FRAP Analysis on Red Alga Reveals the Fluorescence Recovery Is Ascribed to Intrinsic Photoprocesses of Phycobilisomes than Large-Scale Diffusion

BACKGROUND: Phycobilisomes (PBsomes) are the extrinsic antenna complexes upon the photosynthetic membranes in red algae and most cyanobacteria. The PBsomes in the cyanobacteria has been proposed to present high lateral mobility on the thylakoid membrane surface. In contrast, direct measurement of PBsome motility in red algae has been lacking so far. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we investigated the dynamics of PBsomes in the unicellular red alga Porphyridium cruentum in vivo and in vitro, using fluorescence recovery after photobleaching (FRAP). We found that part of the fluorescence recovery could be detected in both partially- and wholly-bleached wild-type and mutant F11 (UTEX 637) cells. Such partial fluorescence recovery was also observed in glutaraldehyde-treated and betaine-treated cells in which PBsome diffusion should be restricted by cross-linking effect, as well as in isolated PBsomes immobilized on the glass slide. CONCLUSIONS/SIGNIFICANCE: On the basis of our previous structural results showing the PBsome crowding on the native photosynthetic membrane as well as the present FRAP data, we concluded that the fluorescence recovery observed during FRAP experiment in red algae is mainly ascribed to the intrinsic photoprocesses of the bleached PBsomes in situ, rather than the rapid diffusion of PBsomes on thylakoid membranes in vivo. Furthermore, direct observations of the fluorescence dynamics of phycoerythrins using FRAP demonstrated the energetic decoupling of phycoerythrins in PBsomes against strong excitation light in vivo, which is proposed as a photoprotective mechanism in red algae attributed by the PBsomes in response to excess light energy

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

The impact of chemotherapy on toxicity and cosmetic outcome in patients receiving whole breast irradiation: an analysis within a state-wide quality consortium

PURPOSE: We investigated whether the use of chemotherapy prior to whole breast irradiation (WBI) using either conventional fractionation (CWBI) or hypofractionation (HWBI) is associated with increased toxicity or worse cosmetic outcome compared to WBI alone. METHODS AND MATERIALS: We identified 6,754 patients who received WBI alone (without a third field covering the superior axillary and supraclavicular nodal regions) with data prospectively collected in a state-wide consortium. We reported rates of four toxicity outcomes: physician-reported acute moist desquamation, patient-reported acute moderate/severe breast pain, a composite acute toxicity measure (including moist desquamation and either patient-reported or physician-reported moderate/significant breast pain), and physician-reported impaired cosmetic outcome at one year following WBI. Successive multivariable models were constructed to estimate the impact of chemotherapy on these outcomes. RESULTS: Rates of moist desquamation, patient-reported pain, composite acute toxicity, and impaired cosmetic outcome were 23%, 34%, 42%, and 10% for 2,859 patients receiving CWBI and 13%, 28%, 31%, and 11% for 3,895 patients receiving HWBI. Receipt of chemotherapy prior to CWBI was not associated with higher rates of patient-reported pain, composite acute toxicity, or impaired cosmetic outcome compared to CWBI without chemotherapy but was associated with more moist desquamation (OR=1.32 [1.07-1.63], p=0.01). Receipt of chemotherapy prior to HWBI was not associated with higher rates of any of the four toxicity outcomes compared to HWBI alone. CONCLUSIONS: In this cohort, use of chemotherapy prior to WBI was generally well tolerated. CWBI with chemotherapy, but not to HWBI with chemotherapy, was associated with higher rates of moist desquamation. Rates of acute breast pain and impaired cosmetic outcome at one year were comparable in patients receiving chemotherapy prior to either CWBI or HWBI. These data support the use of HWBI following chemotherapy

Comparative Effectiveness Analysis of 3D-Conformal Radiation Therapy Versus Intensity Modulated Radiation Therapy (IMRT) in a Prospective Multicenter Cohort of Patients With Breast Cancer

PURPOSE: Simple intensity modulation of radiation therapy reduces acute toxicity compared with 2-dimensional techniques in adjuvant breast cancer treatment, but it remains unknown whether more complex or inverse-planned intensity modulated radiation therapy (IMRT) offers an advantage over forward-planned, 3-dimensional conformal radiation therapy (3DCRT). METHODS AND MATERIALS: Using prospective data regarding patients receiving adjuvant whole breast radiation therapy without nodal irradiation at 23 institutions from 2011 to 2018, we compared the incidence of acute toxicity (moderate-severe pain or moist desquamation) in patients receiving 3DCRT versus IMRT (either inverse planned or, if forward-planned, using ≥5 segments per gantry angle). We evaluated associations between technique and toxicity using multivariable models with inverse-probability-of-treatment weighting, adjusting for treatment facility as a random effect. RESULTS: Of 1185 patients treated with 3DCRT and conventional fractionation, 650 (54.9%) experienced acute toxicity; of 774 treated with highly segmented forward-planned IMRT, 458 (59.2%) did; and of 580 treated with inverse-planned IMRT, 245 (42.2%) did. Of 1296 patients treated with hypofractionation and 3DCRT, 432 (33.3%) experienced acute toxicity; of 709 treated with highly segmented forward-planned IMRT, 227 (32.0%) did; and of 623 treated with inverse-planned IMRT, 164 (26.3%) did. On multivariable analysis with inverse-probability-of-treatment weighting, the odds ratio for acute toxicity after inverse-planned IMRT versus 3DCRT was 0.64 (95% confidence interval, 0.45-0.91) with conventional fractionation and 0.41 (95% confidence interval, 0.26-0.65) with hypofractionation. CONCLUSIONS: This large, prospective, multicenter comparative effectiveness study found a significant benefit from inverse-planned IMRT compared with 3DCRT in reducing acute toxicity of breast radiation therapy. Future research should identify the dosimetric differences that mediate this association and evaluate cost-effectiveness

The Role of Facility Variation on Racial Disparities in Use of Hypofractionated Whole Breast Radiotherapy

INTRODUCTION: Hypofractionated radiotherapy is a less burdensome and less costly approach that is efficacious for most patients with early-stage breast cancer. Concerns about racial disparities in adoption of medical advances motivate investigation of the use of hypofractionated radiation in diverse populations. The goal of our study was to determine whether hypofractionated whole breast radiotherapy after breast conserving surgery was being similarly used across racial groups in the state of Michigan. METHODS AND MATERIALS: A prospectively collected statewide quality consortium database from 25 institutions was queried for breast cancer patients who completed hypofractionated (HF) or conventionally fractionated (CF) whole breast radiotherapy (RT) from 1/2012-12/2018. We used patient-level multivariable modeling to evaluate associations between HF use and race, controlling for patient and facility factors, and multilevel modeling to account for patient clustering within facilities. RESULTS: Of 9,634 patients analyzed, 81% self-reported race as White, 17% as Black and 2% as Asian, similar to statewide and national distributions. 31.7% of Whites were treated at teaching centers compared to 66.7% of Blacks and 64.8% of Asians. In 2018, HF was utilized in 72.7% of Whites versus 56.7% of Blacks and 67.6% of Asians (p=0.0411). On patient-level multivariable analysis, Black and Asian races were significantly associated with a lower likelihood of HF receipt (p\u3c0.001), despite accounting for treatment year, age, laterality, BMI, breast volume, comorbidities, stage, triple-negative status, IMRT use, teaching center treatment, and 2011 ASTRO Hypofractionation Guideline eligibility. On multilevel analysis, race was no longer significantly associated with HF receipt. CONCLUSIONS: We observed that Black and Asian patients receive hypofractionated RT less often than Whites, despite more frequent treatment at teaching centers. Multilevel modeling eliminated this disparity, suggesting that differences in facility-specific HF use appear to have contributed. Further inquiry is needed to determine if reduction of facility-level variation may reduce disparities in accessing HF treatment