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Background Delay in seeking care increases the morbidity and mortality in tuberculosis (TB) patients and leads to increased likelihood of transmission within the community. Stigmatisation is one of the influencing factors causing delay in accessing healthcare when experiencing symptoms of TB. Objective This study aimed to explore the relationship between TB stigmatisation and patient delay among pulmonary tuberculosis (PTB) patients. Methods A mixed methods study was undertaken among 300 randomly sampled participants who completed questionnaire surveys, supplemented by 25 in-depth interviews with purposively-sampled participants identified as delaying access to healthcare. Results Participants reporting higher perceived levels of TB stigmatisation demonstrated a longer duration of patient delay ( p < 0.001). Qualitative data reinforced the view that perceptions of TB as being a disease carrying with it a stigma contributed to a delay in seeking healthcare. This was also exacerbated in patients with HIV-positive. Given that HIV-positive status is still subject to stigma in some parts of society, patients demonstrated additional reluctance to seek out healthcare if they were known to be HIV-positive whilst also experiencing symptoms of TB. Discussion The study highlights that accurate and objective information about TB is required to reduce societal stigmatisation related to the disease, thereby reducing the reluctance of patients to access healthcare.

Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has been a clinical breakthrough for pediatric B-cell acute lymphoblastic leukemia (B-ALL), and loss of the CD19 target antigen on leukemic cells represents a major mechanism of relapse. Previous studies have observed CD19 mutations specific to CD19− relapses, and we sought to clarify and strengthen this relationship using deep whole-exome sequencing in leukemic cells expanded in a patient-derived xenograft. By assessing pre-treatment and relapse cells from 13 patients treated with CAR T-cell therapy, 8 of whom developed CD19− relapse and 5 of whom developed CD19+ relapse, we demonstrate that relapse-specific single-nucleotide variants and small indels with high allele frequency combined with deletions in the CD19 gene in a manner specific to those patients with CD19− relapse. Before CAR T-cell infusion, one patient was found to harbor a pre-existing CD19 deletion in the context of genomic instability, which likely represented the first hit leading to the patient's subsequent CD19− relapse. Across patients, preexisting mutations and genomic instability were not significant predictors of subsequent CD19− relapse across patients, with sample size as a potential limiting factor. Together, our results clarify and strengthen the relationship between genomic events and CD19− relapse, demonstrating this intriguing mechanism of resistance to a targeted cancer immunotherapy.

Protein tyrosine phosphatases constitute an important class of drug targets, whose potential has been limited by the paucity of drug-like small molecule inhibitors. We recently described a class of active site-directed, moderately selective and potent inhibitors of the low molecular weight protein tyrosine phosphatase (LMW-PTP). Here we report our extensive structure-based design and optimization effort that afforded inhibitors with vastly improved potency and specificity. The leading compound inhibits LMW-PTP potently and selectively (K(i) = 1.2 nM, >8,000-fold selectivity). Many compounds exhibit favorable drug-like properties, such as low molecular weight, weak CYP inhibition, high metabolic stability, moderate to high cell permeability (Papp> 0.2 nm/s), and moderate to good oral bioavailability (%F from 23% to 50% in mice) and therefore can be used as in vivo chemical probes to further dissect the complex biological as well as pathophysiological roles of LMW-PTP and for the development of therapeutics targeting LMW-PTP.

Synovial inflammation has known contributions to chronic osteoarthritis (OA) pain, but the potential role in transitions from early to late stages of OA pain is unclear.The slowly progressing surgical destabilization of the medial meniscus (DMM) murine OA model and sham control, was used in male C57BL/6J mice to investigate the interplay between knee inflammation, plasma pro- and anti-inflammatory oxylipins and pain responses during OA progression. Changes in joint histology, macrophage infiltration, chemokine receptor CX3CR1 expression, weight bearing asymmetry, and paw withdrawal thresholds were quantified 4, 8 and 16 weeks after surgery. Plasma levels of multiple bioactive lipid mediators were quantified using liquid chromatography with tandem mass-spectrometry (LC-MS/MS).Structural joint damage was evident at 8 weeks post-DMM surgery onwards. At 16 weeks post-DMM surgery, synovial scores, numbers of CD68 and CD206 positive macrophages and pain responses were significantly increased. Plasma levels of oxylipins were negatively correlated with joint damage and synovitis scores at 4 and 8 weeks post-DMM surgery. Higher circulating levels of the pro-resolving oxylipin pre-cursor 17-HDHA were associated with lower weight bearing asymmetry at week 16.The transition to chronic OA pathology and pain is likely influenced by both joint inflammation and plasma oxylipin mediators of inflammation and levels of pro-resolution molecules.Using a slow progressing surgical model of osteoarthritis we show how the changing balance between local and systemic inflammation may be of importance in the progression of pain behaviours during the transition to chronic osteoarthritis pain.

Purpose: To study the biology and identify markers of severe cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS) in children after chimeric antigen receptor T-cell (CAR T) treatment. Experimental Design: We used comprehensive proteomic profiling to measure over 1,400 serum proteins at multiple serial timepoints in a cohort of patients with B-cell acute lymphoblastic leukemia treated with the CD19-targeted CAR T CTL019 on two clinical trials. Results: We identified fms-like tyrosine kinase 3 (FLT3) and mast cell immunoglobulin-like receptor 1 (MILR1) as preinfusion predictive biomarkers of severe CRS. We demonstrated that CRS is an IFNγ-driven process with a protein signature overlapping with hemophagocytic lymphohistiocytosis (HLH). We identified IL18 as a potentially targetable cytokine associated with the development of ICANS. Conclusions: We identified preinfusion biomarkers that can be used to predict severe CRS with a sensitivity, specificity, and accuracy superior to the current gold standard of disease burden. We demonstrated the fundamental role of the IFNγ pathway in driving CRS, suggesting CRS and carHLH are overlapping rather than distinct phenomena, an observation with important treatment implications. We identified IL18 as a possible targetable cytokine in ICANS, providing rationale for IL18 blocking therapies to be translated into clinical trials in ICANS.

Background Pulmonary tuberculosis (PTB) is a major health problem in Thailand. Delay in getting treatment is an important factor which may worsen the disease and increase TB transmission. Objective This study aimed to investigate the duration and predictors of patient delay among PTB patients in Northeast Thailand. Methods A cross-sectional study was undertaken using a structured questionnaire in nine districts in Nakhon Ratchasima Province from July to September 2018. The duration between the first symptom onset and the first visit to a health facility was determined, with a period of greater than 30 days defined as patient delay. Multiple logistic regression was used to identify predictors of the delay. Results 300 PTB patients participated in the survey, with patient delay identified in 39% of respondents. The median duration of the delay was 35 days among participants overall. Through multivariate analysis, primary education, upper secondary education, previous TB knowledge, TB recognition, TB stigmatisation, weight loss, self-treatment, the number of visits with health providers and using a motorcycle to travel to the hospital were significant predictors of patient delay. Discussion Knowledge needs to be provided to people to increase their recognition and minimise stigmatisation of TB. Education about TB screening needs to be revised and delivered to health providers to increase and improve TB detection processes.

In Pseudomonas aeruginosa , quorum sensing (QS) depends on an interconnected regulatory hierarchy involving the Las, Rhl and Pqs systems, which are collectively responsible for the co-ordinated synthesis of a diverse repertoire of N-acylhomoserine lactones (AHLs) and 2-alkyl-4-quinolones (AQs). Apparent population density-dependent phenomena such as QS may, however, be due to growth rate and/or nutrient exhaustion in batch culture. Using continuous culture, we show that growth rate and population density independently modulate the accumulation of AHLs and AQs such that the highest concentrations are observed at a slow growth rate and high population density. Carbon source (notably succinate), nutrient limitation (C, N, Fe, Mg) or growth at 25 °C generally reduces AHL and AQ levels, except for P and S limitation, which result in substantially higher concentrations of AQs, particularly AQ N-oxides, despite the lower population densities achieved. Principal component analysis indicates that ~26 % variation is due to nutrient limitation and a further 30 % is due to growth rate. The formation of N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) turnover products such as the ring opened form and tetramic acid varies with the limiting nutrient limitation and anaerobiosis. Differential ratios of N-butanoyl-homoserine lactone (C4-HSL), 3OC12-HSL and the AQs as a function of growth environment are clearly apparent. Inactivation of QS by mutation of three key genes required for QS signal synthesis (lasI, rhlI and pqsA) substantially increases the concentrations of key substrates from the activated methyl cycle and aromatic amino acid biosynthesis, as well as ATP levels, highlighting the energetic drain that AHL and AQ synthesis and hence QS impose on P. aeruginosa .

Legends for supplementary figures and tables.

Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has been a clinical breakthrough for pediatric B-cell acute lymphoblastic leukemia (B-ALL), and loss of the CD19 target antigen on leukemic cells represents a major mechanism of relapse. Previous studies have observed CD19 mutations specific to CD19− relapses, and we sought to clarify and strengthen this relationship using deep whole-exome sequencing in leukemic cells expanded in a patient-derived xenograft. By assessing pre-treatment and relapse cells from 13 patients treated with CAR T-cell therapy, 8 of whom developed CD19− relapse and 5 of whom developed CD19+ relapse, we demonstrate that relapse-specific single-nucleotide variants and small indels with high allele frequency combined with deletions in the CD19 gene in a manner specific to those patients with CD19− relapse. Before CAR T-cell infusion, one patient was found to harbor a pre-existing CD19 deletion in the context of genomic instability, which likely represented the first hit leading to the patient's subsequent CD19− relapse. Across patients, preexisting mutations and genomic instability were not significant predictors of subsequent CD19− relapse across patients, with sample size as a potential limiting factor. Together, our results clarify and strengthen the relationship between genomic events and CD19− relapse, demonstrating this intriguing mechanism of resistance to a targeted cancer immunotherapy.

Details of SNVs and small indels detected from deep whole-exome sequencing in CD19 and selected genes associated with genomic instability.

Clinical information regarding the 13 patients involved in this study, including passage numbers of PDX samples.

Supplementary Figure 1-9.

Supplementary Table 1. DNA fragments synthesized and inserted into MSCV-CD19-IRES-DsRedFP via EcoRI/BglII or BglII/XhoI digestion, and later moved into pMX-IRES-Blast via EcoRI/XhoI cloning. Supplementary Table 2. Sets of primers used for PCR amplification and Sanger sequencing of CD19 gene and the upstream -1.2Kb enhancer and promoter regions. Supplementary Table 3. Sets of primers for the analysis of CpG-Methylation status after bisulfite modification of genomic DNA. Supplementary Table 4. Sets of primers used for radioactive semiquantitative PCR analysis of CD19 mRNA isoforms. Supplementary Table 5. Sequence of the mini-gene expanding exon2 and 220nt of its flanking introns that was synthesized (Genewiz) and inserted into pBSKii+ via XhoI/NotI cloning. Supplementary Table 6. Antibodies used for pull down assays for the identification of splicing factors that bind to the CD19-int1-exon2-int2 mini-gene. Supplementary Table 7. Primary and secondary antibodies used for immunoblotting and immunoprecipitations after protein separation by SDS-PAGE and transference to PVDF membrane (N/A, not applicable). Supplementary Table 8. Pairs of oligos for semiquantitative PCR amplification of CD19 cDNA followed by visualization in agarose gel. Same primers were used for sequencing after gel purification of specific bands. Supplementary Table 9. Pairs of oligos used for Real Time-qPCR amplification of cDNA.

The success of chimeric antigen receptor (CAR)–mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells ex vivo for 9 to 14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of ex vivo culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR-targeting CD19 (CART19) exhibited less differentiation and enhanced effector function in vitro when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the antileukemic activity inversely correlated with ex vivo culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale current good manufacturing practice–compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies. Cancer Immunol Res; 6(9); 1100–9. ©2018 AACR.

Supplementary Figure 1. Enhanced skipping of exons 2 and 5-6 in post-CART-19 leukemias. Supplementary Figure 2. Analysis of splicing factors involved in alternative splicing of CD19-exon2.

Cell size of T cells engineered with lentivirus or RNA.

Growth of T cells engineered with lentivirus or RNA.

PDF file - 713K, Figure S1. Kinetics of EL4 killing. Figure S2. iNKT cells kill several CD1d-expressing targets. Figure S3. EL4s express low levels of NKG2D ligands while iNKT and NK cells express NKG2D. Figure S4. Thymic but not splenic NKT cells kill EL4s in a GAg-dependent manner. Figure S5. DN3A4-1.2 NKT hybridoma cells kill GAg-loaded tumors. Figure S6. CD4+ and CD4- 8- liver iNKT cells possess cytotoxic capacity. Figure S7. iNKT cell reconstitution of NSG mice is not associated with in vivo accumulation of contaminating NK cells.

The GD2 ganglioside, which is abundant on the surface of neuroblastoma cells, is targeted by an FDA-approved therapeutic monoclonal antibody and is an attractive tumor-associated antigen for cellular immunotherapy. Chimeric antigen receptor (CAR)–modified T cells can have potent antitumor activity in B-cell malignancies, and trials to harness this cytolytic activity toward GD2 in neuroblastoma are under way. In an effort to enhance the antitumor activity of CAR T cells that target GD2, we generated variant CAR constructs predicted to improve the stability and the affinity of the GD2-binding, 14G2a-based, single-chain variable fragment (scFv) of the CAR and compared their properties in vivo. We included the E101K mutation of GD2 scFv (GD2-E101K) that has enhanced antitumor activity against a GD2+ human neuroblastoma xenograft in vivo. However, this enhanced antitumor efficacy in vivo was concomitantly associated with lethal central nervous system (CNS) toxicity comprised of extensive CAR T-cell infiltration and proliferation within the brain and neuronal destruction. The encephalitis was localized to the cerebellum and basal regions of the brain that display low amounts of GD2. Our results highlight the challenges associated with target antigens that exhibit shared expression on critical normal tissues. Despite the success of GD2-specific antibody therapies in the treatment of neuroblastoma, the fatal neurotoxicity of GD2-specific CAR T-cell therapy observed in our studies suggests that GD2 may be a difficult target antigen for CAR T-cell therapy without additional strategies that can control CAR T-cell function within the CNS. Cancer Immunol Res; 6(1); 36–46. ©2017 AACR.

The adoptive transfer of chimeric antigen receptor (CAR) T cells represents a breakthrough in clinical oncology, yet both between- and within-patient differences in autologously derived T cells are a major contributor to therapy failure. To interrogate the molecular determinants of clinical CAR T-cell persistence, we extensively characterized the premanufacture T cells of 71 patients with B-cell malignancies on trial to receive anti-CD19 CAR T-cell therapy. We performed RNA-sequencing analysis on sorted T-cell subsets from all 71 patients, followed by paired Cellular Indexing of Transcriptomes and Epitopes (CITE) sequencing and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on T cells from six of these patients. We found that chronic IFN signaling regulated by IRF7 was associated with poor CAR T-cell persistence across T-cell subsets, and that the TCF7 regulon not only associates with the favorable naïve T-cell state, but is maintained in effector T cells among patients with long-term CAR T-cell persistence. These findings provide key insights into the underlying molecular determinants of clinical CAR T-cell function.Significance:To improve clinical outcomes for CAR T-cell therapy, there is a need to understand the molecular determinants of CAR T-cell persistence. These data represent the largest clinically annotated molecular atlas in CAR T-cell therapy to date, and significantly advance our understanding of the mechanisms underlying therapeutic efficacy.This article is highlighted in the In This Issue feature, p. 2113

Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we found that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially induced by iNKT cell agonists of varying T-cell receptor (TCR) affinities, such as OCH, α-galactosyl ceramide, and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of TCR signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell–deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T lymphoma. Cancer Immunol Res; 2(1); 59–69. ©2013 AACR.

CD8 IHC on indicated brain structures from mice receiving E101K CAR T cells.

Supplementary Table S3. Gene sets for the TCF7 regulon and interferon response. For each gene set, three annotations are provided: the Ensembl gene id, the Entrez gene id, and HGNC symbol based on annotation from Ensembl version 94.

Supplementary tables 1-4 and figures 1-11

Supplementary Figure Legends from Nature of Tumor Control by Permanently and Transiently Modified GD2 Chimeric Antigen Receptor T Cells in Xenograft Models of Neuroblastoma

Bioluminescent xenograft model of disseminated neuroblastoma.

All supplementary data

Supplemental Figure Legends