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In veterinary medicine, measurement of canine C-reactive protein (cCRP) is used widely to detect inflammatory diseases. We evaluated the precision of Randox and Fuji assays for cCRP, as well as accuracy, correlation, and agreement compared to a reference ELISA. Blood samples from 71 client-owned dogs (20 healthy, 51 diseased) were analyzed with the 3 assays. Inter-assay CVs were ~3.5% with both the Randox and Fuji assays. The mean biases were −1.90% for the Randox and −5.93% for the Fuji test; the targeted biases were ~8.5% for both assays. The CV, bias, and observed total error were acceptable for the 2 assays compared to ASVCP recommendations based on biological variation studies. The Spearman correlation coefficient for cCRP concentration compared with the reference ELISA was 0.83 for the Randox test and 0.92 for the Fuji test. Both assays measured cCRP precisely at intermediate and increased concentrations. Correlation with the reference ELISA was good, and both assays could be used to evaluate cCRP concentrations in veterinary practice. However, the assays did not reach analytical agreement; hence the results obtained by these assays are not interchangeable, and serial monitoring of cCRP requires the use of the same assay.

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Purpose:To expand clinical-grade healthy donor-derived double-negative T cells (DNT) to a therapeutically relevant number and characterize their potential to be used as an “off-the-shelf” adoptive cellular therapy (ACT) against cancers.Experimental Design:We developed methods to expand DNTs under GMP conditions and characterized their surface molecule expression pattern using flow cytometry–based high-throughput screening. We investigated the off-the-shelf potential of clinical-grade DNTs by assessing their cytotoxicity against various cancer types and their off-tumor toxicity in vitro and in xenograft models and determining the effect of cryopreservation under GMP conditions on cell viability and cytotoxicity. Further, we determined the susceptibility of DNTs to conventional allogeneic T cells in vitro and in vivo.Results:Clinical-grade DNTs expanded 1,558 ± 795.5-fold in 17 days with >90% purity. Expanded DNTs showed potent in vitro cytotoxic activity against various cancer types in a donor-unrestricted manner. DNTs enhanced the survival of mice infused with a lethal dose of EBV-LCL and significantly reduced leukemia engraftment in xenograft models. Expanded DNTs cryopreserved using GMP-compliant reagents maintained viability and anticancer functions for at least 600 days. Live allogeneic DNTs did not induce cytotoxicity of alloreactive CD8+ T cells in vitro, and coinfusion of DNTs with peripheral blood mononuclear cells (PBMC) from a different donor into mice resulted in coengraftment of DNTs and PBMC-derived allogeneic conventional T cells in the absence of cytotoxicity toward DNTs, suggesting the lack of host-versus-graft reaction.Conclusions:We have established a method to generate therapeutic numbers of clinical-grade DNTs that fulfill the requirements of an off-the-shelf ACT.

T-cell malignancies are often aggressive and associated with poor prognoses. Adoptive cell therapy has recently shown promise as a new line of therapy for patients with hematological malignancies. However, there are currently challenges in applying adoptive cell therapy to T-cell malignancies. Various approaches have been examined in preclinical and clinical studies to overcome these obstacles. This review aims to provide an overview of the recent progress on adoptive cell therapy for T-cell malignancies. The benefits and drawbacks of different types of adoptive cell therapy are discussed. The potential advantages and current applications of innate immune cell-based adoptive cell therapy for T cell malignancies are emphasized.

Acute myeloid leukemia (AML) remains a devastating disease in need of new therapies to improve patient survival. Targeted adoptive T-cell therapies have achieved impressive clinical outcomes in some B-cell leukemias and lymphomas but not in AML. Double-negative T cells (DNTs) effectively kill blast cells from the majority of AML patients and are now being tested in clinical trials. However, AML blasts obtained from ∼30% of patients show resistance to DNT-mediated cytotoxicity; the markers or mechanisms underlying this resistance have not been elucidated. Here, we used a targeted clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) screen to identify genes that cause susceptibility of AML cells to DNT therapy. Inactivation of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitinating complex components sensitized AML cells to DNT-mediated cytotoxicity. In contrast, CD64 inactivation resulted in resistance to DNT-mediated cytotoxicity. Importantly, the level of CD64 expression correlated strongly with the sensitivity of AML cells to DNT treatment. Furthermore, the ectopic expression of CD64 overcame AML resistance to DNTs in vitro and in vivo. Altogether, our data demonstrate the utility of CRISPR/Cas9 screens to uncover mechanisms underlying the sensitivity to DNT therapy and suggest CD64 as a predictive marker for response in AML patients.

Eukaryotic cells have two separate genomes; nuclear chromosomal DNA and circular mitochondrial DNA. Mitochondrial DNA lacks introns, and encodes 2 rRNAs, 22 t-RNAs and 13 of the 90 proteins that constitute the mitochondrial respiratory chain. To maintain homeostasis, mitochondrial RNA degradation machinery regulates RNA turnover. ATP-dependent helicase, SUV3 (gene SUPV3L1), and exonuclease PNPase (gene PNPT1) function as a complex to degrade mitochondrial dsRNA. By immunoblotting, PNPase and SUV3 proteins were increased in 7/7 AML patient samples and 13/13 of AML cell lines, compared to the normal hematopoietic cells. Analysis of the TARGET AML dataset revealed AML patients with increased expression of SUPV3L1 (p = 0.051, p= 0.045) and PNPT1 (p = 0.0013, p = 0.018) had decreased overall and event free survival. Genetic knockdown or knockout using shRNA or sgRNA against PNPT1 or SUPV3L1 decreased growth and viability of OCI-AML2, TEX, K562, U937, NB4 and OCI-AML 8227 cells. Furthermore, SUPV3L1 & PNPT1 ranked in the top 5.2% and 7.4% of essential genes in 26 leukemia cell lines in CRISPR screens and 2.7% and 4.9% in RNAi screens (depmap.org). Knockdown of PNPT1 & SUPV3L1 also reduced the clonogenic growth of OCI-AML2, TEX and U937 cells and significantly reduced engraftment of TEX cells into the marrow of immune deficient mice, demonstrating the functional importance on leukemia initiating cells in vivo. SUPV3L1 knockdown in primary AML cells reduced engraftment in marrow of immune deficient mice. Bioinformatics analysis to detect processes associated with PNPT1 and SUPV3L1, we identified associations with Response to exogenous dsRNA, Response to virus, and RNA catabolic process ontologies. Consistent with this, we observed knockdown of PNPT1 or SUPV3L1 increased expression of genes (INFgR1, ICAM, IRF7 & JAK/STAT) suggesting an interferon response. As PNPT1 and SUPV3L1 degrade mitochondrial dsRNA, we measured levels of dsRNA after knockdown of these genes. Knockdown of PNPT1 and SUPV3L1 in OCI-AML2 cells increased levels of cytoplasmic/mitochondrial dsRNA 3-4 fold compared to control. Knockdown of PNPT1 and SUPV3L1 also increased dsRNA in 143B cells, but not Rho (0) 143B cells that lack mitochondrial DNA. Upregulation of inflammatory genes leads to viral mimicry and can increase sensitivity to immune mediated killing. We observed enhanced sensitivity to Double Negative T (DNT) cells mediated killing in PNPT1 and SUPV3L1 knockdown OCI-AML2 cells compared to control cells. In summary, RNA degradosome complex proteins SUPV3L1 and PNPT1 are overexpressed in AML, and are essential for the survival of AML cells and AML stem/progenitors. These enzymes regulate levels of mitochondrial dsRNA, and their inhibition leads to increased cytoplasmic dsRNA triggering a viral mimicry response and enhanced sensitivity to immune-mediated killing. Citation Format: Geethu Emily Thomas, Kazem Nouri, Jong Bok Lee, Rose Hurren, Yongran Yan, Neil MacLean, Yulia Jitkova, Li Ma, Xiao Ming Wang, Chaitra Sarathy, Andrea Arruda, Mark D. Minden, Li Zhang, Vito Spadavecchio, Aaron Schimmer. Inhibiting the mitochondrial RNA degradosome complex SUV3 and PNPase increases dsRNA in the cytoplasm, triggers a viral mimicry response and kills AML cells and progenitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4833.

The development of autologous chimeric antigen receptor T (CAR-T) cell therapies has revolutionized cancer treatment. Nevertheless, the delivery of CAR-T cell therapy faces challenges, including high costs, lengthy production times, and manufacturing failures. To overcome this, attempts have been made to develop allogeneic CAR-T cells using donor-derived conventional CD4+or CD8+T cells (Tconvs), but severe graft-versus-host disease (GvHD) and host immune rejection have made this challenging. CD3+CD4−CD8−double-negative T cells (DNTs) are a rare subset of mature T cells shown to fulfill the requirements of an off-the-shelf cellular therapy, including scalability, cryopreservability, donor-independent anticancer function, resistance to rejection, and no observed off-tumor toxicity including GvHD. To overcome the challenges faced with CAR-Tconvs, we evaluated the feasibility, safety, and efficacy of using healthy donor–derived allogeneic DNTs as a CAR-T cell therapy platform. We successfully transduced DNTs with a second-generation anti–CD19-CAR (CAR19) without hampering their endogenous characteristics or off-the-shelf properties. CAR19-DNTs induced antigen-specific cytotoxicity against B cell acute lymphoblastic leukemia (B-ALL). In addition, CAR19-DNTs showed effective infiltration and tumor control against lung cancer genetically modified to express CD19 in xenograft models. CAR19-DNT efficacy was comparable with that of CAR19-Tconvs. However, unlike CAR19-Tconvs, CAR19-DNTs did not cause alloreactivity or xenogeneic GvHD-related mortality in xenograft models. These studies demonstrate the potential of using allogeneic DNTs as a platform for CAR technology to provide a safe, effective, and patient-accessible CAR-T cell treatment option.

Significant improvements in the survival of patients with hematological cancers following hematopoietic stem cell transplantation provide evidence supporting the potency of immune cell-mediated anti-leukemic effects. Studies focusing on immune cell-based cancer therapies have made significant breakthroughs in the last few years. Adoptive cellular therapy (ACT), and chimeric antigen receptor (CAR) T cell therapy, in particular, has significantly increased the survival of patients with B cell acute lymphoblastic leukemia and aggressive B cell lymphoma. Despite antigen-negative relapses and severe toxicities such as cytokine release syndrome after treatment, CAR-T cell therapies have been approved by the FDA in some conditions. Although a number of studies have tried to achieve similar results for acute myeloid leukemia (AML), clinical outcomes have not been as promising. In this review, we summarize recent and ongoing studies on cellular therapies for AML patients, with a focus on antigen-specific versus -nonspecific approaches.

Purpose: To expand clinical-grade healthy donor-derived double-negative T cells (DNT) to a therapeutically relevant number and characterize their potential to be used as an “off-the-shelf” adoptive cellular therapy (ACT) against cancers. Experimental Design: We developed methods to expand DNTs under GMP conditions and characterized their surface molecule expression pattern using flow cytometry–based high-throughput screening. We investigated the off-the-shelf potential of clinical-grade DNTs by assessing their cytotoxicity against various cancer types and their off-tumor toxicity in vitro and in xenograft models and determining the effect of cryopreservation under GMP conditions on cell viability and cytotoxicity. Further, we determined the susceptibility of DNTs to conventional allogeneic T cells in vitro and in vivo. Results: Clinical-grade DNTs expanded 1,558 ± 795.5-fold in 17 days with >90% purity. Expanded DNTs showed potent in vitro cytotoxic activity against various cancer types in a donor-unrestricted manner. DNTs enhanced the survival of mice infused with a lethal dose of EBV-LCL and significantly reduced leukemia engraftment in xenograft models. Expanded DNTs cryopreserved using GMP-compliant reagents maintained viability and anticancer functions for at least 600 days. Live allogeneic DNTs did not induce cytotoxicity of alloreactive CD8+ T cells in vitro, and coinfusion of DNTs with peripheral blood mononuclear cells (PBMC) from a different donor into mice resulted in coengraftment of DNTs and PBMC-derived allogeneic conventional T cells in the absence of cytotoxicity toward DNTs, suggesting the lack of host-versus-graft reaction. Conclusions: We have established a method to generate therapeutic numbers of clinical-grade DNTs that fulfill the requirements of an off-the-shelf ACT.

Background Although the demand for esomeprazole is increasing in veterinary medicine, the pharmacokinetics (PK) and pharmacodynamics of esomeprazole have been described in only a few studies. Objective To determine the PK of 0.5 and 1 mg/kg esomeprazole administered IV q12h and to investigate its effects on intragastric pH in healthy dogs. Animals Six adult Beagles. Methods Open-label, randomized, and crossover design. The dogs received 0.5 or 1 mg/kg esomeprazole IV q12h for 48 hours. Plasma concentrations of esomeprazole were measured by high-performance liquid chromatography-tandem mass spectrometry. Intragastric pH was determined using the Bravo pH monitoring system and recorded as mean percentage time (MPT) for which pH was ≥3 and ≥4 for 24 hours in each group. Results The peak plasma concentration and area under the curve from the time of dosing to the last measurable concentration in the 1 mg/kg group were higher than those in the 0.5 mg/kg group. However, when the dosage normalized, intergroup differences were not significant. The MPTs for which intragastric pH was ≥3 and ≥4 for 48 hours were 88% ± 7% and 81% ± 9% for the 0.5 mg/kg group and 90% ± 9% and 85% ± 11% for the 1 mg/kg group, respectively, with no significant intergroup differences. Conclusions and clinical importance The pharmacokinetic parameters and acid suppressant effect for 0.5 and 1 mg/kg esomeprazole were not significantly different. Furthermore, the efficacy of esomeprazole 0.5 mg/kg IV q12h was sufficient to increase intragastric pH in Beagles.

With recent clinical breakthroughs, immunotherapy has become the fourth pillar of cancer treatment. Particularly, immune cell-based therapies have been envisioned as a promising treatment option with curative potential for leukemia patients. Hence, an increasing number of preclinical and clinical studies focus on various approaches of immune cell-based therapy for treatment of acute leukemia (AL). However, the use of different immune cell lineages and subsets against different types of leukemia and patient disease statuses challenge the interpretation of the clinical applicability and outcome of immune cell-based therapies. This review aims to provide an overview on recent approaches using various immune cell-based therapies against acute B-, T-, and myeloid leukemias. Further, the apparent limitations observed and potential approaches to overcome these limitations are discussed.

To compare the surgical outcomes of medial rectus advancement and lateral rectus recession in postoperative consecutive exotropia with single-stage adjustable suture surgery. Among 1003 patients who underwent bilateral medial rectus recession between November 1996 and March 2013, the patients who required surgery for consecutive exotopia were retrospectively reviewed. Nineteen patients underwent medial rectus advancement and 15 patients underwent lateral rectus recession. All patients underwent single-stage adjustable surgery under topical anesthesia and were followed up for at least 12 months. The mean follow-up duration was 2.4 years. At final follow-up, a successful surgical outcome was found in 12 patients (63.0%) in the medial rectus advancement group and 14 patients (93.3%) in the lateral rectus recession group (P = .039). The change in ocular deviation was correlated with the amount of recession (P = .008) and preoperative angle (P

Purpose: To analyze the efficacy and safety of standard-dose antimuscarinic treatment on male patients with overactive bladder (OAB) symptoms showing poor efficacy after low-dose antimuscarinics. Materials and Methods: We retrospectively reviewed the data of 566 male patients aged ≥40 with OAB symptoms between January 2017 and June 2018. They were treated with low-dose antimuscarinics for at least 4 weeks and showed poor efficacy; therefore, they were switched to standard dose antimuscarinic treatment (5 mg of solifenacin) for ≥12 weeks. The international prostate symptom score (IPSS) and overactive bladder symptom score (OABSS) at baseline (V0), 4 weeks (V1), and 12 weeks (V2) were analyzed. Post void residual urine volume (PVR) was also recorded. Results: The median age, body mass index, and prostate-specific antigen levels were 69.0 years, 24.2 kg/m2, and 1.24 ng/dL, respectively. The mean value of the total IPSS and OABSS significantly decreased between V0 and V2 (from 16.73 to 13.69 and 7.33 to 5.34, respectively, all p

Summary Despite the demonstration of potent immunosuppressive function of T cell receptor (TCR)-αβ+ double-negative regulatory T cells (DN Tregs), scarce numbers and lack of effective expansion method limit their clinical applications. Here we describe an approach that allows for ∼3500-fold ex-vivo expansion of human DN Tregs within 3 weeks with > 97% purity. Ex-vivo-expanded DN Tregs suppress proliferation of polyclonally stimulated autologous T and B cells in vitro through direct cell-to-cell contact. In vivo, we demonstrate for the first time that infusion of human DN Tregs delayed an onset of xenogeneic graft-versus-host disease (GVHD) significantly in a humanized mouse model. Furthermore, preincubation of ex-vivo-expanded DN Tregs with a mechanistic target of rapamycin (mTOR) inhibitor rapamycin enhanced their immune regulatory function further. Taken together, this study demonstrates that human DN Tregs can be expanded ex vivo to therapeutic numbers. The expanded DN Tregs can suppress proliferation of T and B cells and attenuate GVHD, highlighting the potential clinical use of DN Tregs to mitigate GVHD.

As the modernization of the nuclear instrumentation system progresses, research reactors have adopted digital wide-range neutron power measurement (DWRNPM) systems. These systems typically monitor the neutron flux across a range of over 10 decades. Because neutron detectors only measure the local neutron flux at their position, the local neutron flux must be converted to total reactor power through calibration, which involves mapping the local neutron flux level to a reference reactor power. Conventionally, the neutron power range is divided into smaller subranges because the neutron detector signal characteristics and the reference reactor power estimation methods are different for each subrange. Therefore, many factors should be considered when preparing the calibration procedure for DWRNPM channels. The main purpose of this work is to serve as a reference for performing the calibration of DWRNPM systems in research reactors. This work provides a comprehensive overview of the calibration of DWRNPM channels by describing the configuration of the DWRNPM system and by summarizing the theories of operation and the reference power estimation methods with their associated calibration procedure. The calibration procedure was actually performed during the commissioning of an open-pool type research reactor, and the results and experience are documented herein.

Purpose: To explore the potential of ex vivo expanded healthy donor–derived allogeneic CD4 and CD8 double-negative cells (DNT) as a novel cellular immunotherapy for leukemia patients. Experimental Design: Clinical-grade DNTs from peripheral blood of healthy donors were expanded and their antileukemic activity and safety were examined using flow cytometry–based in vitro killing assays and xenograft models against AML patient blasts and healthy donor–derived hematopoietic cells. Mechanism of action was investigated using antibody-mediated blocking assays and recombinant protein treatment assays. Results: Expanded DNTs from healthy donors target a majority (36/46) of primary AML cells, including 9 chemotherapy-resistant patient samples in vitro, and significantly reduce the leukemia load in patient-derived xenograft models in a DNT donor–unrestricted manner. Importantly, allogeneic DNTs do not attack normal hematopoietic cells or affect hematopoietic stem/progenitor cell engraftment and differentiation, or cause xenogeneic GVHD in recipients. Mechanistically, DNTs express high levels of NKG2D and DNAM-1 that bind to cognate ligands preferentially expressed on AML cells. Upon recognition of AML cells, DNTs rapidly release IFNγ, which further increases NKG2D and DNAM-1 ligands’ expression on AML cells. IFNγ pretreatment enhances the susceptibility of AML cells to DNT-mediated cytotoxicity, including primary AML samples that are otherwise resistant to DNTs, and the effect of IFNγ treatment is abrogated by NKG2D and DNAM-1–blocking antibodies. Conclusions: This study supports healthy donor–derived allogeneic DNTs as a therapy to treat patients with chemotherapy-resistant AML and also reveals interrelated roles of NKG2D, DNAM-1, and IFNγ in selective targeting of AML by DNTs. Clin Cancer Res; 24(2); 370–82. ©2017 AACR.

Simple Summary Persistence of infused cells is an important factor that dictates the outcome of adoptive cellular therapy (ACT). DNT therapy is a novel form of ACT with promising result in treating relapsed or refractory AML in preclinical and early clinical studies. However, in vivo kinetics of human DNTs in cancer-bearing host have not been previously investigated. This study was the first to investigate the persistence of DNTs and ways to improve it in patient-derived xenograft models. DNTs persistence was observed up to 50 days in various organs of leukemia-bearing hosts. However, the detected DNT level was low while significant level of persisting AMLs was observed. To improve the in vivo persistence and therapeutic efficacy of DNTs, we expanded DNTs in the presence of an PI3Kδ inhibitor, idelalisib (Ide). Ide treatment of healthy donor-derived DNTs promoted early memory subsets and improved overall fitness, reducing exhaustion while improving viability. These Ide-induced attributes led to prolonged persistence of DNTs, resulting in superior anti-leukemic activity in vivo. Further, Ide-treated DNTs improved the durability of the treatment response. Collectively, our study highlights the importance of DNT persistence and Ide-mediated improvements in the overall fitness of DNTs, which promote longer persistence in vivo and better treatment outcome. Abstract The double negative T cell (DNT) is a unique subset of T cells with potent anti-leukemic potential. Previously, DNT therapy has been shown to effectively target AML cells in patient-derived xenograft (PDX) models. Further, a recently completed phase I/IIa clinical study demonstrated the safety, feasibility, and potential efficacy in AML patients that relapsed after allogeneic hematopoietic stem cell transplantation. However, the persistence and durability of DNT-mediated anti-leukemic response is less well understood. In this study, we characterized the in vivo persistence of DNTs in PDX models. Further, we improved the efficacy and durability of DNT-mediated activity with phosphoinositide 3-kinase delta (PI3Kδ) inhibition. Mechanistically, DNTs treated with the PI3Kδ inhibitor, Idelalisib (Ide), exhibited early memory phenotype with superior viability and proliferative capacity but less cell exhaustion. Collectively, the findings from this study support the use of Ide-treated DNTs to improve its therapeutic outcome.

To compare segmented retinal layer thicknesses between patients with idiopathic infantile nystagmus (IIN) and controls. This retrospective case-control study included 66 patients with IIN and 66 age-matched controls. The retinal layers were examined using spectral domain optical coherence tomography with autosegmentation. Central foveal thickness (CFT), outer nuclear layer (ONL), and outer segment length (OSL) thickness were measured at the fovea center. Mean values for retinal nerve fiber layer, ganglion cell inner plexiform layer (GCIPL), inner nuclear layer, outer plexiform-outer nuclear layer (OPNL) thicknesses were calculated at two measurement points (nasal and temporal hump points at the macula area). There were no significant between-group differences in age, gender, or refraction error. The CFT was thicker in the IIN group compared with the control group (225.0 μm vs. 217.8 μm, P = 0.017) and OSL was shorter in IIN than in controls (40.0 μm vs. 43.7 μm., P

Background Though immune checkpoint blockade (ICB) against PD-1 has shown success in the treatment of lung cancer, not all patients respond. We have previously shown that adoptive transfer of double negative T (DNT) cells expanded from healthy donors can target leukemia but their role in treating established lung cancer is not clear. Here we explore the role of human DNT cells in targeting late-stage established lung cancer either alone or in combination with Nivolumab (anti-PD-1 antibody) and describe underlying mechanisms. Methods DNT cells from resected lung cancer tissue of patients were analyzed by flow cytometry to determine their infiltration and PD-1 expression. Expansion capacity and anti-tumor function of lung cancer patient and healthy donor DNT cells were compared. Late-stage lung cancer xenograft models were developed to determine the anti-tumor effect of DNT cells alone or in combination with anti-PD-1 antibody, and the level of tumor-infiltrating DNT cells was quantified by histology and characterized by flow cytometry. Results Patient-derived tumor infiltrating lymphocytes contained a lower frequency of DNT cells with a higher expression of PD-1 relative to normal lung tissue. Ex vivo expanded patient- and healthy donor-derived DNT cells showed similar levels of cytotoxicity against lung cancer cells in vitro. Healthy donor-derived DNT cells significantly inhibited the growth of late-stage lung cancer xenografts, which was further augmented by anti-PD-1 through increased DNT cell tumor infiltration. Conclusion This study supports the use of DNT cells for adoptive cellular therapy against lung cancer either alone or in combination with anti-PD-1. Electronic supplementary material The online version of this article (10.1186/s13046-019-1126-y) contains supplementary material, which is available to authorized users.

Background The advents of novel immunotherapies have revolutionized the treatment of cancer. Adoptive cellular therapies using chimeric antigen receptor T (CAR-T) cells have achieved remarkable clinical responses in B cell leukemia and lymphoma but the effect on solid tumors including lung cancer is limited. Here we present data on the therapeutic potential of allogeneic CD3+CD4−CD8− double negative T (DNT) cells as a new cellular therapy for the treatment of lung cancer and underlying mechanisms. Methods DNTs were enriched and expanded ex vivo from healthy donors and phenotyped by flow cytometry. Functionally, their cytotoxicity was determined against primary and established non-small-cell lung cancer (NSCLC) cell lines in vitro or through in vivo adoptive transfer into xenograft models. Mechanistic analysis was performed using blocking antibodies against various cell surface and soluble markers. Furthermore, the role of IL-15 on DNT function was determined. Results We demonstrated that ex vivo expanded DNTs can effectively lyse various human NSCLC cells in vitro and inhibit tumor growth in xenograft models. Expanded DNTs have a cytotoxic phenotype, as they express NKp30, NKG2D, DNAM-1, membrane TRAIL (mTRAIL), perforin and granzyme B, and secrete IFNγ and soluble TRAIL (sTRAIL). DNT-mediated cytotoxicity was dependent on a combination of tumor-expressed ligands for NKG2D, DNAM-1, NKp30 and/or receptors for TRAIL, which differ among different NSCLC cell lines. Furthermore, stimulation of DNTs with IL-15 increased expression of effector molecules on DNTs, their TRAIL production and cytotoxicity against NSCLC in vitro and in vivo. Conclusion Healthy donor-derived DNTs can target NSCLC in vitro and in vivo. DNTs recognize tumors via innate receptors which can be up-regulated by IL-15. DNTs have the potential to be used as a novel adoptive cell therapy for lung cancer either alone or in combination with IL-15. Electronic supplementary material The online version of this article (10.1186/s40425-019-0507-2) contains supplementary material, which is available to authorized users.

A 13-year-old castrated male mixed breed dog and a 12-year-old castrated male mixed breed cat were referred to the hospital for the treatment of pulmonary edema and azotemia at the same time. To resolve the pulmonary edema and azotemia, intermittent hemodialysis (HD) was performed using ultrafiltration (UF), and the pulmonary edema, azotemia, other electrolyte and acid-base imbalances were improved. This case study demonstrated that when we encountered pulmonary edema patients with diuretic resistance, severe electrolyte imbalance, and impaired renal function complicated by decongestive therapy using diuretics, UF therapy can be considered a life-saving intervention.

Background While conventional chemotherapy is effective at eliminating the bulk of leukemic cells, chemotherapy resistance in acute myeloid leukemia (AML) is a prevalent problem that hinders conventional therapies and contributes to disease relapse, and ultimately patient death. We have recently shown that allogeneic double negative T cells (DNTs) are able to target the majority of primary AML blasts in vitro and in patient-derived xenograft models. However, some primary AML blast samples are resistant to DNT cell therapy. Given the differences in the modes of action of DNTs and chemotherapy, we hypothesize that DNT therapy can be used in combination with conventional chemotherapy to further improve their anti-leukemic effects and to target chemotherapy-resistant disease. Methods Drug titration assays and flow-based cytotoxicity assays using ex vivo expanded allogeneic DNTs were performed on multiple AML cell lines to identify therapy-resistance. Primary AML samples were also tested to validate our in vitro findings. Further, a xenograft model was employed to demonstrate the feasibility of combining conventional chemotherapy and adoptive DNT therapy to target therapy-resistant AML. Lastly, blocking assays with neutralizing antibodies were employed to determine the mechanism by which chemotherapy increases the susceptibility of AML to DNT-mediated cytotoxicity. Results Here, we demonstrate that KG1a, a stem-like AML cell line that is resistant to DNTs and chemotherapy, and chemotherapy-resistant primary AML samples both became more susceptible to DNT-mediated cytotoxicity in vitro following pre-treatment with daunorubicin. Moreover, chemotherapy treatment followed by adoptive DNT cell therapy significantly decreased bone marrow engraftment of KG1a in a xenograft model. Mechanistically, daunorubicin increased the expression of NKG2D and DNAM-1 ligands on KG1a; blocking of these pathways attenuated DNT-mediated cytotoxicity. Conclusions Our results demonstrate the feasibility and benefit of using DNTs as an immunotherapy after the administration of conventional chemotherapy. Electronic supplementary material The online version of this article (10.1186/s13046-018-0756-9) contains supplementary material, which is available to authorized users.

The purpose of this case report was to present the treatment of continuous renal replacement therapy (CRRT) in dogs with end-stage CKD with uncontrolled uremia. Hemodialysis were carried out 6 patients who failed to improve clinical status with conventional management for CKD. Four dogs with urea reduction ratio (URR) range of 57-72% and 1 dog with URR of 37.3% showed good outcome with decreasing tendency of pre-dialysis Therefore, we suggest that CRRT could be recommended for use in CKD dogs with uncontrolled azotemia or uremia and should be monitor carefully throughout the CRRT.

This study was conducted to survey the epidemiological characteristics and the isolated strains for pathogenic E. coli which was the major causative organisms for food poisoning occurred at school food services in the Gyeonggi-do area during the past three years. We investigated 19 accidents of food-borne disease outbreaks by pathogenic E. coli at school food services from 2010 to 2012. Food-borne disease outbreaks by pathogenic E. coli were usually occurred at direct management type (18 accidents, 95%) and high schools. For the seasonal factors, 13 accidents (65%) were occurred in June to September, especially the end of August and September after the summer holidays. The first patients were occurred on Wednesday (7 accidents, 37%) and Thursday (7 accidents, 37%), and they were mainly reported on Thursday (7 accidents, 37%) and Friday (5 accidents, 26%). The exposure of risk was estimated in Monday (4 accidents, 21%), Tuesday (7 accidents, 37%) and Wednesday (4 accidents, 21%), and kimchi (5 accidents, 50%) was estimated as the food of the high risk responsible for the outbreaks. 98 isolates of pathogenic E. coli consisted of PEC (50%), ETEC (34%), EAEC (15%), and EHEC (1%). The antibiotic resistance of pathogenic E. coli showed in the descending order of ampicilline (40%), nalidixic acid (37%), trimethoprim/sulfamethoxazole (24%), and tetracycline (19%). The antibiotics of second and third generation cephalosporins, cabarpenem, aminoglycosides, and second generation quinolones had antimicrobial susceptibilities and cefalotin, ampicillin/sulbactam and chloramphenicol showed medium resistance at 29%, 25%, and 6% respectively, and 70% of isolates were resistant to more than one antibiotic. By the PFGE analysis, they were classified into nine major groups and 31 profiles with 57% pattern similarity. It was very difficult to find the correlation of antimicrobial susceptibilities and genotype in the small scale-food poisoning, but the similarity of antimicrobial resistance and PFGE patterns in the large scale-food poisoning enabled the outbreaks to estimate the same pathotype of E. coli derived from identical origins.

Bacillus cereus contamination is a major food safety problem for Korean fermented soybean products, but few studies have assessed its potential to cause foodborne illness. The objectives of this study were to investigate the prevalence and characteristics of B. cereus isolated from Korean fermented soybean products. B. cereus was detected in 110 of 162 (67.9%) samples. The highest B. cereus frequency was observed in deonjang (68 of 93 samples, 73.1%) and cheonggukjang (18 of 25, 72.0%); however, nonhemolytic enterotoxin was detected only in 22 of 162 samples (13.6%). Although the tested B. cereus isolates showed diverse pulsotypes according to repetitive sequence-PCR banding patterns, they displayed similar antibiotic sensitivity spectra. The low frequency of enterotoxin detection suggests that the potential risk of B. cereus foodborne illness associated with Korean fermented soybean products is lower than generally presumed. However, considering the prevalence of B. cereus and the high content of fermented soybean products in the Korean diet, it is necessary to constantly monitor the level of contamination with B. cereus and its toxins in such Korean food products.

Aims To compare the surgical outcomes of exotropia patients with cerebral palsy (CP) and controls without CP. Methods 30 patients with exotropia and CP and 60 age-matched controls without CP who underwent bilateral lateral rectus (BLR) recession were retrospectively enrolled. All patients underwent BLR recession according to Parks’ method. Surgical success was defined by esotropia deviation ≤5 PD (prism diopters) and exotropia deviation ≤10 PD. Success rate, cumulative probabilities of success, and postoperative change of angle deviations using a linear mixed model were evaluated. Results Mean postoperative follow-up times were 21.60±8.62 months for the CP group and 25.60±10.82 months for the control group (p=0.081). Mean preoperative deviation was 30.97±10.54 PD in the CP group and 29.75±7.52 PD in the control group (p=0.530), which was reduced to 4.44±7.13 PD and 7.43±7.59 PD, respectively (p=0.093) after BLR recession. Using linear mixed model analysis, the estimated mean postoperative deviation of both groups was in the success range at all times tested. At the final visit, successful surgical outcomes were achieved in 76.7% of the CP group and 56.7% of the control group (p=0.179). Differences in the cumulative probability of surgical success were not statistically significant between the two groups (p=0.106, log rank test). Conclusions CP patients with exotropia showed successful ocular alignment comparable to that of controls after BLR recession of at least 2 years.

To describe the ophthalmological manifestations in patients with childhood onset Leigh syndrome (LS) and investigate the correlation between genotypes and phenotypes in patients with LS.Childhood onset LS was clinically and enzymatically confirmed in a total of 63 patients. Among them, 44 patients who underwent ophthalmologic consultation were included in this study. Patients with LS underwent genotyping for the whole genome of mitochondrial DNA and SURF1 mutations. The clinical demographic and ophthalmologic phenotypes were compared between the good prognosis group and the poor prognosis group.Strabismus (40.9%) was the most frequently observed ophthalmologic manifestation, followed by pigmentary retinopathy (22.5%), optic atrophy (22.5%), ptosis (15.9%), and nystagmus (13.6%). Thirteen patients were exotropes and five patients were esotropes. The mean exodeviation was 29.6±12.5 prism dioptres (PD) and the mean esodeviation was 24.0±8.9 PD. All patients with esotropia reported disease onset at1 year old. Among 26 patients older than 4 years, eight (30.8%) patients had better than 0.4 in the best eye was noted. Eyelid ptosis was a main presenting sign in four patients (9.1%). Among these patients, two patients had m.13513GA mutation in the MT-ND5 gene. Age at onset was 2.47±2.06 years in the good prognosis group and 0.92±0.98 years in the poor prognosis group (p=0.002). Serum lactate peak concentration was 3.23±1.36 mmol/L in the good prognosis group and 4.54±2.31 mmol/L in the poor prognosis group (p=0.051).LS is a group of mitochondrial disorders with variable ophthalmologic manifestations, the most frequent being strabismus in this study. Ptosis could be an initial sign in patients with LS and these patients can be easily misdiagnosed as having juvenile myasthenia gravis.

Purpose: To investigate horizontal image disparity in three-dimensional (3-D) perception using 3-D animations in normal control patients and patients with intermittent exotropia, anisometropic amblyopia, and partially accommodative esotropia. Materials and Methods: A total of 133 subjects were included. Stereopsis was measured using the Titmus Stereo test (Stereo Optical Inc., Chicago, IL, USA) and a 3-D stereopsis test with a 15 inch 3-D display laptop, adjusting 3-D parameters of 0 mm horizontal disparity to 15 mm horizontal disparity. Results: When compared with normal controls, the average threshold of the 3-D stereopsis test was significantly reduced for esotropia patients ( p

To evaluate the impact of myopic keratorefractive surgery on ocular alignment. This prospective study included 194 eyes of 97 myopic patients undergoing laser refractive surgery. All patients received a complete ophthalmic examination with particular attention to ocular alignment before and 3 months after surgery. Patients with a mean age of 26.6 years and a mean refractive error of −4.83 diopters (D) myopia were treated. Asymptomatic ocular misalignment was present preoperatively in 46 (47%) patients: a small-angle heterophoria (1–8 prism diopters, PD) in 36%; and a large-angle heterophoria (>8 PD)/heterotropia in 11%. Postoperatively, the change in angles of 10 PD or greater occurred in 3% for distance and 6% for near fixation: in 7% of the patients with orthophoria, in 3% of those with a small-angle heterophoria, and in 18% of those with a large-angle heterophoria/heterotropia. No patient developed diplopia. The preoperative magnitude of myopia or postoperative refractive status was not related to the change in ocular alignment. The higher anisometropia was associated with a decrease in deviation (P=0.041 for distance and P=0.002 for near fixation), whereas the further near point of convergence tended to be related with an increase in near deviation (P=0.055). Myopic refractive surgery may cause a change in ocular alignment, especially in cases with a large-angle heterophoria/heterotropia. There is also a chance of improvement of misalignment in patients with anisometropia.

This study was conducted to investigate the changes of the residual amount in the leafstalk (end) and the fore-end (upper) portion of lettuce leaves during cultivation period to 14 days, 12 times in total (0, 1, 2, 3, 4, 5, 7, 8, 10, 12, 13 and 14 days) after spraying with boscalid and lufenuron. In the case of boscalid, the initial concentrations at 3 hours (0 day) of the leafstalk and the fore-end portion of lettuce leaves were 18.26 mg/kg and 84.97 mg/kg, respectively and the residual amounts were rapidly decreased to 0.31 mg/kg and 0.37 mg/kg at 14 days after chemical application. In the case of lufenuron, the initial concentrations at 3 hours (0 day) of the leafstalk and the fore-end portion of lettuce leaves were 0.91 mg/kg and 5.21 mg/kg, respectively and the residual amounts were rapidly decreased to 0.06 mg/kg and 0.09 mg/kg at 13 days after chemical application. The variations of the residual concentrations analyzing 12 times after spraying showed that the residual amounts of the leafstalk portion of lettuce leaves were less than its fore-end portion in boscalid and lufenuron. In additon, 9 kinds of pesticide including boscalid in 16 lettuce leaf (found to contain pesticide in 2013) showed that the residual amounts of the leafstalk portion of lettuce leaves were less than its fore-end portion as well.

The safety of fake anti-impotence drugs (fake Viagra : 26 samples, fake Cialis : 25 samples) dis- tributed in Gyeonggi province was studied by monitoring the concentrations of anti-impotence pharmaceutical ingre- dient and their analogues. The concentrations of anti-impotence pharmaceutical ingredient 4 specis and their analogues 17 specis were estimated using by HPLC/PDA, LC-MS/MS. The range of concentration of sildenafil in fake viagra was 40~199 mg/tablet, among them the portion of the concentrations of sildenafil over 150 mg/tablet exceeded 65%. 3 cases in tested samples contained sildenafil and tadalafil. The range of concentration of sildenafil in fake cialis was 102~249 mg/tablet, among them the portion of the concentrations of sildenafil over 150 mg/tablet exceeded 88%. One case in tested samples contained demethylhongdenafil (90 mg/tablet). These results indicate that there were many fake anti-impotence drug contained high level of anti-impotence pharmaceutical ingredients, a sus- tainable monitoring and the blocked distribution of fake anti-impotence drugs recommended.

This study was done to analyze the contents of minerals and to investigate the ratio of measured values to labeled values and to analyze the ratio of calcium to other minerals in 68 specimen with minerals - fortified commercial beverages, noodles, cereals and grain products. Content of calcium, iron and zinc in samples after micro- wave digestion was analyzed with an ICP-OES. The measured values of calcium were ranged 82.2~293.1% of the labeled values in 38 samples composed calcium - fortified commercial beverages, noodles, cereals and grain products. The measured values of iron and zinc were ranged 83.3~301.0%, 90.1~314.1% of the labeled values in minerals - for- tified commercial beverages, noodles, cereals and grain products, 42, 24 samples. The Ca : Fe ratios were 90.55 (50.55~220.64) in fruit & vegetable juice, 850.41 in fruit & vegetable beverage, 553.49 in blended beverage, 179.07 (118.37~238.01) in soy milk, 204.39(41.64~397.52) in noodle, 296.97(121.64~868.88) in fried noodle, 30.89(15.69~ 62.05) in cereal and 7.73(0.22~49.92) in grain product. The Ca : P ratios were 1.44(0.96~1.98) in fruit & vegetable juice, 1.92 in fruit & vegetable beverage, 1.66 in blended beverage, 4.23(2.25~7.72) in soy milk, 1.14(0.28~1.97) in noodle, 1.88(1.17~2.42) in fried noodle, 1.29(0.87~2.92) in cereal and 0.30(0.06~1.57) in grain product. The Ca : Mg ratios were 1.85(0.87~5.04) in fruit & vegetable juice, 28.72 in fruit & vegetable beverage, 2.97 in blended beverage, 5.27(2.93~9.36) in soy milk, 3.97(1.34~7.57) in noodle, 6.77(4.63~10.78) in fried noodle, 4.40(2.30~12.55) in cereal and 1.17(0.23~7.48) in grain product. These results suggest calcium contents and the ratio of calcium contents to other minerals in calcium-fortified food products should be strictly controlled. Moreover, to avoid problems with Excessive nutrition, there must be initiatives for better understanding on food labelling and nutrition for fortified food.

Many studies have reported comparable risk of hard end points between percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) for unprotected left main coronary artery (ULMCA) stenosis. However, there are limited data regarding the morbidity associated with ULMCA revascularization. This study sought to compare the cause and risk of readmissions after PCI and CABG for ULMCA stenosis. We evaluated the unadjusted and adjusted risk of readmissions in 1,352 patients (783 PCI treated and 569 CABG treated) who were consecutively enrolled in a multicenter registry of patients with ULMCA stenosis, named the Premier of Randomized Comparison of Bypass Surgery versus Angioplasty Using Sirolimus-Eluting Stent in Patients with Left Main Coronary Artery Disease trial. Overall, 206 PCI-treated patients (26.3%) experienced at least 1 readmission after the index procedure during 48.7 ± 16.0 months of follow-up compared with 84 CABG-treated patients (14.8%, p