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Supplemental Figure 5. Unfractionated splenocytes derived from WT mice were activated with soluble anti-CD3 +anti-CD28 antibodies in the presence or absence of LNIL. The cells were harvested 5 days after activation and stained for MDSC ( Gr-1+CD11b+), macrophages ( CD11b+F480+) or Dendritic cells ( F480-Cd11C+MHC Class II+) cells and intracellularly stained for TGF-b1. The graph shows the levels of TGF-b ( from both control and LNIL treated cells)expressed as a fold-increase with respect to the control group.

Supplemental Figure 4. Human PBMC's were derived from healthy individuals and stained for CD4 and CD45RO antibodies after which CD4+CD45RO- cells were sorted out and used to generate T-regs ex vivo in the presence or absence of TGF-β1 and L-NIL.

Supplemental Figure 3.1. Splenocytes ( CFSE labelled) from naïve WT mice were activated with soluble anti-CD3 and anti-CD28 antibodies in the presence of RFP+Treg cells sorted (as mentioned above) from control, LNIL and TGF-β groups. After 72 hours, cells were havested and surface stained for CD4 T cells and CFSE dilution profile was measured by flow cytometry.

Supplemental Figure 2. Splenocytes from FOXP3 reporter mice were activated with soluble anti-CD3 and anti-CD28 antibodies in the presence or absence of L-NIL or TGF-β1 or alone for 5 days after which RFP+ cells were sorted from all the groups and used for T-reg suppression assay with CFSE labelled naïve CD4 T cells.

Purpose: Expression of inducible nitric oxide synthase (iNOS) in different cellular compartments may have divergent effects on immune function. We used a syngeneic tumor model to functionally characterize the role of iNOS in regulation of CD4+FOXP3+ regulatory T cells (Treg), and optimize the beneficial effects of iNOS inhibition on antitumor immunity.Experimental Design: Wild-type (WT) or iNOS knockout mice bearing established MT-RET-1 melanoma were treated with the small-molecule iNOS inhibitor L-NIL and/or cyclophosphamide alone or in combination. The effect of iNOS inhibition or knockout on induction of Treg from mouse and human CD4+ T cells in ex vivo culture was determined in parallel in the presence or absence of TGFβ1-depleting antibodies, and TGFβ1 levels were assessed by ELISA.Results: Whereas intratumoral myeloid-derived suppressor cells (MDSC) were suppressed by iNOS inhibition or knockout, systemic and intratumoral FOXP3+ Treg levels increased in tumor-bearing mice. iNOS inhibition or knockout similarly enhanced induction of Treg from activated cultured mouse splenocytes or purified human or mouse CD4+ T cells in a TGFβ1-dependent manner. Although either iNOS inhibition or Treg depletion with low-dose cyclophosphamide alone had little effect on growth of established MT-RET1 melanoma, combination treatment potently inhibited MDSC and Treg, boosted tumor-infiltrating CD8+ T-cell levels, and arrested tumor growth in an immune-dependent fashion.Conclusions: iNOS expression in CD4+ T cells suppresses Treg induction by inhibiting TGFβ1 production. Our data suggest that iNOS expression has divergent effects on induction of myeloid and lymphoid-derived regulatory populations, and strongly support development of combinatorial treatment approaches that target these populations simultaneously. Clin Cancer Res; 20(24); 6439–51. ©2014 AACR.

Supplemental Figure 1. Tumor-free WT or iNOS KO C57/BL6 mice were treated with L-NIL in drinking water or plain drinking water control for 7 days. Spenocytes were harvested and intracellular staining performed to determine FOXP3+ expression in CD4+ cells and analyzed by FACS. S1i) representative FACS plots of CD4+FOXP3+ T-reg from WT (untreated control and L-NIL groups) and iNOS KO (untreated) mice. S1ii) graphical representation of aggregated data from two independent experiments.

Background Nonsurgical rejuvenation of the tear trough area via the use of injectable filler material has become a popular procedure in facial rejuvenation. This procedure offers immediate, albeit temporary, results with minimal recovery time. Objectives The aim of this systematic review was to report on patient satisfaction and complication rates to further guide practitioners. Methods PubMed, Cochrane, and Scopus libraries were queried for articles containing relevant terms. Articles with more than 5 patients who reported on satisfaction and/or complications from the procedure were included for review. In addition to these variables, we noted other aspects of injection, including filler material, technique, and needle or cannula delivery. Studies that did not otherwise fulfill inclusion criteria for statistical analysis but reported on intravascular injection–related complications were cited. Results Initial query resulted in 1655 studies which were assessed for duplicates and inclusion/exclusion criteria. After screening, 28 articles were included for analysis. In total, 1956 patients were captured who had been injected with 1 of 4 materials: hyaluronic acid (1535), calcium hydroxyapatite (376), autologous fibroblast/keratin gel (35), and collagen-based filler (10). Short- and long-term satisfaction rates were 84.4% and 76.7%, respectively. Minor complications were common (44%). Secondarily, we found the use of cannula for filler injection of this region to be associated with a lower rate of ecchymosis (7% vs 17%, P Conclusions Filler injection volumization of tear trough deformity is an effective technique for facial rejuvenation associated with high patient satisfaction. Multiple filler materials offer acceptable satisfaction and complication profiles. Level of Evidence: 4

Many organisms, ranging from plants to mammals, contain phospholipase A2 enzymes (PLA2s), which catalyze the production of lysophospholipids and fatty acid proinflammatory mediators. PLA2s are also common constituents of animal venoms, including bees, scorpions and snakes, and they cause a wide variety of toxic effects including neuro-, myo-, cyto-, and cardio-toxicity, anticoagulation and edema. The aim of this study was to develop a generic method for profiling enzymatically active PLA2s in snake venoms after chromatographic separation. For this, low-volume high-throughput assays for assessment of enzymatic PLA2 activity were evaluated and optimized. Subsequently, the assays were incorporated into a nanofractionation platform that combines high-resolution fractionation of crude venoms by liquid chromatography (LC) with bioassaying in 384-well plate format, and parallel mass spectrometric (MS) detection for toxin identification. The miniaturized assays developed are based on absorbance or fluorescence detection (respectively, using cresol red or fluorescein as pH indicators) to monitor the pH drop associated with free fatty acid formation by enzymatically active PLA2s. The methodology was demonstrated for assessment of PLA2 activity profiles of venoms from the snake species Bothrops asper, Echis carinatus, Echis coloratus, Echis ocellatus, Oxyuranus scutellatus and Daboia russelii russelii.

Preoperative corticosteroids have been shown to improve surgical visibility and intraoperative blood loss for chronic rhinosinusitis with nasal polyposis (CRSwNP) patients undergoing endoscopic sinus surgery (ESS). However, there is no consensus on the optimal dosing regimen.A randomized, controlled trial was conducted to compare low, medium, and high dose corticosteroids prior to ESS. Patients with CRSwNP refractory to medical management were randomized to low (N = 8), medium (N = 10), or high (N = 5) dosing regimens of corticosteroids prior to ESS. Baseline disease severity was measured with the 22-item Sino-nasal Outcome Test and Lund-Mackay scores. Modified Lund-Kennedy endoscopic scores (MLKES) were measured at baseline and after corticosteroid treatment. Intraoperative parameters were measured including Boezaart surgical visibility score, intraoperative blood loss, and operative time.Medium dose corticosteroids demonstrated a superior surgical visibility score to low dose and comparable results to high dose, but these results were not significant (p = 0.33). No significant difference was observed between groups for total blood loss (p = 0.15), operative time (p = 0.87), or change in MLKES (p = 0.27).Current recommendations include the use of preoperative corticosteroids in patients with CRSwNP undergoing ESS, but there is no consensus on dose or duration. We did not find a statistically significant difference in surgical field visibility, intraoperative blood loss, or operative time between different dosing regimens. Further studies are needed to evaluate the efficacy of a low-dose preoperative regimen with the goal of reducing cumulative patient exposure to systemic corticosteroids.

Many organisms, ranging from plants to mammals, contain phospholipase A

Background: Inferior turbinate hypertrophy is often encountered by plastic surgeons who perform rhinoplasty. Many treatment options are available to treat the inferior turbinate. The objective of this study was to systematically review outcomes of available techniques and provide guidance to surgical turbinate management. Methods: A MEDLINE search was performed for means of treating inferior turbinate hypertrophy. Studies selected focused on treatment of the inferior turbinate in isolation and excluding patients with refractory allergic rhinitis, vasomotor rhinitis, or hypertrophic rhinitis. Results: Fifty-eight articles were identified, collectively including the following surgical treatments of inferior turbinate hypertrophy: total turbinectomy, partial turbinectomy, submucosal resection, laser surgery, cryotherapy, electrocautery, radiofrequency ablation, and turbinate outfracture. Outcomes and complications were collected from all studies. Procedures such as turbinectomy (partial/total) and submucosal resection showed crusting and epistaxis at comparatively higher rates, whereas more conservative treatments such as cryotherapy and submucous diathermy failed to provide long-term results. Submucosal resection and radiofrequency ablation were shown to decrease nasal resistance and preserve mucosal function. No literature exists to support the belief that turbinate outfracture alone is an effective treatment for turbinate hypertrophy. Conclusions: Treatment of inferior turbinate hypertrophy is best accomplished with modalities that provide long-lasting results, preservation of turbinate function, and low complication rates. Submucosal resection and radiofrequency ablation appear to best fulfill these criteria. Turbinate outfracture should only be considered in combination with tissue-reduction procedures.

Postoperative pain management is controversial as there are no current guidelines to direct clinical practice. The purpose of this study was to demonstrate prescribing patterns for pain management after functional endoscopic sinus surgery (FESS).A 15-item web-based survey was electronically distributed to 1770 members of the American Rhinologic Society (ARS). Linear regression analysis was used to determine associations between providers and prescribing patterns.The survey was completed by 168 members (9.49%), representing all regions of the United States. The most commonly prescribed medications were opioid/non-opioid combination pills. One hundred fifty-seven of 168 members (94.05%) prescribed at least one kind of opioid after FESS, with an average of 27.38 pills. The majority of surveyed members worked in private or academic settings. Academic physicians were less likely to prescribe ibuprofen (p = 0.0407), and nonsteroidal anti-inflammatory drugs (NSAIDs) in general (p = 0.032). Physicians in private practice were less likely to refer patients to pain management (p = 0.0117), but more likely to refer patients to nontraditional forms of pain management (p = 0.0164). Academic physicians were more likely to refer patients to pain management (p = 0.00121). There was no association between perception of pain control and the prescription of NSAIDs or with number of opioid pills prescribed.Most providers prescribed opioids after FESS. There was no significant difference in the number of opioids prescribed based on geography or practice setting. There was significant heterogeneity in the adjuvant pain management strategy between academic and private practitioners. Most members provided patient education and few reported poor pain control. However, there was a gap in understanding of appropriate medication disposal and evidence-based postoperative pain management.

Prolonged length of stay (pLOS), disease-related complications, and 30-day readmissions are important quality metrics under the Affordable Care Act. The purpose of our study was to investigate the effect of patient-level and hospital-level factors on these outcomes for patients admitted for transsphenoidal pituitary surgery.The Statewide Planning and Research Cooperative System (SPARCS) database was queried to investigate 30-day readmissions and pLOS for transsphenoidal pituitary surgery in New York from 1995 to 2015. Multivariate logistic regression, adjusting for patient and hospital characteristics, was performed to assess the effect of these variables on the outcomes of interest.A total of 9950 patients underwent transsphenoidal pituitary surgery; 7122 (72%), 2394 (24%), and 434 (4%) patients were treated at high-volume, medium-volume, and low-volume centers, respectively. Patient factors associated with treatment at high-volume centers (HVCs) included: top income quartile, private insurance, urban residence, and white or Asian race (p0.05). Patient variables associated with treatment at low-volume centers (LVCs) included: age65 years, elevated Charlson comorbidity index (CCI) scores, bottom income quartile, Medicaid and Medicare insurance, rural residence, black race, and Hispanic ethnicity (p0.05). Variables predictive of prolonged hospitalizations in our multivariable model included black race, Hispanic ethnicity, Medicaid insurance, low income, female gender, LVC, and comorbidities (panhypopituitarism, hypothyroidism, diabetes insipidus [DI], visual disturbances, CCI) while predictors of readmissions included Asian race, female gender, and comorbidities (Cushing syndrome, DI, CCI).Patients undergoing transsphenoidal pituitary surgery at HVCs have shorter hospitalizations, fewer postoperative electrolyte abnormalities, and lower charges; however, socioeconomic factors may influence access to quality care.