Independent analysis of the flagellum surface and matrix proteomes provides insight into flagellum signaling in mammalian-infectious Trypanosoma brucei.

The flagellum of African trypanosomes is an essential and multifunctional organelle that functions in motility, cell morphogenesis, and host-parasite interaction. Previous studies of the trypanosome flagellum have been limited by the inability to purify flagella without first removing the flagellar membrane. This limitation is particularly relevant in the context of studying flagellum signaling, as signaling requires surface-exposed proteins in the flagellar membrane and soluble signaling proteins in the flagellar matrix. Here we employ a combination of genetic and mechanical approaches to purify intact flagella from the African trypanosome, Trypanosoma brucei, in its mammalian-infectious stage. We combined flagellum purification with affinity-purification of surface-exposed proteins to conduct independent proteomic analyses of the flagellum surface and matrix fractions. The proteins identified encompass a broad range of molecular functionalities, including many predicted to function in signaling. Immunofluorescence and RNA interference studies demonstrate flagellum localization and function for proteins identified and provide insight into mechanisms of flagellum attachment and motility. The flagellum surface proteome includes many T. brucei-specific proteins and is enriched for proteins up-regulated in the mammalian-infectious stage of the parasite life-cycle. The combined results indicate that the flagellum surface presents a diverse and dynamic host-parasite interface that is well-suited for host-parasite signaling.

Collision Risk Mitigation of Varian TrueBeam Linear Accelerator With Supplemental Live-View Cameras.

BACKGROUND: Noncoplanar radiation therapy techniques such as 4π have potential dosimetric advantages but introduce complexities in treatment delivery that increase the risk for collision. Direct or remote visual confirmation of clearance is a safeguard against collisions of the gantry, couch, and patient. With our institution's Varian TrueBeam system, we identified configurations that cannot be visualized with the included closed-circuit television cameras. At our practice, electronic, portal imaging device (EPID) collision risk also exists because of the routine deployment to capture exit-dose images for treatment quality assurance. We propose a simple, cost-effective solution using network cameras to help eliminate blind spots that permits safe, noncoplanar arrangements with an EPID-acquired exit dose. METHODS AND MATERIALS: Two Panasonic cameras were installed overhead while a third Panasonic camera was mounted onto the pedestal to monitor the couch undersurface. Live views from each camera were accessed with a web-based client. The EPID and gantry were visually assessed at 52 couch and gantry rotational angle configurations at 6 couch translational positions. Visibility was compared for the standard and supplemental camera setups at each configuration (χ2 test). RESULTS: Of the 294 assessable couch-gantry configurations, the standard camera setup had limited visibility of either gantry or EPID for 146 configurations compared with 72 configurations with additional cameras (51% blind-spot reduction; P < .01). An 87% blind-spot reduction was observed for our laterally centered, cranial-based, couch translational position (P < .01). CONCLUSIONS: The supplemental cameras were simple, effective additions for collision detection, especially for noncoplanar radiation therapy with EPID-acquired, exit-dose imaging. Over half of the assessable noncoplanar configurations had blind spots using standard cameras, which was reduced to <25% with additional cameras. In practice, there were almost no blind spots for patients with brain tumors who were treated with our templated beam arrangements. Using live-view camera feeds, vault re-entry to visually confirm clearance was reduced approximately 10-fold, which increased the treatment efficiency. In the most recent 12 months, no collision or near-collision events have been reported.

Lens Dose-Response Prediction Modeling and Cataract Incidence in Patients With Retinoblastoma After Lens-Sparing or Whole-Eye Radiation Therapy.

PURPOSE: We retrospectively assessed the incidence of cataracts in patients with retinoblastoma (Rb) treated with either lens-sparing radiation therapy (LSRT) or whole-eye radiation therapy (WERT). A secondary aim of this study was to model the dose-response risk of cataract. METHODS AND MATERIALS: We reviewed 65 patients with Rb treated with radiation therapy (RT) at Children's Hospital, Los Angeles from 1997 to 2015. Eyes that were enucleated before RT or lacked follow-up eye examinations were excluded. All patients underwent computed tomography simulation, and mean lens dose data were collected. Follow-up ophthalmologic examinations and intraocular lens implant history were reviewed for cataracts. The primary event-free survival (EFS) outcome was cataract development. Eyes without cataracts were censored on the last date of eye examination or post-RT enucleation, if applicable. Kaplan-Meier estimates were used to compare EFS outcomes, and dose response was projected with Cox regression and logistic regression models. RESULTS: Sixty-one patients (94 eyes) were analyzed with a median follow-up of 51.8 months. For eyes treated with WERT, cataracts developed in 71.7% versus 35.3% for LSRT. Median EFS for WERT and LSRT were 20.8 and 67.9 months, respectively. Compared with WERT, a significant EFS benefit was demonstrated for LSRT (P < .001). Mean lens dose had a significant effect on cataracts in both Cox regression and logistic regression models (P < .01). The mean lens dose of 7 Gy was projected to have a 5-year cataract incidence of 20% and 25% with the logistic and Cox regression models, respectively. CONCLUSIONS: We report the first clinical data demonstrating significantly improved EFS in patients with Rb treated with LSRT. Through lens dose-response modeling, we validate a mean lens dose threshold of 7 Gy to keep cataract risk below 25%. Although RT is used less often for Rb owing to advances in chemotherapy delivery options, these findings are relevant for refining lens dose constraints, particularly in children who have received radiation dose near the orbit.

Evaluation of weekly paclitaxel plus carboplatin followed by anthracycline chemotherapy on the neoadjuvant treatment of patients with triple-negative breast cancer.

OBJECTIVE: To evaluate the effectiveness and tolerability of neoadjuvant chemotherapy with weekly paclitaxel in combination with weekly carboplatin area under curve 2 followed by anthracycline chemotherapy. PATIENTS AND METHODS: This is a retrospective review of electronic medical records of patients (N = 32) with stage 1c-III triple-negative breast cancer. Patients received neoadjuvant chemotherapy with paclitaxel 80 mg/m2 once per week for 12 weeks in combination with carboplatin area under curve 2 once per week for 12 weeks (wP + wCb), followed by a standard anthracycline regimen including either doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2 every 2 or 3 weeks, or epirubicin 90 mg/m2 and cyclophosphamide 600 mg/m2 every 3 weeks for four cycles with myeloid growth factor support. RESULTS: Most patients (91%) received all 12 cycles of wP + wCb, and 88% received all four planned cycles of anthracycline chemotherapy. Of the patients, 84% completed all planned therapies. The complete pathologic response rate was 60%. In terms of hematologic toxicity, 96% of the patients experienced grade ≥3 leucopenia, 40% grade ≥3 anemia, and 15% grade ≥3 thrombocytopenia, and neutropenic fever was seen in 22% of the patients. CONCLUSION: The combination of neoadjuvant chemotherapy with wP + wCb before anthracycline chemotherapy can be tolerated by patients with triple-negative breast cancer. Complete pathologic response rates were comparable with those historically seen. Careful selection of patients is fundamental as this regimen is associated with a high incidence of hematologic toxicity.

Impressive Response to Concomitant Platinum-based Chemotherapy and Yttrium-90 in a Patient with Heavily Pretreated Triple-negative Breast Cancer Widely Metastasized to the Liver.

Metastatic triple-negative breast cancer (TNBC) constitutes a heterogeneous group of diseases with systemic treatment options limited to cytotoxic chemotherapy at the time being. The disease tends to affect visceral organs more frequently when compared to hormone receptor-positive breast cancer. The prognoses of patients with heavily pretreated disease affecting the liver are very dismal. We present the response to radioembolization and systemic chemotherapy in a seriously ill patient who had undergone previous lines of chemotherapy for TNBC with extensive liver metastases.

Stereotactic Radiosurgery and Ipilimumab Versus Stereotactic Radiosurgery Alone in Melanoma Brain Metastases.

Benefits of stereotactic radiosurgery (SRS) have been well established in melanoma brain metastases (MBM). Immunotherapy agents such as ipilimumab (ipi) have recently demonstrated clinical efficacy in advanced disease as well. The theoretical synergistic effects of combining these therapies in MBM have not been explored in detail, however, we conducted a systematic review with meta-analysis of studies that compared combined SRS and ipi versus SRS alone in MBM. Medical Literature Analysis and Retrieval System Online (MEDLINE) and Central databases were used for our literature search, which was conducted by three reviewers. We included studies that examined SRS and ipilimumab compared to SRS alone in MBM. Pertinent results were tabulated in a standardized spreadsheet. Newcastle-Ottawa Scale (NOS) Risk of Bias Assessment and Grading of Recommendations, Assessment, Development and Evaluation (GRADE) method for rating evidence quality were used for qualitative analysis. Review Manager was used for statistical analysis. We identified four cohort studies that compared SRS plus ipi versus SRS alone in MBM. As per the GRADE criteria, we found low-quality evidence for survival benefits associated with combined treatment. Meta-analysis confirmed a significant benefit in survival for SRS and ipilimumab (hazard ratio 0.38, 95% confidence interval 0.28 - 0.52, p < 0.01). There were no significant differences between comparison groups for local control, distant brain control, radiation necrosis, or intracranial bleeding. We conclude that low-quality evidence exists for superior overall survival in MBM treated with SRS and ipilimumab compared to SRS without ipilimumab. There is also no increased risk of radiation necrosis and/or intracranial bleeding with combining radiation and immunotherapy in this setting.

Pulmonary Embolism Secondary to Testosterone-Enhancing Herbal Supplement Use.

Decreased testosterone levels in men are often a normal sign of aging. Testosterone replacement therapy (TRT) is a well-established option for those with symptomatic hypogonadism related to low testosterone levels. Conversely, designer herbal supplements in the context of testosterone supplementation are poorly studied, yet remain popular among aging men who seek the well-known, often enhancing, effects of testosterone that involve muscle mass and sexual function/drive. In 2014, the Food and Drug Administration (FDA) issued a warning about the significant risk of venous clots secondary to testosterone product use. Testosterone-induced polycythemia is one of the proposed mechanisms for this increased clotting propensity. Increased thromboxane A2 receptor density on platelets and increased platelet aggregation have also been linked to testosterone treatment in men. Fenugreek extract is a common active ingredient in commercially available herbal supplements that are often marketed as testosterone enhancers. It is thought that certain fenugreek compounds inhibit aromatase and 5-alpha-reductase activity, leading to diminished testosterone breakdown. However, the efficacy and safety profile of this agent in its use for boosting testosterone levels are unclear. In this case report, we present a patient with new-onset, bilateral pulmonary embolism possibly associated with the daily use of fenugreek-containing testosterone supplements.

A novel AhR ligand, 2AI, protects the retina from environmental stress.

Various retinal degenerative diseases including dry and neovascular age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy are associated with the degeneration of the retinal pigmented epithelial (RPE) layer of the retina. This consequently results in the death of rod and cone photoreceptors that they support, structurally and functionally leading to legal or complete blindness. Therefore, developing therapeutic strategies to preserve cellular homeostasis in the RPE would be a favorable asset in the clinic. The aryl hydrocarbon receptor (AhR) is a conserved, environmental ligand-dependent, per ARNT-sim (PAS) domain containing bHLH transcription factor that mediates adaptive response to stress via its downstream transcriptional targets. Using in silico, in vitro and in vivo assays, we identified 2,2'-aminophenyl indole (2AI) as a potent synthetic ligand of AhR that protects RPE cells in vitro from lipid peroxidation cytotoxicity mediated by 4-hydroxynonenal (4HNE) as well as the retina in vivo from light-damage. Additionally, metabolic characterization of this molecule by LC-MS suggests that 2AI alters the lipid metabolism of RPE cells, enhancing the intracellular levels of palmitoleic acid. Finally, we show that, as a downstream effector of 2AI-mediated AhR activation, palmitoleic acid protects RPE cells from 4HNE-mediated stress, and light mediated retinal degeneration in mice.

Vectored immunoprophylaxis protects humanized mice from mucosal HIV transmission.

The vast majority of new HIV infections result from relatively inefficient transmission of the virus across mucosal surfaces during sexual intercourse. A consequence of this inefficiency is that small numbers of transmitted founder viruses initiate most heterosexual infections. This natural bottleneck to transmission has stimulated efforts to develop interventions that are aimed at blocking this step of the infection process. Despite the promise of this strategy, clinical trials of preexposure prophylaxis have had limited degrees of success in humans, in part because of lack of adherence to the recommended preexposure treatment regimens. In contrast, a number of existing vaccines elicit systemic immunity that protects against mucosal infections, such as the vaccines for influenza and human papilloma virus. We recently demonstrated the ability of vectored immunoprophylaxis (VIP) to prevent intravenous transmission of HIV in humanized mice using broadly neutralizing antibodies. Here we demonstrate that VIP is capable of protecting humanized mice from intravenous as well as vaginal challenge with diverse HIV strains despite repeated exposures. Moreover, animals receiving VIP that expresses a modified VRC07 antibody were completely resistant to repetitive intravaginal challenge by a heterosexually transmitted founder HIV strain, suggesting that VIP may be effective in preventing vaginal transmission of HIV between humans.

Administration of human umbilical cord blood cells produces interleukin-10 (IL-10) in IL-10 deficient mice without immunosuppression.

Recent studies from our laboratory have shown that intravenous administration of human umbilical cord blood (HUCB) mononuclear cells to mice improved blood glucose levels, survival, atherosclerosis and prostate cancer. In this study, we examined the effect of HUCB cells on the production of IL-10 levels in IL-10 knockout mice. It has been proposed that administration of IL-10 may be beneficial in the treatment of inflammatory bowl disease. The results show that mice treated with HUCB cells (100 x 10(6)) produce IL-10, as demonstrated by both qualitative and quantitative analyses, and that the levels of this cytokine persisted until the mice were sacrificed (5.5 months after administration). Immunohistochemical staining of the intestine using HuNu antibody cocktail demonstrated the presence of HUCB cells in the knockout mouse. Although the mice did not receive any immunosuppression, there was no evidence of graft-versus-host disease. Our data suggest that HUCB cells are capable of producing IL-10, and the use of these cells or HUCB may be indicated in the treatment of certain human diseases.