Recommendations for Effective Integration of Ethics and Responsible Conduct of Research (E/RCR) Education into Course-Based Undergraduate Research Experiences: A Meeting Report.

Advancement of the scientific enterprise relies on individuals conducting research in an ethical and responsible manner. Educating emergent scholars in the principles of ethics/responsible conduct of research (E/RCR) is therefore critical to ensuring such advancement. The recent impetus to include authentic research opportunities as part of the undergraduate curriculum, via course-based undergraduate research experiences (CUREs), has been shown to increase cognitive and noncognitive student outcomes. Because of these important benefits, CUREs are becoming more common and often constitute the first research experience for many students. However, despite the importance of E/RCR in the research process, we know of few efforts to incorporate E/RCR education into CUREs. The Ethics Network for Course-based Opportunities in Undergraduate Research (ENCOUR) was created to address this concern and promote the integration of E/RCR within CUREs in the biological sciences and related disciplines. During the inaugural ENCOUR meeting, a four-pronged approach was used to develop guidelines for the effective integration of E/RCR in CUREs. This approach included: 1) defining appropriate student learning objectives; 2) identifying relevant curriculum; 3) identifying relevant assessments; and 4) defining key aspects of professional development for CURE facilitators. Meeting outcomes, including the aforementioned E/RCR guidelines, are described herein.

Collaborative Posters Develop Students' Ability to Communicate about Undervalued Scientific Resources to Nonscientists.

Scientists are increasingly called upon to communicate with the public, yet most never receive formal training in this area. Public understanding is particularly critical to maintaining support for undervalued resources such as biological collections, research data repositories, and expensive equipment. We describe activities carried out in an inquiry-driven organismal biology laboratory course designed to engage a diverse student body using biological collections. The goals of this cooperative learning experience were to increase students' ability to locate and comprehend primary research articles, and to communicate the importance of an undervalued scientific resource to nonscientists. Our results indicate that collaboratively created, research-focused informational posters are an effective tool for achieving these goals and may be applied in other disciplines or classroom settings.

Burden of Psychosocial and Cognitive Impairment in Patients With Atrial Fibrillation.

BACKGROUND: Impairments in psychosocial status and cognition relate to poor clinical outcomes in patients with atrial fibrillation (AF). However, how often these conditions co-occur and associations between burden of psychosocial and cognitive impairment and quality of life (QoL) have not been systematically examined in patients with AF. METHODS: A total of 218 patients with symptomatic AF were enrolled in a prospective study of AF and psychosocial factors between May 2013 and October 2014 at the University of Massachusetts Medical Center. Cognitive function, depression, and anxiety were assessed at baseline and AF-specific QoL was assessed 6 months after enrollment using validated instruments. Demographic and clinical information were obtained from a structured interview and medical record review. RESULTS: The mean age of the study participants was 63.5 ± 10.2 years, 35% were male, and 81% had paroxysmal AF. Prevalences of impairment in 1, 2, and 3 psychosocial/cognitive domains (eg, depression, anxiety, or cognition) were 75 (34.4%), 51 (23.4%), and 16 (7.3%), respectively. Patients with co-occurring psychosocial/cognitive impairments (eg, >1 domain) were older, more likely to smoke, had less education, and were more likely to have heart failure (all P < 0.05). Compared with participants with no psychosocial/cognitive impairments, AF-specific QoL at 6 months was significantly poorer among participants with baseline impairment in 2 (B = -13.6, 95% CI: -21.7 to -5.4) or 3 (B = -15.1, 95% CI: -28.0 to -2.2) psychosocial/cognitive domains. CONCLUSION: Depression, anxiety, and impaired cognition were common in our cohort of patients with symptomatic AF and often co-occurred. Higher burden of psychosocial/cognitive impairment was associated with poorer AF-specific QoL.

Association of Left Atrial Function Index With Late Atrial Fibrillation Recurrence after Catheter Ablation.

INTRODUCTION: Although catheter ablation (CA) for atrial fibrillation (AF) is commonly used to improve symptoms, AF recurrence is common and new tools are needed to better inform patient selection for CA. Left atrial function index (LAFI), an echocardiographic measure of atrial mechanical function, has shown promise as a noninvasive predictor of AF. We hypothesized that LAFI would relate to AF recurrence after CA. METHODS AND RESULTS: All AF patients undergoing index CA were enrolled in a prospective institutional AF Treatment Registry between 2011 and 2014. LAFI was measured post hoc from pre-ablation clinical echocardiographic images in 168 participants. Participants were mostly male (33% female), middle-aged (60 ± 10 years), obese and had paroxysmal AF (64%). Mean LAFI was 25.9 ± 17.6. Over 12 months of follow-up, 78 participants (46%) experienced a late AF recurrence. In logistic regression analyses adjusting for factors known to be associated with AF, lower LAFI remained associated with AF recurrence after CA [OR 0.04 (0.01-0.67), P = 0.02]. LAFI discriminated AF recurrence after CA slightly better than CHADS2 (C-statistic 0.60 LAFI, 0.57 CHADS2). For participants with persistent AF, LAFI performed significantly better than CHADS2 score (C statistic = 0.79 LAFI, 0.56 CHADS2, P = 0.02). CONCLUSION: LAFI, an echocardiographic measure of atrial function, is associated with AF recurrence after CA and has improved ability to discriminate AF recurrence as compared to the CHADS-2 score, especially among persistent AF patients. Since LAFI can be calculated using standard 2D echocardiographic images, it may be a helpful tool for predicting AF recurrence.

Relation of Atrial Fibrillation in Acute Myocardial Infarction to In-Hospital Complications and Early Hospital Readmission.

Atrial fibrillation (AF) is a common complication of acute myocardial infarction (AMI) and contributes to high rates of in-hospital adverse events. However, there are few contemporary studies examining rates of AF in the contemporary era of AMI or the impact of new-onset AF on key in-hospital and postdischarge outcomes. We examined trends in AF in 6,384 residents of Worcester, Massachusetts, who were hospitalized with confirmed AMI during 7 biennial periods between 1999 and 2011. Multivariate logistic regression analysis was used to examine associations between occurrence of AF and various in-hospital and postdischarge complications. The overall incidence of AF complicating AMI was 10.8%. Rates of new-onset AF increased from 1999 to 2003 (9.8% to 13.2%), and decreased thereafter. In multivariable adjusted models, patients developing new-onset AF after AMI were at a higher risk for in-hospital stroke (odds ratio [OR] 2.5, 95% confidence interval [CI] 1.6 to 4.1), heart failure (OR 2.0, 95% CI 1.7 to 2.4), cardiogenic shock (OR 3.7, 95% CI 2.8 to 4.9), and death (OR 2.3, 95% CI 1.9 to 3.0) than patients without AF. Development of AF during hospitalization for AMI was associated with higher rates of readmission within 30 days after discharge (21.7% vs 16.0%), but no significant difference was noted in early postdischarge 30-day all-cause mortality rates (8.3% vs 5.1%). In conclusion, new-onset AF after AMI is strongly related to in-hospital complications of AMI and higher short-term readmission rates.

PULSE-SMART: Pulse-Based Arrhythmia Discrimination Using a Novel Smartphone Application.

BACKGROUND: Atrial fibrillation (AF) is a common and dangerous rhythm abnormality. Smartphones are increasingly used for mobile health applications by older patients at risk for AF and may be useful for AF screening. OBJECTIVES: To test whether an enhanced smartphone app for AF detection can discriminate between sinus rhythm (SR), AF, premature atrial contractions (PACs), and premature ventricular contractions (PVCs). METHODS: We analyzed two hundred and nineteen 2-minute pulse recordings from 121 participants with AF (n = 98), PACs (n = 15), or PVCs (n = 15) using an iPhone 4S. We obtained pulsatile time series recordings in 91 participants after successful cardioversion to sinus rhythm from preexisting AF. The PULSE-SMART app conducted pulse analysis using 3 methods (Root Mean Square of Successive RR Differences; Shannon Entropy; Poincare plot). We examined the sensitivity, specificity, and predictive accuracy of the app for AF, PAC, and PVC discrimination from sinus rhythm using the 12-lead EKG or 3-lead telemetry as the gold standard. We also administered a brief usability questionnaire to a subgroup (n = 65) of app users. RESULTS: The smartphone-based app demonstrated excellent sensitivity (0.970), specificity (0.935), and accuracy (0.951) for real-time identification of an irregular pulse during AF. The app also showed good accuracy for PAC (0.955) and PVC discrimination (0.960). The vast majority of surveyed app users (83%) reported that it was "useful" and "not complex" to use. CONCLUSION: A smartphone app can accurately discriminate pulse recordings during AF from sinus rhythm, PACs, and PVCs.

Addition of B-Type Natriuretic Peptide to Existing Clinical Risk Scores Enhances Identification of Patients at Risk for Atrial Fibrillation Recurrence After Pulmonary Vein Isolation.

INTRODUCTION: Predicting which patients will be free from atrial fibrillation (AF) after pulmonary vein isolation (PVI) remains challenging. Clinical risk prediction scores show modest ability to identify patients at risk for AF recurrence after PVI. B-type natriuretic peptide (BNP) is associated with risk for incident and recurrent AF but is not currently included in existing AF risk scores. We sought to evaluate the incremental benefit of adding preoperative BNP to existing risk scores for predicting AF recurrence during the 6 months after PVI. METHODS: One hundred sixty-one patients with paroxysmal or persistent AF underwent an index PVI procedure between 2010 and 2013; 77 patients (48%) had late AF recurrence after PVI (>3 months post-PVI) over the 6-month follow-up period. RESULTS: A BNP greater than or equal to 100 pg/dL (P=0.01) and AF recurrence within 3 months after PVI (P<0.001) were associated with late AF recurrence in multivariate analyses. Addition of BNP to existing clinical risk scores significantly improved the areas under the curve for each score, with an integrated discrimination improvement of 0.08 (P=0.001) and a net reclassification improvement of 60% (P=0.001) for all risk scores. CONCLUSIONS: Circulating BNP levels are independently associated with late AF recurrence after PVI. Inclusion of BNP significantly improves the discriminative ability of CHADS2, CHA2DS2-VASc, R2CHADS2, and the HATCH score in predicting clinically significant, late AF recurrence after PVI and should be incorporated in decision-making algorithms for management of AF. B-R2CHADS2 is the best score model for prediction of late AF recurrence.

OX40 is a potent immune-stimulating target in late-stage cancer patients.

OX40 is a potent costimulatory receptor that can potentiate T-cell receptor signaling on the surface of T lymphocytes, leading to their activation by a specifically recognized antigen. In particular, OX40 engagement by ligands present on dendritic cells dramatically increases the proliferation, effector function, and survival of T cells. Preclinical studies have shown that OX40 agonists increase antitumor immunity and improve tumor-free survival. In this study, we performed a phase I clinical trial using a mouse monoclonal antibody (mAb) that agonizes human OX40 signaling in patients with advanced cancer. Patients treated with one course of the anti-OX40 mAb showed an acceptable toxicity profile and regression of at least one metastatic lesion in 12 of 30 patients. Mechanistically, this treatment increased T and B cell responses to reporter antigen immunizations, led to preferential upregulation of OX40 on CD4(+) FoxP3(+) regulatory T cells in tumor-infiltrating lymphocytes, and increased the antitumor reactivity of T and B cells in patients with melanoma. Our findings clinically validate OX40 as a potent immune-stimulating target for treatment in patients with cancer, providing a generalizable tool to favorably influence the antitumor properties of circulating T cells, B cells, and intratumoral regulatory T cells.

Dual anti-OX40/IL-2 therapy augments tumor immunotherapy via IL-2R-mediated regulation of OX40 expression.

The provision of T cell co-stimulation via members of the TNFR super-family, including OX40 (CD134) and 4-1BB (CD137), provides critical signals that promote T cell survival and differentiation. Recent studies have demonstrated that ligation of OX40 can augment T cell-mediated anti-tumor immunity in pre-clinical models and more importantly, OX40 agonists are under clinical development for cancer immunotherapy. OX40 is of particular interest as a therapeutic target as it is not expressed on naïve T cells but rather, is transiently up-regulated following TCR stimulation. Although TCR engagement is necessary for inducing OX40 expression, the downstream signals that regulate OX40 itself remain unclear. In this study, we demonstrate that OX40 expression is regulated through a TCR and common gamma chain cytokine-dependent signaling cascade that requires JAK3-mediated activation of the downstream transcription factors STAT3 and STAT5. Furthermore, combined treatment with an agonist anti-OX40 mAb and IL-2 augmented tumor immunotherapy against multiple tumor types. Dual therapy was also able to restore the function of anergic tumor-reactive CD8 T cells in mice with long-term well-established (>5 wks) tumors, leading to increased survival of the tumor-bearing hosts. Together, these data reveal the ability of TCR/common gamma chain cytokine signaling to regulate OX40 expression and demonstrate a novel means of augmenting cancer immunotherapy by providing dual anti-OX40/common gamma chain cytokine-directed therapy.

A 30-year perspective (1975-2005) into the changing landscape of patients hospitalized with initial acute myocardial infarction: Worcester Heart Attack Study.

BACKGROUND: The effects of lifestyle changes and evolving treatment practices on coronary disease incidence rates, demographic and clinical profile, and the short-term outcomes of patients hospitalized with acute myocardial infarction have not been well characterized. The purpose of this study was to examine multidecade-long trends (1975-2005) in the incidence rates, demographic and clinical characteristics, treatment practices, and hospital outcomes of patients hospitalized with an initial acute myocardial infarction from a population-based perspective. METHODS AND RESULTS: Residents of the Worcester, Mass, metropolitan area (median age, 37 years; 89% white) hospitalized with an initial acute myocardial infarction (n=8898) at all greater-Worcester medical centers during 15 annual periods between 1975 and 2005 comprised the sample of interest. The incidence rates of initial acute myocardial infarction were lower in 2005 (209 of 100,000 population) than in 1975 (277 of 100,000), although these trends varied inconsistently over time. Patients hospitalized during the most recent study years were significantly older (mean age, 64 years in 1975; 71 years in 2005), more likely to be women (38% in 1975; 48% in 2005), and have a greater prevalence of comorbidities. Hospitalized patients were increasingly more likely to receive effective cardiac medications and coronary interventional procedures for the period under investigation. Hospital survival rates improved significantly over time (81% survived in 1975; 91% survived in 2005), although varying trends were observed in the occurrence of clinically important complications. CONCLUSIONS: The results of this community-wide investigation provide insight into the changing magnitude, characteristics, management practices, and outcomes of patients hospitalized with a first myocardial infarction.

Classification and atherosclerosis distribution in patients with left main coronary disease.

OBJECTIVE: The purpose of this investigation was to characterize clinical variables and angiographic distribution of coronary atherosclerosis to classify patients with de novo left main (LM) disease in a real-world population presenting for coronary angiography. BACKGROUND: Limited quantitative and angiographic published data exist that provide detailed quantitative information to classify potential target population for elective LM percutaneous coronary intervention (PCI) and guide development of dedicated LM PCI platforms. METHODS: Medical history and clinical presentation were prospectively collected on 177 consecutive patients with LM stenosis > or =50% by coronary angiography. Blinded quantitative coronary angiography (QCA) was performed on all LM stenoses to classify LM-A (ostial), LM-B (nonostial, non-bifurcation), and LM-C (bifurcation involvement). QCA was performed on the left anterior descending (LAD), left circumflex (LCx), and right coronary arteries (RCA) and branches (> or =2.5 mm) to identify lesions with > or =60% stenosis or occlusion. RESULTS: No differences in baseline clinical history or presentation discriminated the distribution patterns of LM stenosis. QCA revealed 66% of LM stenoses were LM-C. Mean LM reference vessel diameter was 4.65 mm and average lesion length was 11.12 mm. Around 88.7% of patients had at least one lesion > or =60% in a major epicardial artery and 32.2% of patients had RCA chronic total occlusion. Right-to-left coronary collateralization was only identified in patients with obstructive stenosis in the LAD or LCx in addition to the LM stenosis. CONCLUSION: Dedicated LM stent platforms may need to be developed to accommodate larger vessel size and bifurcation distributions. A majority of patients with LM stenosis will require adjunctive epicardial vessel PCI to achieve complete anatomic revascularization.

Characterization of the class I-restricted gp100 melanoma peptide-stimulated primary immune response in tumor-free vaccine-draining lymph nodes and peripheral blood.

PURPOSE: The aim of this study was to characterize the primary gp100(209-2M)-specific T-cell response in vaccine-draining, metastases-free lymph nodes and peripheral blood of peptide-vaccinated stage I to III melanoma patients. EXPERIMENTAL DESIGN: After two or three gp100(209-2M) vaccinations, sentinel lymph nodes that drained both the primary tumor and adjacent vaccine sites were excised concomitant with wide excision of the tumor. Comparative 7-color flow cytometry phenotype analysis was done on gp100 tetramer-positive CD8(+) T cells from sentinel lymph nodes, closely proximate time-related peripheral blood mononuclear cells (PBMC) collected 2 to 4 weeks after sentinel lymph node excision, and on PBMC collected 6 months later after 7 or 11 more immunizations. Lymph node and peripheral blood T cells were tested for proliferative response, functional avidity, and tumor cell-induced CD107 mobilization. RESULTS: The frequencies of gp100-specific CD8(+) T cells from time-related PBMC and sentinel lymph nodes were comparable and were similar to those reported for virus-specific memory T cells. Their respective in vitro proliferation responses were also equivalent but statistically higher than proliferation responses of peripheral blood T cells collected after completion of the entire vaccine regimen. By contrast, functional avidity and CD107 responses were significantly higher in circulating T cells. Sentinel lymph node-derived, gp100-specific CD8(+) T cells predominantly expressed central and effector memory phenotype signatures, whereas there were higher frequencies of effector T cells in the peripheral blood. CONCLUSION: Priming immunization with gp100(209-2M) without coadministration of CD4(+) helper T cell-restricted antigens induced the effective expansion of peptide-specific central and effector memory CD8(+) T cells with high proliferation potential in vaccine-draining lymph nodes of stage I to III melanoma patients. Lymph node memory T cells gave rise to circulating gp100-specific effector T cells exhibiting increased functional maturation.

Phenotype and functional characterization of long-term gp100-specific memory CD8+ T cells in disease-free melanoma patients before and after boosting immunization.

PURPOSE: Effective cancer vaccines must both drive a strong CTL response and sustain long-term memory T cells capable of rapid recall responses to tumor antigens. We sought to characterize the phenotype and function of gp100 peptide-specific memory CD8+ T cells in melanoma patients after primary gp100(209-2M) immunization and assess the anamnestic response to boosting immunization. EXPERIMENTAL DESIGN: Eight-color flow cytometry analysis of gp100-specific CD8+ T cells was done on peripheral blood mononuclear cells collected shortly after the primary vaccine regimen, 12 to 24 months after primary vaccination, and after boosting immunization. The anamnestic response was assessed by comparing the frequency of circulating gp100-specific T cells before and after boosting. Gp100 peptide-induced in vitro functional avidity and proliferation responses and melanoma-stimulated T-cell CD107 mobilization were compared for cells from all three time points for multiple patients. RESULTS: The frequency of circulating gp100-specific memory CD8+ T cells was comparable with cytomegalovirus-specific and FLU-specific T cells in the same patients, and the cells exhibited anamnestic proliferation after boosting. Their phenotypes were not unique, and individual patients exhibited one of two distinct phenotype signatures that were homologous to either cytomegalovirus-specific or FLU-specific memory T cells. Gp100-specific memory T cells showed some properties of competent memory T cells, such as heightened in vitro peptide-stimulated proliferation and increase in central memory (TCM) differentiation when compared with T-cell responses measured after the primary vaccine regimen. However, they did not acquire enhanced functional avidity usually associated with competent memory T-cell maturation. CONCLUSIONS: Although vaccination with class I-restricted melanoma peptides alone can break tolerance to self-tumor antigens, it did not induce fully competent memory CD8+ T cells--even in disease-free patients. Data presented suggest other vaccine strategies will be required to induce functionally robust long-term memory T cells.

Polychromatic flow cytometry: a rapid method for the reduction and analysis of complex multiparameter data.

BACKGROUND: Recent advances in flow cytometry have resulted in the development of reliable techniques for performing polychromatic (5-17 color) flow cytometry analysis. However, the data reduction and analysis involved in the resolution of hundreds of possible cellular subphenotypes identified, using a single polychromatic flow cytometry staining panel, presents a major obstacle to the successful application of this technology. METHODS: To generate two distinct collections of T cell populations with differentially expressed surface markers, cryopreserved lymph node cells from 5 melanoma patients vaccinated with the modified gp100(209-2M) melanoma peptide were stimulated with cognate peptide and cultured in either IL-21 + low-dose IL-2 or IL-15 + low-dose IL-2. In vitro stimulated (IVS) cells were interrogated using 8-color flow cytometry. Data were analyzed using Winlist Hyperlog and FCOM software, and 32 T cell subsets were resolved for each culture condition. Hierarchical clustering analysis was applied to the relative percentages of each subphenotype for both IVS conditions to determine if unique cell surface marker expression signatures were produced for each IVS culture. RESULTS: Sequential data analysis using Hyperlog and FCOM demonstrated that lymphocytes cultured in IL-21 + IL-2 had a distinctively different set of subphenotype signatures compared to cells grown in IL-15 + IL-2 for all 5 patients. Importantly, subsequent cluster analysis of all 32 subphenotype frequencies in each IVS test condition for all 5 patients reproducibly demonstrated that cellular subphenotypes produced after IL-21 + IL-2 IVS partitioned separately from subphenotypes produced by IL-15 + IL-2 IVS. CONCLUSIONS: The integrated sequential use of Hyperlog and FCOM software with cluster analysis algorithms for the reduction and analysis of polychromatic flow cytometry data produces an effective, rapid technique for the assessment of complex patterns of subphenotype expression between and within multiple test samples. This approach to data analysis may enhance the use of polychromatic flow cytometry for both research and clinical applications.

Age-based differences of percutaneous coronary intervention in the drug-eluting stent era.

INTRODUCTION: Limited data are available on contemporary percutaneous coronary intervention (PCI) practice patterns and outcomes in elderly patients. The objective of this study was to evaluate "real-world" PCI in elderly and nonelderly patients during the first year of availability of drug-eluting stents (DES) in the United States market (May 1, 2003-April 30, 2004). METHODS: One thousand one hundred sixty-six consecutive patients (272 elderly [age > or =75 years] and 894 nonelderly [age <75 years]) having PCI for de novo coronary artery disease (CAD) at Dartmouth-Hitchcock Medical Center were included in this study. Primary outcome measures of this study were in-hospital major adverse cardiac events (MACE-death, new MI, urgent revascularization). Secondary end points included acute renal failure, respiratory failure, and vascular complications. RESULTS: Elderly patients had higher MACE (8.5% vs 1.5%, P < or = 0.001), unadjusted in-hospital mortality (7.4% vs 0.8%, P < or = 0.001), in-hospital cardiac arrest (1.5% vs 0.3%, P = 0.03), requirements for assisted blood pressure support (13.2% vs 7.0%, P = 0.0001), respiratory failure (2.2% vs 0.9%, P = 0.08), acute renal failure (2.9% vs 0.8%, P = 0.005), and vascular complications (10.3% vs 5.5%, P = 0.005) than their nonelderly counterparts. Higher MACE rates persisted in the elderly despite correction for baseline differences using multivariate regression modeling. CONCLUSIONS: Advanced age remains a predictor of adverse outcomes attending PCI even in the contemporary era in which DES are available. This study highlights the need for further progress and investigation to optimize outcomes of PCI in the elderly.

gp100(209-2M) peptide immunization of human lymphocyte antigen-A2+ stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8+ T cells.

Thirty-five HLA-A2(+) patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100(209-2M), emulsified in Montanide adjuvant. Direct ex vivo gp100(209-2M) tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer(+) CD8(+) T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05-8.9%). Ex vivo IFN-gamma cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100(209-2M) in vitro activation showed that for all of the patients studied tetramer(+) CD8(+) T cells produced IFN-gamma; however, some patients had significant numbers of tetramer(+) IFN-gamma(-) CD8(+)T cells suggesting functional anergy. Additionally, 8 day gp100(209-2M) in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer(+) CD8(+) T cells from postvaccine cells for 34 patients, and these IVS tetramer(+) CD8(+) T cells were functionally responsive by IFN-gamma cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8(+) T cells demonstrated the proliferation of functionally anergic gp100(209-2M)- tetramer(+) CD8(+) T cells in several patients and also indicated interpatient variability of gp100(209-2M) stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer(+) CD8+ T cells (78.1 +/- 4.2%) had either an "effector" (CD45 RA(+)/CCR7(-)) or an "effector-memory" phenotype (CD45RA(-)/CCR7(-)). Notably, analysis of PBMCs collected 12-24 months after vaccine therapy demonstrated the durable presence of gp100(209-2M)-specific memory CD8(+) T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8(+) T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.

Immunological monitoring of patients with melanoma after peptide vaccination using soluble peptide/HLA-A2 dimer complexes.

To facilitate the immunologic monitoring of a peptide vaccine trial, a novel, empty dimeric HLA-A2 molecule (A2 dimer) that could be loaded with peptides was produced. The dimer comprises the extracellular domain of HLA-A2 noncovalently linked to a fusion protein consisting of human beta2-microglobulin joined to the human IgG1 Fc domain. Peptide-loaded dimer complexes were used to assess the function of peptide-specific T cells. HLA-A2 gp100 peptide dimers stimulated interferon (IFN)-gamma production by the gp100-specific TIL-1520 cell line. Gp100/A2 dimer stimulation in combination with intracellular cytokine staining was used to analyze peptide-specific T-cell responses in patients with melanoma after vaccination with the modified gp100: 209-2M peptide in adjuvant. Titration analysis of the amount of peptide-loaded dimer required to stimulate gp100-specific T cells was used to estimate the functional avidity of effector/memory CD8+ T lymphocytes. The number of peptide-specific T cells detected in the peripheral blood of vaccinated patients using this assay was comparable to the number determined by staining with fluoresceinated gp100: 209-2M HLA-A2 tetramers. IFN-gamma production by T cells was comparable after stimulation with peptide-pulsed dimers, T2 cells, or autologous dendritic cells. Peptide-loaded A2 dimers could also be used directly to stimulate T cells in the ELISPOT assay.

Adaptation of commercial immunoassays to analysis of complex biological substances.

An Intracellular Adhesion Molecule I (ICAM-1) immunoassay from R and D Systems, and a Melanoma Inhibitory Activity (MIA) immunoassay from Roche Diagnostics were tested for accurate quantitation within complex biological substances such as cell lysates. Prior to assay, lysates of melanoma cells were treated with detergents to obtain soluble antigens. Maximum ICAM-1 and maximum MIA were detected after treatment using 0.8% Triton X-100. Two key aspects of assay accuracy were: 1) determining the dilutions of test sample that provided accurate quantitation (sample range), and 2) performing spiking experiments at these dilutions to determine absence or presence of a "matrix" effect due to biological complexity of the sample. A high degree of accuracy was found by diluting this particular cellular extract 50-fold prior to ICAM-1 assay, or only 5-fold prior to MIA assay. In addition, the bicinchoninic acid protein assay was analyzed to test the accuracy of protein quantitation of cellular lysates. Precision, limits of detection, and quantitation, robustness, linearity, and specificity also were tested for the immunoassays.