Examining the impact of COVID-19 disruptions on population-based breast cancer screening in Ireland.

OBJECTIVE: Many population-based breast screening programmes temporarily suspended routine screening following the COVID-19 pandemic onset. This study aimed to describe screening mammography utilisation and the pattern of screen-detected breast cancer diagnoses following COVID-19-related screening disruptions in Ireland. METHODS: Using anonymous aggregate data from women invited for routine screening, three time periods were examined: (1) January-December 2019, (2) January-December 2020, and (3) January-December 2021. Descriptive statistics were conducted and comparisons between groups were performed using chi-square tests. RESULTS: In 2020, screening mammography capacity fell by 67.1% compared to 2019; recovering to 75% of mammograms performed in 2019, during 2021. Compared to 2019, for screen-detected invasive breast cancers, a reduction in Grade 1 (14.2% vs. 17.2%) and Grade 2 tumours (53.4% vs. 58.0%) and an increase in Grade 3 tumours (32.4% vs. 24.8%) was observed in 2020 (p = 0.03); whereas an increase in Grade 2 tumours (63.3% vs. 58.0%) and a reduction in Grade 3 tumours (19.6% vs. 24.8%) was found in 2021 (p = 0.02). No changes in oestrogen receptor-positive or nodal-positive diagnoses were observed; however the proportion of oestrogen/progesterone receptor-positive breast cancers significantly increased in 2020 (76.2%; p < 0.01) and 2021 (78.7%; p < 0.001) compared to 2019 (67.8%). CONCLUSION: These findings demonstrate signs of a grade change for screen-detected invasive breast cancers early in the pandemic, with recovery evident in 2021, and without an increase in nodal positivity. Future studies are needed to determine the COVID-19 impact on long-term breast cancer outcomes including mortality.

Assessment of Plasma Proteomics Biomarker's Ability to Distinguish Benign From Malignant Lung Nodules: Results of the PANOPTIC (Pulmonary Nodule Plasma Proteomic Classifier) Trial.

BACKGROUND: Lung nodules are a diagnostic challenge, with an estimated yearly incidence of 1.6 million in the United States. This study evaluated the accuracy of an integrated proteomic classifier in identifying benign nodules in patients with a pretest probability of cancer (pCA) ≤ 50%. METHODS: A prospective, multicenter observational trial of 685 patients with 8- to 30-mm lung nodules was conducted. Multiple reaction monitoring mass spectrometry was used to measure the relative abundance of two plasma proteins, LG3BP and C163A. Results were integrated with a clinical risk prediction model to identify likely benign nodules. Sensitivity, specificity, and negative predictive value were calculated. Estimates of potential changes in invasive testing had the integrated classifier results been available and acted on were made. RESULTS: A subgroup of 178 patients with a clinician-assessed pCA ≤ 50% had a 16% prevalence of lung cancer. The integrated classifier demonstrated a sensitivity of 97% (CI, 82-100), a specificity of 44% (CI, 36-52), and a negative predictive value of 98% (CI, 92-100) in distinguishing benign from malignant nodules. The classifier performed better than PET, validated lung nodule risk models, and physician cancer probability estimates (P < .001). If the integrated classifier results were used to direct care, 40% fewer procedures would be performed on benign nodules, and 3% of malignant nodules would be misclassified. CONCLUSIONS: When used in patients with lung nodules with a pCA ≤ 50%, the integrated classifier accurately identifies benign lung nodules with good performance characteristics. If used in clinical practice, invasive procedures could be reduced by diverting benign nodules to surveillance. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01752114; URL: www.clinicaltrials.gov).

The building blocks of successful translation of proteomics to the clinic.

Recently, the first two multiplexed tests using selective reaction monitoring (SRM-MS) mass spectrometry have entered clinical practice. Despite different areas of indication, risk stratification in lung cancer and preterm birth, they share multiple steps in their development strategies. Here we review these strategies and their implications for successful translation of biomarkers to clinical practice. We believe that the identification of blood protein panels for the identification of disease phenotypes is now a reproducible and standard (albeit complex) process.

An integrated risk predictor for pulmonary nodules.

It is estimated that over 1.5 million lung nodules are detected annually in the United States. Most of these are benign but frequently undergo invasive and costly procedures to rule out malignancy. A risk predictor that can accurately differentiate benign and malignant lung nodules could be used to more efficiently route benign lung nodules to non-invasive observation by CT surveillance and route malignant lung nodules to invasive procedures. The majority of risk predictors developed to date are based exclusively on clinical risk factors, imaging technology or molecular markers. Assessed here are the relative performances of previously reported clinical risk factors and proteomic molecular markers for assessing cancer risk in lung nodules. From this analysis an integrated model incorporating clinical risk factors and proteomic molecular markers is developed and its performance assessed on a subset of 222 lung nodules, between 8mm and 20mm in diameter, collected in a previously reported prospective study. In this analysis it is found that the molecular marker is most predictive. However, the integration of clinical and molecular markers is superior to both clinical and molecular markers separately. CLINICAL TRIAL REGISTRATION: Registered at ClinicalTrials.gov (NCT01752101).

Protein catalyzed capture agents with tailored performance for in vitro and in vivo applications.

We report on peptide-based ligands matured through the protein catalyzed capture (PCC) agent method to tailor molecular binders for in vitro sensing/diagnostics and in vivo pharmacokinetics parameters. A vascular endothelial growth factor (VEGF) binding peptide and a peptide against the protective antigen (PA) protein of Bacillus anthracis discovered through phage and bacterial display panning technologies, respectively, were modified with click handles and subjected to iterative in situ click chemistry screens using synthetic peptide libraries. Each azide-alkyne cycloaddition iteration, promoted by the respective target proteins, yielded improvements in metrics for the application of interest. The anti-VEGF PCC was explored as a stable in vivo imaging probe. It exhibited excellent stability against proteases and a mean elimination in vivo half-life (T1/2 ) of 36 min. Intraperitoneal injection of the reagent results in slow clearance from the peritoneal cavity and kidney retention at extended times, while intravenous injection translates to rapid renal clearance. The ligand competed with the commercial antibody for binding to VEGF in vivo. The anti-PA ligand was developed for detection assays that perform in demanding physical environments. The matured anti-PA PCC exhibited no solution aggregation, no fragmentation when heated to 100°C, and > 81% binding activity for PA after heating at 90°C for 1 h. We discuss the potential of the PCC agent screening process for the discovery and enrichment of next generation antibody alternatives.

Development and validation of a spontaneous preterm delivery predictor in asymptomatic women.

BACKGROUND: Preterm delivery remains the leading cause of perinatal mortality. Risk factors and biomarkers have traditionally failed to identify the majority of preterm deliveries. OBJECTIVE: To develop and validate a mass spectrometry-based serum test to predict spontaneous preterm delivery in asymptomatic pregnant women. STUDY DESIGN: A total of 5501 pregnant women were enrolled between 17(0/7) and 28(6/7) weeks gestational age in the prospective Proteomic Assessment of Preterm Risk study at 11 sites in the United States between 2011 and 2013. Maternal blood was collected at enrollment and outcomes collected following delivery. Maternal serum was processed by a proteomic workflow, and proteins were quantified by multiple reaction monitoring mass spectrometry. The discovery and verification process identified 2 serum proteins, insulin-like growth factor-binding protein 4 (IBP4) and sex hormone-binding globulin (SHBG), as predictors of spontaneous preterm delivery. We evaluated a predictor using the log ratio of the measures of IBP4 and SHBG (IBP4/SHBG) in a clinical validation study to classify spontaneous preterm delivery cases (<37(0/7) weeks gestational age) in a nested case-control cohort different from subjects used in discovery and verification. Strict blinding and independent statistical analyses were employed. RESULTS: The predictor had an area under the receiver operating characteristic curve value of 0.75 and sensitivity and specificity of 0.75 and 0.74, respectively. The IBP4/SHBG predictor at this sensitivity and specificity had an odds ratio of 5.04 for spontaneous preterm delivery. Accuracy of the IBP4/SHBG predictor increased using earlier case-vs-control gestational age cutoffs (eg, <35(0/7) vs ≥35(0/7) weeks gestational age). Importantly, higher-risk subjects defined by the IBP4/SHBG predictor score generally gave birth earlier than lower-risk subjects. CONCLUSION: A serum-based molecular predictor identifies asymptomatic pregnant women at risk of spontaneous preterm delivery, which may provide utility in identifying women at risk at an early stage of pregnancy to allow for clinical intervention. This early detection would guide enhanced levels of care and accelerate development of clinical strategies to prevent preterm delivery.

Clinical Utility of a Plasma Protein Classifier for Indeterminate Lung Nodules.

Evaluation of indeterminate pulmonary nodules is a complex challenge. Most are benign but frequently undergo invasive and costly procedures to rule out malignancy. A plasma protein classifier was developed that identifies likely benign nodules that can be triaged to CT surveillance to avoid unnecessary invasive procedures. The clinical utility of this classifier was assessed in a prospective-retrospective analysis of a study enrolling 475 patients with nodules 8-30 mm in diameter who had an invasive procedure to confirm diagnosis at 12 sites. Using this classifier, 32.0 % (CI 19.5-46.7) of surgeries and 31.8 % (CI 20.9-44.4) of invasive procedures (biopsy and/or surgery) on benign nodules could have been avoided. Patients with malignancy triaged to CT surveillance by the classifier would have been 24.0 % (CI 19.2-29.4). This rate is similar to that described in clinical practices (24.5 % CI 16.2-34.4). This study demonstrates the clinical utility of a non-invasive blood test for pulmonary nodules.

Maintaining an open trauma intensive care unit bed for rapid admission can be cost-effective.

BACKGROUND: In 2012, we implemented a ready open trauma intensive care unit (TICU) bed process. Our hypothesis was that this process would decrease emergency department (ED) length of stay (LOS) in a cost-effective manner without worsening clinical outcomes. METHODS: We developed a charge nurse without a patient assignment to facilitate this open bed. We also provided team training for early ICU resuscitation. All Level 1 activations admitted directly to the TICU before and after the implementation were examined. Patients taken directly to the operating room from the ED, deaths within 24 hours of admission, and patients with nonsurvivable head injuries were excluded. Cost-effectiveness of the position was examined. RESULTS: Age (mean [SD], 45.78 [18.71] years), sex (74.7% male), and Injury Severity Score (ISS) (mean [SD], 17.27 [9.26]) were not significantly different. Median ED LOS for the postimplementation group decreased from 230 minutes to 66 minutes (p < 0.001). Median ICU LOS (from 3.29 to 2.98 days, p = 0.13) and total median hospital LOS (from 10.71 to 7.98 days, p = 0.06) decreased but were not statistically significant. Controlling for age, ISS, sex, and mechanism of injury the postimplementation group had a 29% reduction in ICU LOS (2.12 days), a 28% reduction in hospital LOS (4.34), and a 54% reduction in ED LOS (154 minutes). The LOS decreased despite a small increase in ISS (from 15.89 to 18.37). Observed/expected mortality did not differ between the groups, preimplementation/postimplementation of 0.87 and 0.92. Nursing productivity increased one nurse after implementation at a cost of $624 per day. The ICU LOS decrease of 1.6 days at a rate of $1,144 average ICU daily cost of room and board totaled $1,830 per patient. The decreased ICU LOS dollars minus the increase nurse pay resulted in an overall savings of $1,206 per patient. CONCLUSION: Rapid access to the TICU made possible by the charge nurse without a direct assignment and team training has a potential cost savings without adversely affecting patient outcomes. LEVEL OF EVIDENCE: Cost analysis, level III.

Management of Pulmonary Nodules by Community Pulmonologists: A Multicenter Observational Study.

BACKGROUND: Pulmonary nodules (PNs) are a common reason for referral to pulmonologists. The majority of data for the evaluation and management of PNs is derived from studies performed in academic medical centers. Little is known about the prevalence and diagnosis of PNs, the use of diagnostic testing, or the management of PNs by community pulmonologists. METHODS: This multicenter observational record review evaluated 377 patients aged 40 to 89 years referred to 18 geographically diverse community pulmonary practices for intermediate PNs (8-20 mm). Study measures included the prevalence of malignancy, procedure/test use, and nodule pretest probability of malignancy as calculated by two previously validated models. The relationship between calculated pretest probability and management decisions was evaluated. RESULTS: The prevalence of malignancy was 25% (n = 94). Nearly one-half of the patients (46%, n = 175) had surveillance alone. Biopsy was performed on 125 patients (33.2%). A total of 77 patients (20.4%) underwent surgery, of whom 35% (n = 27) had benign disease. PET scan was used in 141 patients (37%). The false-positive rate for PET scan was 39% (95% CI, 27.1%-52.1%). Pretest probability of malignancy calculations showed that 9.5% (n = 36) were at a low risk, 79.6% (n = 300) were at a moderate risk, and 10.8% (n = 41) were at a high risk of malignancy. The rate of surgical resection was similar among the three groups (17%, 21%, 17%, respectively; P = .69). CONCLUSIONS: A substantial fraction of intermediate-sized nodules referred to pulmonologists ultimately prove to be lung cancer. Despite advances in imaging and nonsurgical biopsy techniques, invasive sampling of low-risk nodules and surgical resection of benign nodules remain common, suggesting a lack of adherence to guidelines for the management of PNs.

An integrated quantification method to increase the precision, robustness, and resolution of protein measurement in human plasma samples.

BACKGROUND: Current quantification methods for mass spectrometry (MS)-based proteomics either do not provide sufficient control of variability or are difficult to implement for routine clinical testing. RESULTS: We present here an integrated quantification (InteQuan) method that better controls pre-analytical and analytical variability than the popular quantification method using stable isotope-labeled standard peptides (SISQuan). We quantified 16 lung cancer biomarker candidates in human plasma samples in three assessment studies, using immunoaffinity depletion coupled with multiple reaction monitoring (MRM) MS. InteQuan outperformed SISQuan in precision in all three studies and tolerated a two-fold difference in sample loading. The three studies lasted over six months and encountered major changes in experimental settings. Nevertheless, plasma proteins in low ng/ml to low µg/ml concentrations were measured with a median technical coefficient of variation (CV) of 11.9% using InteQuan. The corresponding median CV using SISQuan was 15.3% after linear fitting. Furthermore, InteQuan surpassed SISQuan in measuring biological difference among clinical samples and in distinguishing benign versus cancer plasma samples. CONCLUSIONS: We demonstrated that InteQuan is a simple yet robust quantification method for MS-based quantitative proteomics, especially for applications in biomarker research and in routine clinical testing.

Validation of a multiprotein plasma classifier to identify benign lung nodules.

INTRODUCTION: Indeterminate pulmonary nodules (IPNs) lack clinical or radiographic features of benign etiologies and often undergo invasive procedures unnecessarily, suggesting potential roles for diagnostic adjuncts using molecular biomarkers. The primary objective was to validate a multivariate classifier that identifies likely benign lung nodules by assaying plasma protein expression levels, yielding a range of probability estimates based on high negative predictive values (NPVs) for patients with 8 to 30 mm IPNs. METHODS: A retrospective, multicenter, case-control study was performed using multiple reaction monitoring mass spectrometry, a classifier comprising five diagnostic and six normalization proteins, and blinded analysis of an independent validation set of plasma samples. RESULTS: The classifier achieved validation on 141 lung nodule-associated plasma samples based on predefined statistical goals to optimize sensitivity. Using a population based nonsmall-cell lung cancer prevalence estimate of 23% for 8 to 30 mm IPNs, the classifier identified likely benign lung nodules with 90% negative predictive value and 26% positive predictive value, as shown in our prior work, at 92% sensitivity and 20% specificity, with the lower bound of the classifier's performance at 70% sensitivity and 48% specificity. Classifier scores for the overall cohort were statistically independent of patient age, tobacco use, nodule size, and chronic obstructive pulmonary disease diagnosis. The classifier also demonstrated incremental diagnostic performance in combination with a four-parameter clinical model. CONCLUSIONS: This proteomic classifier provides a range of probability estimates for the likelihood of a benign etiology that may serve as a noninvasive, diagnostic adjunct for clinical assessments of patients with IPNs.

Factors that influence physician decision making for indeterminate pulmonary nodules.

RATIONALE: Pulmonologists frequently encounter indeterminate pulmonary nodules in practice, but it is unclear what clinical factors they rely on to guide the diagnostic evaluation. OBJECTIVES: To assess the current approach to the management of indeterminate pulmonary nodules and to determine the extent to which the addition of a hypothetical diagnostic blood test will influence clinical decision making. METHODS: Selected pulmonologists practicing in the United States were invited to participate in a conjoint exercise based on 20 randomly generated cases of varying age, smoking history, and nodule size. Some cases included the result of a hypothetical blood test. Each respondent chose from among three diagnostic options for a patient: noninvasive monitoring (i.e., serial CT or positron emission tomography scan), a minor procedure (i.e., biopsy or bronchoscopy), or a major procedure (i.e., video-assisted thorascopic surgery or thoracotomy). Multivariate logistic regression was used to assess the impact of the three risk factors and the diagnostic blood test on decision making. MEASUREMENTS AND MAIN RESULTS: Four hundred nineteen physicians participated (response rate, 10%). One hundred fifty-three physician surveys met predetermined criteria and were analyzed (4% of all invitees). A diagnostic procedure was recommended for 23% of 6-mm nodules, versus 54, 66, 77, and 84% of nodules 10, 14, 18, and 22 mm, respectively (P < 0.001). Older age limited recommendations for invasive testing: 54% of 80-year-olds versus 61, 64, 63, and 61% of patients 71, 62, 53, and 44 years of age, respectively (P < 0.001). In multivariate analyses, nodule size, smoking history, age, and the blood test each influenced decision making (P < 0.001). CONCLUSIONS: The pulmonologists who participated in this survey were more likely to proceed with invasive testing, instead of observation or additional imaging, as the size of the nodule increased. The use of a hypothetical blood test resulted in significant alterations in the decision to pursue invasive testing.

Informed choice in bowel cancer screening: a qualitative study to explore how adults with lower education use decision aids.

BACKGROUND: Offering informed choice in screening is increasingly advocated, but little is known about how evidence-based information about the benefits and harms of screening influences understanding and participation in screening. OBJECTIVE: We aimed to explore how a bowel cancer screening decision aid influenced decision making and screening behaviour among adults with lower education and literacy. METHODS: Twenty-one men and women aged 55-64 years with lower education levels were interviewed about using a decision aid to make their screening decision. Participants were purposively selected to include those who had and had not made an informed choice. RESULTS: Understanding the purpose of the decision aid was an important factor in whether participants made an informed choice about screening. Participants varied in how they understood and integrated quantitative risk information about the benefits and harms of screening into their decision making; some read it carefully and used it to justify their screening decision, whereas others dismissed it because they were sceptical of it or lacked confidence in their own numeracy ability. Participants' prior knowledge and beliefs about screening influenced how they made sense of the information. DISCUSSION AND CONCLUSIONS: Participants valued information that offered them a choice in a non-directive way, but were concerned that it would deter people from screening. Healthcare providers need to be aware that people respond to screening information in diverse ways involving a range of literacy skills and cognitive processes.

A blood-based proteomic classifier for the molecular characterization of pulmonary nodules.

Each year, millions of pulmonary nodules are discovered by computed tomography and subsequently biopsied. Because most of these nodules are benign, many patients undergo unnecessary and costly invasive procedures. We present a 13-protein blood-based classifier that differentiates malignant and benign nodules with high confidence, thereby providing a diagnostic tool to avoid invasive biopsy on benign nodules. Using a systems biology strategy, we identified 371 protein candidates and developed a multiple reaction monitoring (MRM) assay for each. The MRM assays were applied in a three-site discovery study (n = 143) on plasma samples from patients with benign and stage IA lung cancer matched for nodule size, age, gender, and clinical site, producing a 13-protein classifier. The classifier was validated on an independent set of plasma samples (n = 104), exhibiting a negative predictive value (NPV) of 90%. Validation performance on samples from a nondiscovery clinical site showed an NPV of 94%, indicating the general effectiveness of the classifier. A pathway analysis demonstrated that the classifier proteins are likely modulated by a few transcription regulators (NF2L2, AHR, MYC, and FOS) that are associated with lung cancer, lung inflammation, and oxidative stress networks. The classifier score was independent of patient nodule size, smoking history, and age, which are risk factors used for clinical management of pulmonary nodules. Thus, this molecular test provides a potential complementary tool to help physicians in lung cancer diagnosis.

An acute care surgery service generates a positive contribution margin in an appropriately staffed hospital.

BACKGROUND: Acute care surgery (ACS) includes trauma, surgical critical care, and emergent general surgery. There is a national shortage of institutions that can provide for patients needing access to emergency surgical care. Inability to fund ACS surgeons can be a barrier. We hypothesize that an ACS service, in an appropriately staffed hospital, generates a positive contribution margin (CM). STUDY DESIGN: Fiscal data for 2009 were retrospectively reviewed at the University of Kentucky, a Level I trauma center with an ACS service. Contribution margin (ie, net revenue minus direct costs) and mean length of stay were calculated for all patients admitted to the ACS service. Inpatient data were stratified by trauma vs general surgery, emergent vs elective, and by payor mix. RESULTS: Annual CM associated with the 5 ACS faculty was $21,799,000. Trauma generated higher CM than general surgery. General surgery had a greater CM, more if emergent than if elective ($9,500 vs $5,500; p < 0.01). Self-payment was lower with emergent general surgery vs trauma (20% vs 25%; p = 0.02). CONCLUSIONS: Acute care surgery generates a positive CM. Emergent general surgery generates a greater CM than elective general surgery because of increased case mix index. These data suggest that hospital subsidization of acute care surgeons is financially feasible and might address the surgical workforce shortage and the critical problem of access to emergency surgical services.

An acute care surgery rotation contributes significant general surgical operative volume to residency training compared with other rotations.

BACKGROUND: Surgical resident rotations on trauma services are criticized for little operative experience and heavy workloads. This has resulted in diminished interest in trauma surgery among surgical residents. Acute care surgery (ACS) combines trauma and emergency/elective general surgery, enhancing operative volume and balancing operative and nonoperative effort. We hypothesize that a mature ACS service provides significant operative experience. METHODS: A retrospective review was performed of ACGME case logs of 14 graduates from a major, academic, Level I trauma center program during a 3-year period. Residency Review Committee index case volumes during the fourth and fifth years of postgraduate training (PGY-4 and PGY-5) ACS rotations were compared with other service rotations: in total and per resident week on service. RESULTS: Ten thousand six hundred fifty-four cases were analyzed for 14 graduates. Mean cases per resident was 432 ± 57 in PGY-4, 330 ± 40 in PGY-5, and 761 ± 67 for both years combined. Mean case volume on ACS for both years was 273 ± 44, which represented 35.8% (273 of 761) of the total experience and exceeded all other services. Residents averaged 8.9 cases per week on the ACS service, which exceeded all other services except private general surgery, gastrointestinal/minimally invasive surgery, and pediatric surgery rotations. Disproportionately more head/neck, small and large intestine, gastric, spleen, laparotomy, and hernia cases occurred on ACS than on other services. CONCLUSIONS: Residents gain a large operative experience on ACS. An ACS model is viable in training, provides valuable operative experience, and should not be considered a drain on resident effort. Valuable ACS rotation experiences as a resident may encourage graduates to pursue ACS as a career.

Strangulated appendix after transabdominal preperitoneal (TAPP) inguinal hernia repair.

INTRODUCTION: Laparoscopic inguinal hernia repair can be performed totally extraperitoneal or transabdominal preperitoneal (TAPP). Both repairs are associated with mesh-related complications. This is the first report of a mesh-strangulated appendix, with subsequent necrosis and perforation, after TAPP inguinal hernia repair. CASE: A 48-year-old male, 10-years status post-bilateral TAPP inguinal hernia repair presented with acute right groin bulge, pain, nausea, emesis, and fever. He was found to have a large, tender, nonreducible right groin mass. He was taken to the operating room for right groin exploration and found to have gross purulent material but no evidence of a recurrent hernia. A laparoscope was inserted into the abdomen where the appendix was found strangulated between the mesh and the transversalis fascia. CONCLUSIONS: Mesh-related complications after TAPP inguinal hernia repair are rare. This is the first report of a strangulated appendix secondary to mesh entrapment after TAPP repair.

Red blood cell arginase suppresses Jurkat (T cell) proliferation by depleting arginine.

BACKGROUND: Transfusion of packed red blood cells (PRBC) suppresses immunity, but the mechanisms are incompletely understood. PRBCs contain arginase, an enzyme which converts arginine to ornithine and depletes arginine in vitro. Arginine depletion suppresses proliferation of Jurkat T cells in other models. We hypothesize that PRBC arginase-mediated arginine depletion will suppress proliferation of T cells. METHODS: A transfusion model was designed adding PRBC to culture RPMI media with or without an irreversible arginase blocker (nor-NOHA), incubating for 6-48 hours and then removing the PRBCs. Amino acid concentrations in the media were measured using liquid chromatography mass spectrometry. T cells were then added to the pre-conditioned media, cultured for 24 hours, and proliferation was measured. RESULTS: PRBC depleted arginine significantly and increased ornithine in media compared to baseline PRBC treated wells and significantly decreased T cell proliferation. These effects were enhanced with volume of PRBC exposure. Nor-NOHA inhibition of arginase restored T cell proliferation in PRBC treated cultures. CONCLUSIONS: Jurkat T cell proliferation was impaired by PRBC in clinically relevant volumes. The mechanism influencing T cell impairment appears to result from arginine depletion by arginase. Arginine depletion by PRBC arginase may be a novel mechanism for immunosuppression after transfusion.

Modulation of the phagosome proteome by interferon-gamma.

Macrophages are immune cells that function in the clearance of infectious particles. This process involves the engulfment of microbes into phagosomes where these particles are lysed and degraded. In the current study, we used a large scale quantitative proteomics approach to analyze the changes in protein abundance induced on phagosomes by interferon-gamma (IFN-gamma), an inflammatory cytokine that activates macrophages. Our analysis identified 167 IFN-gamma-modulated proteins on phagosomes of which more than 90% were up-regulated. The list of phagosomal proteins regulated by IFN-gamma includes proteins expected to alter phagosome maturation, enhance microbe degradation, trigger the macrophage immune response, and promote antigen loading on major histocompatibility complex (MHC) class I molecules. A dynamic analysis of IFN-gamma-sensitive proteins by Western blot indicated that newly formed phagosomes display a delayed proteolytic activity coupled to an increased recruitment of the MHC class I peptide-loading complex. These phagosomal conditions may favor antigen presentation by MHC class I molecules on IFN-gamma-activated macrophages.

An evaluation of multidimensional fingerprinting in the context of clinical proteomics.

Multidimensional fingerprinting (MDF) utilizes measurable peptide characteristics to identify proteins. In this study, 3-D fingerprinting, namely, parent protein molecular weight, peptide mass, and peptide retention time on RPLC, is used to identify 331 differentially expressed proteins between normal and human colon cancer plasma membrane samples. A false discovery rate (FDR) procedure is introduced to evaluate the performance of MDF on the colon cancer dataset. This evaluation establishes a false protein identification rate below 15% for this dataset. Western blot analysis is performed to validate the differential expression of the MDF-identified protein VDAC1 on the original tissue samples. The limits of MDF are further assessed by a simulation study where key parameters such as database size, query size, and mass accuracy are varied. The results of this simulation study demonstrate that fingerprinting with three dimensions yields low FDR values even for large queries on the complete human proteome without the need for prior peptide sequencing by tandem mass spectrometry. Specifically, when mass accuracy is 10 ppm or lower, full human proteome searches can achieve FDR values of 10% or less.