverse events, including the emergence of secondary tumors and drug resistance, have been reported. Studies have revealed that undesirable RAF dimerization induced by inhibitors promotes these adverse effects. Here, we developed highly sensitive biosensors of RAF dimerization in cells utilizing the split enhanced click beetle luciferase (Emerald Luc, ELuc) complementation technique. We demonstrated that our biosensor system works effectively for high-throughput screens in the microplate format. A comprehensive analysis of commercially available RAF inhibitors performed using this assay system revealed that the inhibitors exhibit various potencies in inducing the dimerization of RAF isoforms, and their dimerization potencies do not always correlate with the RAF enzyme inhibition. This sensitive assay system will become a powerful tool to discover next-generation BRAF inhibitors with safer profiles.

Background Various studies have reported poorer graft survival among individuals displaying T-cell-positive flow cytometry crossmatches (FCXM). Good outcomes have been observed in immunologically high-risk patients with the use of rituximab, plasmapheresis, and γ-globulin. Because the relevance of FCXM B-cell–positivity (BCXM (+)) alone remains controversial, we examined its impact on living donor renal transplantations. Patients and Methods We retrospectively studied 146 adult renal transplantation recipients from April 2007 to June 2012, dividing the patients into BCXM (+) ( n = 31) versus BCXM (−) recipients ( n = 115). We examined patient and graft survivals as well as rejection rates at 0 to 3, 3 to 12, and 12 to 24 months. We also determined the incidence of infectious diseases. We performed stepwise multivariate regression to identify risk factors contributing rejection episodes. Results One-year patient and graft survivals were 100% in both groups. The BCXM (−) group have a 16.8% rejection probability whereas the BCXM (+) group, 33.2% ( P = .201). There were no significantly differences in the incidence of infectious diseases. Only the rate of a sensitizing history was an independent risk factor for a rejection episode. Conclusion BCXM (+) showed only a tendency but not a significant impact on rejection episodes compared with BCXM (−); short-term graft survivals were similar.

Vasculitides are a group of diseases in which inflammation occurs in various vascular walls of the whole body, and ischemic symptoms are caused by stenoses and occlusions of blood vessels. Various parts of blood vessels of the whole body are affected, and the clinical manifestations are diverse. In the Chapel Hill Consensus Conference (CHCC) 2012, vasculitides are classified into seven categories. Takayasu arteritis and giant cell arteritis are included in large-vessel vasculitis. Large-vessel arteritis is defined as vasculitis affecting the aorta and its major branches more often than other vasculitides, but any sized artery may be affected. Ultrasonography has been progressing rapidly, so we can easily depict vessels of the surface of the body, in 0.1-mm units, and indicate the blood flow noninvasively. Ultrasonography has been used for the diagnosis of and estimation of the treatment for large-vessel vasculitis, and its importance has been increasing.

Selective serotonin reuptake inhibitors (SSRIs) are widely used to treat both anxiety disorders and depressive disorders. However, nonadherence to SSRIs is a major issue in recurrence. In the present study, we investigated paroxetine adherence in depressed patients by monitoring the plasma paroxetine concentrations between patients with rapid and those with a late response to paroxetine treatment. Twenty inpatients in our university hospital, who met the DSM-IV-TR diagnosis of major depressive disorder in a single episode, were enrolled in the study. Twelve patients (M/F: 7/13, age: 37.4 +/- 10.4 years) were treated with paroxetine (40 mg/day), and all achieved remission (HAMD < or = 7) within at least 12 weeks. We divided the patients into two groups, an early-remission group (HAMD < or = 7 within 4 weeks) and a late-remission group (HAMD < or = 7 within 8-12 weeks). Their dosages of paroxetine were constant because of no emerging adverse effects. Blood samples were obtained on the day the subjects were discharged (B) and 12 weeks after discharge. The paroxetine concentrations in the early-remission group were significantly decreased 12 weeks after discharge, and no difference was found between the early- and late-remission groups. These results suggest that adherence to paroxetine was independent of the duration of the depressive state suffered by the patients. Clinicians always take their cautions for the adherence to paroxetine regardless of the clinical time courses the patients recovering from their depressive symptoms.

Reactive oxygen species (ROS) have been attracting attention as mediators of various cell-signaling pathways. Nox-family NADPH oxidases have proven to be a major source of ROS production in various cell types and have crucial roles in various physiological and pathological processes. In this study, we show that Nox4, a member of Nox family, is prominently expressed in various neuroepithelial tumors by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical studies. We quantified Nox4 mRNA expression by real-time PCR in tumor specimens from 58 patients with astrocytomas and found that the expression levels of Nox4 mRNA in glioblastomas (WHO grade IV) were significantly higher than those in other astrocytomas (WHO grade II and III). In addition, we show that specific knockdown of Nox4 expression by RNA interference results in cell-growth inhibition and enhances induction of apoptosis by chemotherapeutic agents, such as cisplatin, in cultured glioma cell lines. Based on these observations, enhanced expression of Nox4 appears to be involved in cell proliferation and survival in glioma cells. © 2008 Wiley-Liss, Inc.

The aims of this study were to investigate the expression of pentraxin-3 in inflamed gastrointestinal tissue in patients with inflammatory bowel diseases and to elucidate the usefulness of plasma pentraxin-3 level as an inflammation marker in patients with inflammatory bowel diseases. Pentraxin-3 immunoreactivity was found in infiltrating neutrophils and vessels in the inflamed gut. Plasma pentraxin-3 concentration in patients with active inflammatory bowel diseases was significantly higher than that of normal subjects and patients with inactive inflammatory bowel diseases. Significant positive correlations of clinical disease activity with plasma pentraxin-3 concentration and serum CRP concentration were found in patients with inflammatory bowel diseases. Pentraxin-3 is directly produced from the inflamed gut in inflammatory bowel diseases. In conclusion, plasma pentraxin-3 concentration is a useful marker for understanding the disease activity in patients with inflammatory bowel diseases.

Pentraxin 3 (PTX3) is a prototype protein of long pentraxin. PTX3 is produced by various cells, such as monocytes/macrophages (Mphis) in response to lipopolysaccharide (LPS) and proinflammatory signals. We performed immunoblotting, immunohistochemical staining, reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA) of PTX3 in human monocyte-derived Mphis and neutrophils. PTX3 expression was observed in the cytoplasm of both GM-CSF induced monocyte-derived Mphi (GM-Mphi) and M-CSF induced monocyte-derived Mphi (M-Mphi). PTX3 level in both Mphis was up-regulated at 24 h after LPS stimulation. Moreover, we confirmed PTX3 expression in freshly isolated neutrophils, and PTX3 level was distinctly up-regulated at 6 and 24 h after LPS stimulation. These findings suggested that PTX3 expression, not only in Mphis, but also in neutrophils, may reflect the role of PTX3 in inflammation. We believe that PTX3 can contribute as a diagnostic tool to evaluate inflammation at peripheral sites.

Medulloblastoma (MB) is the most common malignant neuroepithelial tumor of childhood. The DNA topoisomerase II (Topo II) inhibitor etoposide has been widely used for the treatment of MBs; however, it remains unknown whether MB cells are more sensitive to etoposide than other malignant neuroepithelial tumor cells. In this study, we tested the chemosensitivities of malignant neuroepithelial tumors (26 glioblastomas, 9 anaplastic astrocytomas, and 5 MBs) to etoposide and vincristine using the succinate dehydrogenase inhibition test and found that MB cells are more sensitive to etoposide and more resistant to vincristine than other tumor cells. We performed quantitative reverse-transcription polymerase chain reaction to evaluate the expression of genes related to etoposide sensitivity, and found co-overexpression of DNA topoisomerase II (Topo II) alpha and beta mRNA in MBs. In addition, the levels of Topo IIalpha and beta mRNA in these tumors correlated with etoposide sensitivity. Immunohistochemical studies using surgical samples of these tumors demonstrated that the percentages of Topo IIalpha immunopositive cells (Topo IIalpha labeling index) correlated with those of Ki-67 immunopositive cells (MIB-1 labeling index); however, neither the Topo IIalpha nor the MIB-1 labeling index correlated with the levels of Topo IIalpha mRNA or etoposide sensitivity. Based on these observations, Topo IIalpha and beta mRNA expression, but not the Topo IIalpha labeling index, might be a useful marker for sensitivity to etoposide in human malignant neuroepithelial tumors.

血液型不適合骨髄移植を行う際は, 輸注に伴う有害事象を避けるため, 骨髄液中の赤血球・血漿除去が必要である. 今回われわれは, 血液成分分離装置COBE Spectra により, 骨髄液から単核球を分離することで, 骨髄細胞濃縮を行った血液型不適合骨髄移植20例において, その有用性について検討した. COBE Spectra を用いた骨髄濃縮により, 赤血球量は98.4%除去され, 最終赤血球量は4.2±2.4mlであった. 有核細胞回収率は34.0±8.38%, CD34陽性細胞回収率は112.3±36.3%であった. 20症例全例で, 移植後の溶血反応および生着不全は認めず, 移植後造血回復は速やかであった. COBE Spectra を用いることで, 清潔な無菌閉鎖回路内において骨髄濃縮が可能となり, 処理時間も短縮された. 種々の細胞免疫療法の用途で広く普及しているCOBE Spectra を用いた骨髄濃縮法は, 血液型不適合骨髄移植における骨髄濃縮の標準的方法として有用であると考えられた.

Complement-dependent cytotoxic crossmatch is an important indicator for kidney transplant. However, there is controversy about treatment for flow cytometry crossmatch-positive cases.This was a retrospective study of 127 living-donor kidney transplant recipients from May 2007 to July 2011. We divided patients into 115 flow cytometry crossmatch T-cell and B-cell-negative cases, and 12 T-cell and B-cell-positive cases. Both groups were given 20 mg basiliximab the day of surgery and 4 days after surgery. Common oral immunosuppressive agents used were tacrolimus, mycophenolate mofetil, and methylprednisolone. Flow cytometry crossmatch T-cell and B-cell-negative recipients started immunosuppression 7 days before surgery, T-cell and B-cell-positive recipients started immunosuppression 14 days before surgery. T-cell and B-cell-positive patients also received 200 mg rituximab 1 week before surgery, had 3 plasma exchange sessions before transplant, and received intravenous immunoglobulin 20 g/day during surgery and after surgery for 5 days. We measured flow-panel reactive antibodies of T-cell and B-cell-positive patients just before surgery to check desensitization efficiency. We evaluated patient survival, graft survival, graft function, and frequency of rejection and infectious diseases.Patient survival and graft survival were 100% in both groups. Flow cytometry crossmatch T-cell and B-cell-positive cases had no rejection events, but T-cell and B-cell-negative groups developed rejection. There was no statistical difference in the incidence of infection and graft function. Flow-panel reactive antibody demonstrated improvement in all T-cell and B-cell-positive cases.In living-donor kidney transplant, flow cytometry crossmatch T-cell and B-cell-positive patients are still considered to be at high risk. Although this is a short-term outcome, all T-cell and B-cell-positive patients in this study achieved excellent results with appropriate preoperative and postoperative treatment.

Objective: To investigate the effect of pitavastatin on asymptomatic atherosclerosis in patients with hypercholesterolemia.Methods: Thirty-five outpatients with hypercholesterolemia (61.5±12.8 yr) were administered 2 mg oral pitavastatin daily for 6 months. Plasma pentraxin 3 (PTX3), a novel inflammatory marker of atherosclerosis, was measured together with the serum hsCRP and carotid-artery intima-media thickness (IMT).Results: Significant improvement of the LDL-C/HDL-C and log (TG/HDL-C) ratios began to be observed from 1 month after using pitavastatin. Significant correlation of the initial PTX3 value was observed with the initial plaque score (PS) (p=0.038, r=0.246), but not between the hsCRP and plasma PTX3 or PS. When patients were divided into 3 groups based on the initial PTX3 values, a significant decrease of the plasma PTX3 was obtained in the highest PTX3 group alone (p=0.034). The change in the plasma PTX3 value (ΔPTX3) was significantly correlated with the Δ mean IMT during the study period (p=0.008, r=0.456).Conclusion: Pitavastatin significantly reduced the elevated plasma levels of PTX3 in patients with hypercholesterolemia by its pleiotropic effect against atherosclerotic inflammation. This study showed for the first time that the plasma PTX3 might be a useful blood parameter for direct detection of active atherosclerotic change.

In the present study, we investigated the relationship between the serotonin transporter gene linked polymorphic regions (5-HTTLPR) or plasma paroxetine levels and clinical response to paroxetine in depressed patients. Sixty patients who met the DSM-IV criteria for major depressive disorder were enrolled in the study. Twenty-two were male and 38 were female, with ages ranging from 25 to 71 (mean +/- SD = 42 +/- 16) years. The clinical improvement of patients was assessed using the Hamilton rating scale for depression (Ham-D) before and every week after paroxetine administration. According to the data reported previously, patients with an at least 50% decrease in their Ham-D score were classified as responders. The results showed that the plasma paroxetine levels at 4 weeks were significantly higher in responders (rapid responders) than in nonresponders. On the other hand, no significant associations were found between the L genotype (L/L, L/S) or S genotype (S/S) and the response rates either at 4 weeks or 8 weeks. These results suggest that patients with higher plasma levels at 4 weeks might respond rapidly to paroxetine treatment, but the final response rate at 8 weeks will be independent of the plasma paroxetine levels and the 5-HTTLPR L/S genotype.

Elevated plasma levels of long pentraxin 3 (PTX3), high-sensitive C-reactive protein (hsCRP) and B-type natriuretic peptide (BNP) are found in acute coronary syndromes (ACS). The aim of this study was to assess the prognostic value of PTX3 as compared to BNP and hsCRP, and their combination, as a prognostic marker of mortality in acute chest pain patients. PTX3 was measured in EDTA plasma with a new, high-sensitive ELISA method (PPMX, Tokyo, Japan). BNP was analysed in EDTA plasma using the Microparticle Enzyme Immunoassay (MEIA) Abbott AxSYM®. HsCRP was measured with the use of an immunoturbidimetric assay (Tinaquant® C-reactive protein (latex) high sensitive assay, Roche Diagnostics). The blood samples were taken on admission in 795 patients. The patients were followed for 24 months concerning mortality. For statistical analysis, the study cohort was divided into quartiles (Q1–4) according to PTX3 levels. A multiple logistic regression was fitted which included standard risk measures. At 24 months follow-up, 121 of the 784 patients included in the model had died. The odds ratio for comparing Q4 versus Q1 for PTX3, BNP and hsCRP were 4.34, 3.35 and 0.52 (p=0.001, p=0.024 and p=0.096), respectively, and the combination of PTX3 and BNP showed an incremental prognostic value (figure ). PTX3 is a new independent marker that strongly predicts long-term all-cause mortality in patients with acute chest pain and the combination of this marker with BNP adds substantially to the prognostic value as compared to either marker alone. Figure The number of biomakers greater than the median for % death in 24 months (p

Long Pentraxin 3 (PTX3) is a newly identified member of the Pentraxin protein family that includes C-reactive protein (CRP). Unlike CRP, PTX3 is produced by the major cell types involved in atherosclerotic lesions in response to inflammatory stimuli. Increased PTX3 levels are found in acute coronary syndromes (ACS). Its role in long-term prediction of clinical outcome is, however, unknown. The aim of the current study was to assess the predictive value of PTX3 concerning all-cause mortality in patients hospitalized for acute chest pain. Plasma PTX3 was measured with a new, high-sensitive ELISA method (PPMX, Tokyo, Japan) in blood samples taken on admission in 784 patients admitted for acute chest pain suggestive of ACS. The patients were followed for 24 months concerning clinical outcome. For statistical analysis, the study cohort was divided into quartiles according to PTX3 levels. A multiple logistic regression model was fitted to include standard risk measures. At 24 months follow-up 121 patients had died. By logistic regression, the odds Odds Ratio for death among patients with highest PTX3 levels was 3.13 as compared to those with lowest levels (p=0.007) (table). Long Pentraxin 3 is a new, independent marker that strongly predicts long-term all-cause mortality in patients with acute chest pain.

Reactive oxygen species (ROS) have been attracting attention as mediators of various cell-signaling pathways. Nox-family NADPH oxidases have proven to be a major source of ROS production in various cell types and have crucial roles in various physiological and pathological processes. In this study, we show that Nox4, a member of Nox family, is prominently expressed in various neuroepithelial tumors by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical studies. We quantified Nox4 mRNA expression by real-time PCR in tumor specimens from 58 patients with astrocytomas and found that the expression levels of Nox4 mRNA in glioblastomas (WHO grade IV) were significantly higher than those in other astrocytomas (WHO grade II and III). In addition, we show that specific knockdown of Nox4 expression by RNA interference results in cell-growth inhibition and enhances induction of apoptosis by chemotherapeutic agents, such as cisplatin, in cultured glioma cell lines. Based on these observations, enhanced expression of Nox4 appears to be involved in cell proliferation and survival in glioma cells.

In the present study, we examined the effects of acute treatment with paroxetine on the consumption of cigarette smoking and caffeine in 65 patients who met the DSM-IV criteria for major depressive disorder (M/F: 28/37, age: 48 +/- 15 years). Plasma levels of cotinine or caffeine were analysed using high-performance liquid chromatography (HPLC). The amount of cigarette smoking and plasma levels of cotinine, but not caffeine, decreased 4 weeks after paroxetine treatment. There was no difference between smokers and nonsmokers with respect to their response to paroxetine treatment. In addition, plasma 3-methoxy-4-hydroxyphenylglycol (MHPG) levels in responders to paroxetine treatment was higher than those in nonresponders, and there was a negative correlation between the changes in plasma MHPG levels and the changes in Hamilton rating scale for depression (Ham-D) scores before and 4 weeks after paroxetine administration. These results suggest that paroxetine has the potential to reduce the amount of cigarette smoking in depressed smokers, and we reconfirmed our previous results that depressed patients with higher plasma MHPG levels had better response to paroxetine treatment than those with lower plasma MHPG levels using larger depressed samples.

Objective—Plasma pentraxin 3 (PTX3) levels are increased in patients with acute myocardial infarction, yet its involvement in unstable angina pectoris (UAP) remains unclear. To critically evaluate the role of PTX3 in UAP, a sensitive and precise measurement of PTX3 concentration is needed.Methods and Results—We established a high sensitive plasma ELISA assay system for the detection of PTX3 using monoclonal antibodies. The lower limit of detection of our ELISA was 0.1 ng/mL, sensitivity far greater than the current commercially available kit. Plasma samples were obtained from 162 consecutive patients treated for hypertension, hyperlipidemia, diabetes mellitus, or cardiovascular disease at a physician’s office. PTX3 was not associated with any known coronary risk factors. Additionally, we collected plasma samples from 252 consecutive subjects admitted to a university hospital for coronary artery assessment by coronary angiography. PTX3 was significantly increased in patients in whom coronary intervention was performed. We further analyzed the plasma level of PTX3 in 52 patients with effort angina (EAP) and 16 patients with UAP. Compared with the control group, PTX3 were significantly higher in the UAP group.Conclusions—The levels of plasma PTX3 were increased in patients with arterial inflammation, especially UAP. This PTX3 detection system will be useful for the prediction of UAP.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental contaminant that can exert developmental toxicity. To investigate the stage-specific effects of TCDD on preimplantation embryos, we exposed mouse embryos to TCDD at different stages (1-, 2-, and 8-cell) and collected them at different stages of development (the 1- or 2-, 8-cell, and blastocyst stage, respectively). Semiquantitative RT-PCR revealed increased constitutive gene expression of the arylhydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) at the 1-cell stage, decreased expression at the 2- to 8-cell stage, and increased expression again at the blastocyst stage, and addition of TCDD to media did not affect their mRNA levels. Interestingly, no cytochrome P4501A1 (CYP1A1) mRNA was detected in embryos at the 1-, 2-, and 8-cell stages after exposure to 10 nM TCDD for 12 or 24 h, whereas CYP1A1 mRNA was significantly increased at the blastocyst stage in response to TCDD, and its induction was found to be concentration-dependent on TCDD exposure from 0.01 to 10 nM for 24 h. In addition, no significant differences in development rate of preimplantation embryos, cell number of blastocyst embryos, or apoptotic indices, such as TUNEL-positive cell number or Bax/Bcl-2 expression ratios were observed at the blastocyst stage between TCDD-exposed groups and non-exposed group. These results suggest that the sensitivity to TCDD differs with the embryonic stage, which may reflect an ability of embryos to adapt to environmental stressors, such as dioxins.