Enhanced recruitment of genetically modified CX3CR1-positive human T cells into Fractalkine/CX3CL1 expressing tumors: importance of the chemokine gradient.

BACKGROUND: Adoptive T-cell based immunotherapies constitute a promising approach to treat cancer, however, a major problem is to obtain effective and long-lasting anti-tumor responses. Lack of response may be due to insufficient trafficking of specific T cells to tumors. A key requirement for efficient migration of cytotoxic T cells is that they express chemokine receptors that match the chemokines produced by tumor or tumor-associated cells. METHODS: In this study, we investigated whether the in vivo tumor trafficking of activated T cells could be enhanced by the expression of the chemokine receptor CX3CR1. Two human colorectal cancer cell lines were used to set up a xenograft tumor model in immunodeficient mice; the NCI-H630, constitutively expressing the chemokine ligand CX3CL1 (Fractalkine), and the RKO cell line, transduced to express CX3CL1. RESULTS: Human primary T cells were transduced with the receptor CX3CR1-eGFP. Upon in vivo adoptive transfer of genetically modified CX3CR1-T cells in mice bearing NCI-H630 tumors, enhanced lymphocyte migration and tumor trafficking were observed, compared to mice receiving Mock-T cells, indicating improved homing ability towards ligand-expressing tumor cells. Furthermore, significant inhibition of tumor growth was found in mice receiving modified CX3CR1-T cells. In contrast, tumors formed by RKO cells transduced with the ligand (RKO-CX3CL1) were not affected, nor more infiltrated upon transfer of CX3CR1-T lymphocytes, likely because high levels of the chemokine were shed by tumor cells in the systemic circulation, thus nullifying the blood-tissue chemokine gradient. CONCLUSIONS: This study demonstrates that ectopic expression of CX3CR1 enhanced the homing of adoptively transferred T cells towards CX3CL1-producing tumors, resulting in increased T cell infiltration in tumor tissues and decreased tumor growth. Our results also establish that a correct chemokine gradient between the systemic circulation and the tumor is an essential requirement in adoptive T-cell based immunotherapy to efficiently recruit T cell effectors at the correct sites

Enhanced recruitment of genetically modified CX3CR1-positive human T cells into Fractalkine/CX3CL1 expressing tumors: Importance of the chemokine gradient

Background: Adoptive T-cell based immunotherapies constitute a promising approach to treat cancer, however, a major problem is to obtain effective and long-lasting anti-tumor responses. Lack of response may be due to insufficient trafficking of specific T cells to tumors. A key requirement for efficient migration of cytotoxic T cells is that they express chemokine receptors that match the chemokines produced by tumor or tumor-associated cells. Methods: In this study, we investigated whether the in vivo tumor trafficking of activated T cells could be enhanced by the expression of the chemokine receptor CX3CR1. Two human colorectal cancer cell lines were used to set up a xenograft tumor model in immunodeficient mice; the NCI-H630, constitutively expressing the chemokine ligand CX3CL1 (Fractalkine), and the RKO cell line, transduced to express CX3CL1. Results: Human primary T cells were transduced with the receptor CX3CR1-eGFP. Upon in vivo adoptive transfer of genetically modified CX3CR1-T cells in mice bearing NCI-H630 tumors, enhanced lymphocyte migration and tumor trafficking were observed, compared to mice receiving Mock-T cells, indicating improved homing ability towards ligand-expressing tumor cells. Furthermore, significant inhibition of tumor growth was found in mice receiving modified CX3CR1-T cells. In contrast, tumors formed by RKO cells transduced with the ligand (RKO-CX3CL1) were not affected, nor more infiltrated upon transfer of CX3CR1-T lymphocytes, likely because high levels of the chemokine were shed by tumor cells in the systemic circulation, thus nullifying the blood-tissue chemokine gradient. Conclusions: This study demonstrates that ectopic express

Structural equation modelling analysis on relationships of job demands and resources with work engagement, burnout and work ability: an observational study among physicians in Dutch hospitals

Objective: To investigate to what extent work engagement mediates the relationships of job resources with work ability, and to what extent burnout mediates the relationships of job demands and resources with work ability. Design: Multicentre observational study. Setting: Academic and non-academic hospitals in the Netherlands. Participants: Physicians (n=385) participated in this study. Primary and secondary outcome measures: We measured work ability with selected items from the validated Questionnaire of Experience and Evaluation of Work 2.0 (QEEW V.2.0), work engagement with the Utrecht Work Engagement Scale and burnout with the exhaustion subscale of the Oldenburg Burnout Inventory. The job demand ‘workload’ and job resources ‘development opportunities’, ‘participation in decision-making’, ‘inspirational leadership’ and ‘relationships with colleagues’ were measured using the QEEW V.2.0. The job demand ‘bureaucratic burden’ was measured with the Three Item Red Tape scale. A structural equation model was built to answer our research question. Results: Work engagement mediated relationships of job resources with physicians’ work ability, and burnout mediated relationships of job resources and demands with work ability. Development opportunities (β=0.39, SE=0.12, p<0.001), participation in decision-making (β=0.18, SE=0.08, p=0.028) and relationships with colleagues (β=0.19, SE=0.19, p=0.002) were positively related to work engagement. Development opportunities (β=−0.20, SE=0.08, p=0.004) were negatively related and workload (β=0.51, SE=0.19, p<0.001) was positively related to burnout. Work engagement (β=0.22, SE=0.04, p<0.001) was positively related and burnout (β=−0.56, SE=0.06, p<0.001) was negatively related to work ability. Conclusions: Physicians’ work engagement and burnout mediated the relationships of various job demands and resources with their work ability. More work-engaged and less burned-out physicians reported better work ability. Hospitals may attenuate excessive workloads and facilitate development opportunities, participation in decision-making and good collegial relationships to enhance physicians’ occupational well-being and performance

Immunotherapy in Glioblastoma: Current Shortcomings and Future Perspectives

Glioblastomas are aggressive, fast-growing primary brain tumors. After standard-of-care treatment with radiation in combination with temozolomide, the overall prognosis of newly diagnosed patients remains poor, with a 2-year survival rate of less than 20%. The remarkable survival benefit gained with immunotherapy in several extracranial tumor types spurred a variety of experimental intervention studies in glioblastoma patients. These ranged from immune checkpoint inhibition to vaccinations and adoptive T cell therapies. Unfortunately, almost all clinical outcomes were universally disappointing. In this perspective, we provide an overview of immune interventions performed to date in glioblastoma patients and re-evaluate their performance. We argue that shortcomings of current immune therapies in glioblastoma are related to three major determinants of resistance, namely: low immunogenicity; immune privilege of the central nervous system; and immunosuppressive micro-environment. In this perspective, we propose strategies that are guided by exact shortcomings to sensitize glioblastoma prior to treatment with therapies that enhance numbers and/or activation state of CD8 T cells

Doctors' alertness, contentedness and calmness before and after night shifts: a latent profile analysis

Background While night shifts are crucial for patient care, they threaten doctors’ well-being and performance. Knowledge of how the impact of night shifts differs for doctors is needed to attenuate the adverse effects of night shifts. This study aimed to obtain more precise insight into doctors’ feelings surrounding night shift by: identifying profiles based on doctors’ alertness, contentedness and calmness scores before and after night shifts (research question (RQ) 1); assessing how doctors’ pre- and post-shift profiles change (RQ2); and determining associations of doctors’ demographics and shift circumstances with alertness, contentedness and calmness change (RQ3). Methods Latent Profile Analysis using doctors’ pre- and post-shift self-rated alertness, contentedness and calmness scores was employed to identify pre- and post-shift profiles (RQ1). A cross-tabulation revealed pre- and post-shift profile changes (RQ2). Multiple regressions determined associations of demographics (i.e. age, sex, specialty) and night shift circumstances (i.e. hours worked pre-call, hours awake pre-call, shift duration, number of consecutive shifts, total hours of sleep) with alertness, contentedness and calmness change (RQ3). Results In total, 211 doctors participated with a mean age of 39.8 ± 10 years; 47.4% was male. The participants included consultants (46.4%) and trainees (53.6%) of the specialties surgery (64.5%) and obstetrics/gynaecology (35.5%). Three pre-shift (Indifferent, Ready, Engaged) and four post-shift profiles (Lethargic, Tired but satisfied, Excited, Mindful) were found. Most doctors changed from Ready to Tired but satisfied, with alertness reducing most. Age, specialty, sleep, shift duration and the number of consecutive shifts associated with alertness, contentedness and calmness changes. Conclusions The results provided nuanced insight into doctors’ feelings before and after night shifts. Future research may assess whether specific subgroups benefit from tailored interventions

Lack of B and T cell reactivity towards IDH1(R132H) in blood and tumor tissue from LGG patients

Purpose Mutations in the isocitrate dehydrogenase-1 gene (IDH1) occur at high frequency in grade II–III gliomas (LGGs). IDH1 mutations are somatic, missense and heterozygous afecting codon 132 in the catalytic pocket of the enzyme. In LGG, most mutations (90%) result in an arginine to histidine substitution (IDH1R132H) providing a neo-epitope that is expressed in all tumor cells. To assess the immunogenic nature of this epitope, and its potential use to develop T cell treatments, we measured IDH1R132H-specifc B and T cell reactivity in blood and tumor tissue of LGG patients. Methods Sera from IDH1R132H-mutated LGG patients (n=27) were assayed for the presence of a neo-specifc antibody response using ELISA. In addition, PBMCs (n=36) and tumor-infltrating lymphocytes (TILs, n=10) were measured for T cell activation markers and IFN-γ production by fow cytometry and ELISA. In some assays, frequencies of CD4 T cells specifc for mutated peptide presented by HLA-DR were enriched prior to T cell monitoring assays. Results Despite high sensitivity of our assay, we failed to detect IDH1R132H-specifc IgG in sera of LGG patients. Similarly, we did not observe CD4 T cell reactivity towards IDH1R132H in blood, neither did we observe such reactivity following preenrichment of frequencies of IDH1R132H-specifc CD4 T cells. Finally, we did not detect IDH1R132H-specifc CD4 T cells among TILs. Conclusions The absence of both humoral and cellular responses in blood and tumors of LGG patients indicates that IDH1R132H is not sufciently immunogenic and devaluates its further therapeutic exploitation, at least in the majority of LGG patients

Treatment of early-stage breast cancer with percutaneous thermal ablation, an open-label randomised phase 2 screening trial:rationale and design of the THERMAC trial

INTRODUCTION: Breast cancer is the most frequently diagnosed malignancy worldwide but almost half of the patients have an excellent prognosis with a 5-year survival rate of 98%–99%. These patients could potentially be treated with thermal ablation to avoid surgical excision, reduce treatment-related morbidity and increase patients’ quality of life without jeopardising treatment effectiveness. Previous studies showed highest complete ablation rates for radiofrequency, microwave and cryoablation. However, due to heterogeneity among studies, it is unknown which of these three techniques should be selected for a phase 3 comparative study. METHODS AND ANALYSIS: The aim of this phase 2 screening trial is to determine the efficacy rate of radiofrequency, microwave and cryoablation with the intention to select one treatment for further testing in a phase 3 trial. Additionally, exploratory data are obtained for the phase 3 trial. The design is a multicentre open-label randomised phase 2 screening trial. Patients with unifocal, invasive breast cancer with a maximum diameter of 2 cm without lymph node or distant metastases are included. Triple negative, Bloom-Richardson grade 3 tumours and patients with an indication for neoadjuvant chemotherapy will be excluded. Included patients will be allocated to receive one of the three thermal ablation techniques. Three months later surgical excision will be performed to determine the efficacy of thermal ablation. Treatment efficacy in terms of complete ablation rate will be assessed with CK 8/18 and H&E staining. Secondary outcomes include feasibility of the techniques in an outpatient setting, accuracy of MRI for complete ablation, patient satisfaction, adverse events, side effects, cosmetic outcome, system usability and immune response. ETHICS AND DISSEMINATION: This study protocol was approved by Medical Research Ethics Committee of the Erasmus Medical Center, Rotterdam, the Netherlands. Study results will be submitted for publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NL9205 (www.trialregister.nl); Pre-results

Reactivity of Biarylazacyclooctynones in Copper-Free Click Chemistry

The 1,3-dipolar cycloaddition of cyclooctynes with azides, also called "copper-free click chemistry", is a bioorthogonal reaction with widespread applications in biological discovery. The kinetics of this reaction are of paramount importance for studies of dynamic processes, particularly in living subjects. Here we performed a systematic analysis of the effects of strain and electronics on the reactivity of cyclooctynes with azides through both experimental measurements and computational studies using a density functional theory (DFT) distortion/interaction transition state model. In particular, we focused on biarylazacyclooctynone (BARAC) because it reacts with azides faster than any other reported cyclooctyne and its modular synthesis facilitated rapid access to analogues. We found that substituents on BARAC's aryl rings can alter the calculated transition state interaction energy of the cycloaddition through electronic effects or the calculated distortion energy through steric effects. Experimental data confirmed that electronic perturbation of BARAC's aryl rings has a modest effect on reaction rate, whereas steric hindrance in the transition state can significantly retard the reaction. Drawing on these results, we analyzed the relationship between alkyne bond angles, which we determined using X-ray crystallography, and reactivity, quantified by experimental second-order rate constants, for a range of cyclooctynes. Our results suggest a correlation between decreased alkyne bond angle and increased cyclooctyne reactivity. Finally, we obtained structural and computational data that revealed the relationship between the conformation of BARAC's central lactam and compound reactivity. Collectively, these results indicate that the distortion/interaction model combined with bond angle analysis will enable predictions of cyclooctyne reactivity and the rational design of new reagents for copper-free click chemistry