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Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC), and provides a target for a dendritic cell (DC) vaccine. CD137 ligand (CD137L) expressed on antigen presenting cells, costimulates CD137-expressing T cells, and reverse CD137L signaling differentiates monocytes to CD137L-DC, a type of DC, which is more potent than classical DC in stimulating T cells. In this phase I study, patients with locally recurrent or metastatic NPC were administered CD137L-DC pulsed with EBV antigens (CD137L-DC-EBV-VAX). Of the 12 patients treated, 9 received full 7 vaccine doses with a mean administered cell count of 23.9 × 106 per dose. Treatment was well tolerated with only 4 cases of grade 1 related adverse events. A partial response was obtained in 1 patient, and 4 patients are still benefitting from a progression free survival (PFS) of currently 2–3 years. The mean pre-treatment neutrophil: lymphocyte ratio was 3.4 and a value of less than 3 was associated with prolonged median PFS. Progressors were characterized by a high frequency of naive T cells but a low frequency of CD8+ effector T cells while patients with a clinical benefit (CB) had a high frequency of memory T cells. Patients with CB had lower plasma EBV DNA levels, and a reduction after vaccination. CD137L-DC-EBV-VAX was well tolerated. The use of CD137L-DC-EBV-VAX is demonstrated to be safe. Consistent results were obtained from all 12 patients, indicating that CD137L-DC-EBV-VAX induces an anti-EBV and anti-NPC immune response, and warranting further studies in patients post effective chemotherapy. Precis. The first clinical testing of CD137L-DC, a new type of monocyte-derived DC, finds that CD137L-DC are safe, and that they can induce an immune response against Epstein-Barr virus-associated nasopharyngeal carcinoma that leads to tumor regression or prevents tumor progression.

Simulated Infective Protocol (SIP) is an ex-vivo culture system modeled after the temporal changes of essential cytokines in an acute infection, and previously proven successful in converting T lymphocytes harvested and activated from peripheral blood of normal donors, to revertant CD45RA + Central Memory T lymphocytes (Tcmra) demonstrating properties akin to T Memory Stem Cells (Tscm). In this study, we applied similar SIP on tumor infiltrating lymphocytes (TIL) from bone marrow of patients diagnosed with acute myeloid leukemia (AML), and replicated the feasibility to convert activated TILs into Tcmra phenotype. These revertant Tcmra lymphocytes re-expressed CD45RA+, CCR7+, CD62L + and CD127+, shown improved survivability with longer telomere length, expressed memory properties including higher Eomes to Tbet ratio, and exhibited cytotoxicity against autologous AML blast cells.

Background Timely blood delivery to patients with critical bleeding poses logistic challenges. A modern, high speed hospital pneumatic tube system (PTS) is one solution, but blood units may be subjected to high-speed torque and acceleration/deceleration forces. Objective To validate a new PTS system for potential use at our 1,400-bed hospital in Singapore. Method Our validation included red blood cells, platelets, thawed plasma, and cryoprecipitate units transported from the blood bank for a distance of 820 meters (PTS track), at a velocity of 3–6 meters per second. Transit time, temperature, bag integrity, and blood quality were assessed visually and through analytical testing on pre- and post-PTS specimens. Results Blood units arrived physically intact in less than 8 minutes. The temperature for each was within the acceptable range. Comparative testing of pre-PTS and post-PTS specimens showed no significant difference in physical quality and analyzed parameters (P> .05). Conclusions High speed PTS transportation of blood components has satisfactory fidelity and speed, without significant impact on quality. As a result, we incorporated PTS blood delivery into the hospital massive-transfusion protocol and successfully operationalized that new system.

ObjectiveTo determine the clinical outcomes of older COVID-19 patients who received DMB compared to those who did not. We hypothesized that fewer patients administered DMB would require oxygen therapy and/or intensive care support than those who did not.MethodologyCohort observational study of all consecutive hospitalized COVID-19 patients aged 50 and above in a tertiary academic hospital who received DMB compared to a recent cohort who did not. Patients were administered oral vitamin D3 1000 IU OD, magnesium 150mg OD and vitamin B12 500mcg OD (DMB) upon admission if they did not require oxygen therapy. Primary outcome was deterioration post-DMB administration leading to any form of oxygen therapy and/or intensive care support.ResultsBetween 15 January and 15 April 2020, 43 consecutive COVID-19 patients aged ≥50 were identified. 17 patients received DMB and 26 patients did not. Baseline demographic characteristics between the two groups was significantly different in age. In univariate analysis, age and hypertension showed significant influence on outcome while DMB retained protective significance after adjusting for age or hypertension separately in multivariate analysis. Fewer DMB patients than controls required initiation of oxygen therapy during their hospitalization (17.6% vs 61.5%, P=0.006). DMB exposure was associated with odds ratios of 0.13 (95% CI: 0.03 – 0.59) and 0.20 (95% CI: 0.04 – 0.93) for oxygen therapy and/or intensive care support on univariate and multivariate analyses respectively.ConclusionsDMB combination in older COVID-19 patients was associated with a significant reduction in proportion of patients with clinical deterioration requiring oxygen support and/or intensive care support. This study supports further larger randomized control trials to ascertain the full benefit of DMB in ameliorating COVID-19 severity.

We wish to propose an infection model to address some of the unique and unusual features observed in coronavirus disease 2019 (COVID-19), e.g., why were severe patients mounting more intense immune response compared to the mild cases, and why did severe patients demonstrate higher and more protracted viral shedding when their immune responses were more intense. In this exposition, we will first construct a pathogenic model for COVID-19, and followed with a discussion on how it may influence critical aspects of COVID-19 management. By extrapolating from this model, we will highlight important and potential therapeutic strategies. This proposed model is intended to provide a generalized framework, but it requires validation from further clinical studies.

Highlights • Use of a combination of vitamin D, magnesium, and vitamin B12 (DMB) in patients with coronavirus disease (COVID-19) was studied. • Fewer patients ≥50 y of age with COVID-19 on DMB suffered clinical deterioration. • Further studies are warranted to ascertain the full benefit of DMB in patients with COVID-19., Objectives The aim of this study was to determine clinical outcomes of older patients with coronavirus (COVID-19) who received a combination of vitamin D, magnesium, and vitamin B12 (DMB) compared with those who did not. We hypothesized that fewer patients administered this combination would require oxygen therapy, intensive care support, or a combination of both than those who did not. Methods This was a cohort observational study of all consecutive hospitalized patients ≥50 y of age with COVID-19 in a tertiary academic hospital. Before April 6, 2020, no patients received the (DMB) combination. After this date, patients were administered 1000 IU/d oral vitamin D3, 150 mg/d oral magnesium, and 500 mcg/d oral vitamin B12 upon admission if they did not require oxygen therapy. Primary outcome was deterioration leading to any form of oxygen therapy, intensive care support, or both. Results Between January 15 and April 15, 2020, we identified 43 consecutive patients ≥50 y of age with COVID-19. Seventeen patients received DMB before onset of primary outcome and 26 patients did not. Baseline demographic characteristics between the two groups were significantly different by age. In univariate analysis, age and hypertension had a significant influence on outcome. After adjusting for age or hypertension separately in a multivariate analysis, the intervention group retained protective significance. Fewer treated patients than controls required initiation of oxygen therapy during hospitalization (17.6 vs 61.5%, P = 0.006). DMB exposure was associated with odds ratios of 0.13 (95% confidence interval [CI], 0.03–0.59) and 0.20 (95% CI, 0.04–0.93) for oxygen therapy, intensive care support, or both on univariate and multivariate analyses, respectively. Conclusions A vitamin D / magnesium / vitamin B12 combination in older COVID-19 patients was associated with a significant reduction in the proportion of patients with clinical deterioration requiring oxygen support, intensive care support, or both. This study supports further larger randomized controlled trials to ascertain the full benefit of this combination in ameliorating the severity of COVID-19.

6535 Background: Epstein-Barr virus (EBV) is associated with non-keratinising (NK) NPC, a disease prevalent in Southeast Asia, and provides a potential target for dendritic cell (DC) vaccine therapy. CD137 ligand (CD137L) expressed on antigen presenting cells costimulates CD137 expressing T cells upon receptor/ligand interaction. CD137L signalling differentiates monocytes to CD137L-DC, a novel type of DC, which are more potent than classical DC in stimulating autologous T cells. Here, we explore the safety and efficacy of autologous CD137L-DC pulsed with EBV peptides spanning Epstein Barr nuclear antigen 1, latent membrane protein 1 (LMP1) and LMP2 (CD137L-DC-EBV-VAX) in patients with locally recurrent or metastatic NPC. Methods: In this single centre, phase I study, eligible patients (pts) with locally recurrent or metastatic NK-NPC and clinical benefit (CB) from their prior treatment (stable disease [SD], partial [PR] or complete response[CR]), underwent apheresis to isolate monocytes which were differentiated to CD137L-DC through CD137L agonist exposure. CD137L-DC were pulsed with EBV antigens during maturation to obtain CD137L-DC-EBV-VAX which was administered intradermally every 2 weeks (w) for up to 7 injections following site preconditioning with Tetanus and Diphtheria vaccine. Results: 14 pts were enrolled of which 2 progressed rapidly and did not begin treatment. Mean age was 58 years. Median lines of prior treatment for metastatic NPC was 1 (range 1-6), the most common being cisplatin and gemcitabine. 9 pts received 7 vaccine doses (range 2-7) with a mean administered cell count of 23.9x106. CB was seen in 5 cases (42%) with 1 PR and 4 SD beyond 1 year. Median progression free survival (mPFS) was 26w (95% CI, 23-43). The lowest PFS (8w) was in a pt with 6 prior lines of treatment including a checkpoint inhibitor. Mean pretreatment neutrophil: lymphocyte ratio (NLR) was 3.4 and a value of less than 3 was associated with prolonged mPFS (42 vs 14w, p = 0.01). Enzyme linked immune absorbent spot (ELISPOT) analysis in 5 pts with CB showed a rise in interferon-γ secreting peripheral T cells prior to the 3rd vaccine versus baseline. Treatment was well tolerated with only 4 cases of grade 1 related adverse events reported, most commonly injection site reaction (3pts). Conclusions: CD137L-DC-EBV-VAX is safe and exhibits promising efficacy when administered following CB from chemotherapy. A rise in activated peripheral blood mononuclear cells after 2 vaccinations in selected patients showing benefit suggests immunological correlates with efficacy. Clinical trial information: NCT03282617 .

Point-of-care testing (POCT) of the international normalised ratio (INR) is recognised as a rapid and reliable alternative to venous INR measurement for titration and monitoring of patients anticoagulated with vitamin K antagonists.1–4 Besides empowering patient self-testing and self-management,5–7 POCT coagulometers are now widely used in healthcare institutions for bedside anticoagulation management and have replaced venous INR sampling in many anticoagulation clinics (ACCs).8–10 For the past decade, our institution has been using the CoaguChek XS system (Roche Diagnostics, Basel, Switzerland) for INR testing at our outpatient ACC and inpatient anticoagulation service. This followed a validation study conducted by our group which showed excellent correlation with venous INR below a reading of 3.5.11 A tendency for a higher INR on the POCT platform above this threshold prompted us to introduce a mechanism that rejected results above 3.5 on the CoaguChek XS system and mandating repeat testing with venous INR. This test protocol remains in effect today. Recently, two new fourth-generation coagulometers were introduced on the CoaguChek platform. The CoaguChek Pro II is designed for institutional use with the added capability of measuring activated partial thromboplastin time while the CoaguChek INRange is targeted at patients performing home INR monitoring. These machines have the same detection system as the CoaguChek XS devices but uses the new CoaguChek PT Test strip which has a broader intended use compared with the XS devices’ test strip.12 We evaluated the performance of these two coagulometers for INR testing with a validation study comparing their performance against the old CoaguChek XS Pro and standard venous INR. The results were intended for us to conduct a review of our current POCT INR test protocol. This paper reports the findings of our efforts. ### Study design and study population This prospective validation study comparing three different POCT coagulometers against venous INR was …

Adoptive cell therapy is an emerging treatment strategy for a number of serious diseases. Regulatory T (Treg) cells represent 1 cell type of particular interest for therapy of inflammatory conditions, as they are responsible for controlling unwanted immune responses. Initial clinical trials of adoptive transfer of Treg cells in patients with graft-versus-host disease were shown to be safe. However, obtaining sufficient numbers of highly pure and functional Treg cells with minimal contamination remains a challenge. We developed a novel approach to isolate "untouched" human Treg cells from healthy donors on the basis of negative selection using the surface markers CD49d and CD127. This procedure, which uses an antibody cocktail and magnetic beads for separation in an automated system (RoboSep), was scaled up and adapted to be compatible with good manufacturing practice conditions. With this setup we performed 9 Treg isolations from large-scale leukapheresis samples in a good manufacturing practice facility. These runs yielded sufficient numbers of "untouched" Treg cells for immediate use in clinical applications. The cell preparations consisted of viable highly pure FoxP3-positive Treg cells that were functional in suppressing the proliferation of effector T cells. Contamination with CD4 effector T cells was

CD137 ligand (CD137L), a member of the tumour necrosis factor family, is expressed as a cell surface molecule. Engagement of CD137L on haematopoietic progenitor cells induces monocytic differentiation, and in peripheral monocytes CD137L signalling promotes differentiation to mature dendritic cells. We hypothesized that CD137L signalling would also induce differentiation in transformed myeloid cells. Here we show that recombinant CD137 protein, which crosslinks CD137L and initiates reverse CD137L signalling in myeloid cells, induces morphological changes (adherence, spreading), loss of progenitor markers (CD117), expression of maturation markers (CD11b, CD13) and secretion of cytokines that are indicative of myeloid differentiation. Under the influence of CD137L signalling, acute myeloid leukaemia (AML) cells acquired expression of co-stimulatory molecules (CD80, CD86, CD40), the dendritic cell marker CD83 and dendritic cell activities, enabling them to stimulate T cells. CD137L signalling induced differentiation in 71% (15 of 21) of AML samples, irrespective of French-American-British classification and CD137L expression level. However, the type of response varied with the AML subtype and patient sample. In summary, this study demonstrated that CD137L signalling induced differentiation in malignant cells of AML patients, and suggests that it may be worthwhile to investigate treatment with recombinant CD137 protein as a potential novel therapeutic approach for AML.

Central memory T lymphocytes were reported to develop after acute but not chronic infection, which prompted this feasibility study on generating long-term CD8 T cells ex vivo, by applying a culture condition that simulates an acute infection. During 35 d of culture, naive T cells (CD45RA(+), CD127(+), CCR7(+), CD62L(+), CXCR3(+)) first developed into effector T cells (CD45RA(+/-), CD127(+/-), CCR7(+/-), CD62L(+), CXCR3(+)), followed by three intermediate stages: intermediate T cells 1 (CD45RO(+), CD127(+/-), CCR7(+), CD62L(+), CXCR3(+)), intermediate T cells 2 (CD45RO(+), CD127(-), CCR7(-), CD62L(+), CXCR3(+)), and intermediate T cells 3 (CD45RO(+/-), CD127(+), CCR7(+), CD62L(-), CXCR3(+)) before reverting to stable CD45RA(+) central memory T cells (CD45RA(+), CD127(+), CCR7(+), CD62L(+), CXCR3(+)). If both anti-CD3 and the inflammatory milieu persisted beyond day 10, intermediate T cells 2 gradually developed into effector memory T cells (CD45RO(+), CD127(-), CCR7(-), CD62L(-), CXCR3(+)). Furthermore, intermediate T cells 2 or effector memory T cells, when cultured in persistent inflammatory cytokines devoid of anti-CD3, were converted to central memory T cells (CD45RO(+), CCR7(+), CD62L(+)). Overall, these results support ex vivo memory-like T lymphocyte production and favor a developmental pathway including both divergent and linear relationships.

Background Hemophilia A treatment relies on costly factor VIII (FVIII) replacement that may transmit iatrogenic viral diseases. Viral vectors and cell implants are being developed as improvements. We investigated in vivo electroporation of naked DNA as a safe and simple method for correcting FVIII deficiency. Methods B-domain-deleted murine FVIII cDNA expression plasmids were constructed with CMV and elongation factor 1α promoters for characterisation in murine C2C12 myoblasts. The construct conferring highest in vitro FVIII secretion was electroporated into skeletal muscle of FVII null mice in vivo for phenotypic correction using a protocol that minimised tissue injury. Results B-domain-deleted murine FVIII cDNA plasmids induced FVIII secretion from stably transfected C2C12 myoblasts (0.54 ± 0.20 mU/day/105 cells). Phenotypic correction of hemophilic mice was more consistently achieved using a protocol for in vivo electroporation of gastrocnemius muscle with FVIII cDNA that reduced tissue injury by the use of plate electrodes, hyaluronidase pre-treatment and lower field strength. This technique was associated with

Relapsed and refractory disease shortens the survival of acute myeloid leukemia (AML) patients. Therefore, additional therapy apart from conventional chemotherapy and stem cell transplantation is urgently needed, especially in eradicating residual leukemic stem cells. The identification of leukemia associated antigens, and the observation that administration of allogeneic T cells may mediate graft versus leukemia effect paved the way to develop various immunotherapy strategies. Effective provision of both tumor antigen and co-stimulation are essential for strategies aimed at enrolling cytotoxic T- lymphocytes to eradicate leukemic cells. In this study, we attempted to up-regulate co- stimulators on leukemic cells (termed “dendritisation” to mimic dendritic cells in providing strong co-stimulation) and couple with inherent leukemic antigens to generate specific antileukemic T-lymphocytes against autologous blast cells. Our study shows evidence of distinct cytotoxicity of CD4 positive T lymphocytes against autologous blast cells. Dendritised AML blasts were able to function with antigen presenting capability, while still phenotypically existing as blast cells. This was also achievable for a wide range of subtypes of AML, making it a potential immunotherapy against AML.