Results of a randomized, double-blind phase II clinical trial of NY-ESO-1 vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in participants with high-risk resected melanoma.

BACKGROUND: To compare the clinical efficacy of New York Esophageal squamous cell carcinoma-1 (NY-ESO-1) vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in a randomized, double-blind phase II study in participants with fully resected melanoma at high risk of recurrence. METHODS: Participants with resected stage IIc, IIIb, IIIc and IV melanoma expressing NY-ESO-1 were randomized to treatment with three doses of NY-ESO-1/ISCOMATRIX or ISCOMATRIX adjuvant administered intramuscularly at 4-week intervals, followed by a further dose at 6 months. Primary endpoint was the proportion free of relapse at 18 months in the intention-to-treat (ITT) population and two per-protocol populations. Secondary endpoints included relapse-free survival (RFS) and overall survival (OS), safety and NY-ESO-1 immunity. RESULTS: The ITT population comprised 110 participants, with 56 randomized to NY-ESO-1/ISCOMATRIX and 54 to ISCOMATRIX alone. No significant toxicities were observed. There were no differences between the study arms in relapses at 18 months or for median time to relapse; 139 vs 176 days (p=0.296), or relapse rate, 27 (48.2%) vs 26 (48.1%) (HR 0.913; 95% CI 0.402 to 2.231), respectively. RFS and OS were similar between the study arms. Vaccine recipients developed strong positive antibody responses to NY-ESO-1 (p≤0.0001) and NY-ESO-1-specific CD4+ and CD8+ responses. Biopsies following relapse did not demonstrate differences in NY-ESO-1 expression between the study populations although an exploratory study demonstrated reduced (NY-ESO-1)+/Human Leukocyte Antigen (HLA) class I+ double-positive cells in biopsies from vaccine recipients performed on relapse in 19 participants. CONCLUSIONS: The vaccine was well tolerated, however, despite inducing antigen-specific immunity, it did not affect survival endpoints. Immune escape through the downregulation of NY-ESO-1 and/or HLA class I molecules on tumor may have contributed to relapse

Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients

<p>Abstract</p> <p>Background</p> <p>HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis.</p> <p>Methods</p> <p>Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100_209(210M)and MART-1_26–35(27L), IFNγ real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100₂₀₉, gp100_pool, MART-1_27–35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established.</p> <p>Results</p> <p>The validation process demonstrated that the HLA-A2 tetramer, IFNγ real time RT-PCR, and IFNγ ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545–1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000–1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNγ response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods.</p> <p>Conclusion</p> <p>Our results demonstrated the tetramer flow cytometry assay, IFNγ real-time RT-PCR, and INFγ ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses.</p

The Winter Worries of Bats : Past and Present Perspectives on Winter Habitat and Management of Cave Hibernating Bats

Winter is a time of fascinating changes in biology for cave-hibernating bats, but it is also a time of vulnerability. Unsurprisingly, assessments of winter habitat for these mammals and how it can be managed have been a focus of many researchers involved with the North American Society for Bat Research over the last 50 years. Over this time, a paradigm shift has occurred in the way scientists think about factors driving selection of winter habitat, especially temperature. To illustrate this change, we review three hypotheses seeking to explain microclimate selection in cavernicolous bats. The first, which we call the “Colder is Better Hypothesis,” posits that bats should select cold microclimates that minimize energy expenditure. The “Hibernation Optimization Hypothesis” suggests that bats should select microclimates that reduce expression of torpor to balance energy conservation against non-energetic costs of hibernation. Finally, the “Thrifty Female Hypothesis” asserts that females should select colder microclimates than males to conserve energy for reproduction. We discuss these hypotheses and the shift from viewing hibernation as a phenomenon driven solely by the need to conserve energy in the context of hibernacula management in North America. We focus on both historical and recent conservation threats, most notably alteration of thermal regimes and the disease white-nose syndrome. We urge against returning to an over-simplified view of winter habitat selection in response to our current conservation challenges.Peer reviewe

Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients

<p>Abstract</p> <p>Background</p> <p>HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis.</p> <p>Methods</p> <p>Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100_209(210M)and MART-1_26–35(27L), IFNγ real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100₂₀₉, gp100_pool, MART-1_27–35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established.</p> <p>Results</p> <p>The validation process demonstrated that the HLA-A2 tetramer, IFNγ real time RT-PCR, and IFNγ ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545–1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000–1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNγ response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods.</p> <p>Conclusion</p> <p>Our results demonstrated the tetramer flow cytometry assay, IFNγ real-time RT-PCR, and INFγ ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses.</p

Bioavailability in soils

The consumption of locally-produced vegetables by humans may be an important exposure pathway for soil contaminants in many urban settings and for agricultural land use. Hence, prediction of metal and metalloid uptake by vegetables from contaminated soils is an important part of the Human Health Risk Assessment procedure. The behaviour of metals (cadmium, chromium, cobalt, copper, mercury, molybdenum, nickel, lead and zinc) and metalloids (arsenic, boron and selenium) in contaminated soils depends to a large extent on the intrinsic charge, valence and speciation of the contaminant ion, and soil properties such as pH, redox status and contents of clay and/or organic matter. However, chemistry and behaviour of the contaminant in soil alone cannot predict soil-to-plant transfer. Root uptake, root selectivity, ion interactions, rhizosphere processes, leaf uptake from the atmosphere, and plant partitioning are important processes that ultimately govern the accumulation ofmetals and metalloids in edible vegetable tissues. Mechanistic models to accurately describe all these processes have not yet been developed, let alone validated under field conditions. Hence, to estimate risks by vegetable consumption, empirical models have been used to correlate concentrations of metals and metalloids in contaminated soils, soil physico-chemical characteristics, and concentrations of elements in vegetable tissues. These models should only be used within the bounds of their calibration, and often need to be re-calibrated or validated using local soil and environmental conditions on a regional or site-specific basis.Mike J. McLaughlin, Erik Smolders, Fien Degryse, and Rene Rietr

Multiple Determinants of Whole and Regional Brain Volume among Terrestrial Carnivorans

Mammalian brain volumes vary considerably, even after controlling for body size. Although several hypotheses have been proposed to explain this variation, most research in mammals on the evolution of encephalization has focused on primates, leaving the generality of these explanations uncertain. Furthermore, much research still addresses only one hypothesis at a time, despite the demonstrated importance of considering multiple factors simultaneously. We used phylogenetic comparative methods to investigate simultaneously the importance of several factors previously hypothesized to be important in neural evolution among mammalian carnivores, including social complexity, forelimb use, home range size, diet, life history, phylogeny, and recent evolutionary changes in body size. We also tested hypotheses suggesting roles for these variables in determining the relative volume of four brain regions measured using computed tomography. Our data suggest that, in contrast to brain size in primates, carnivoran brain size may lag behind body size over evolutionary time. Moreover, carnivore species that primarily consume vertebrates have the largest brains. Although we found no support for a role of social complexity in overall encephalization, relative cerebrum volume correlated positively with sociality. Finally, our results support negative relationships among different brain regions after accounting for overall endocranial volume, suggesting that increased size of one brain regions is often accompanied by reduced size in other regions rather than overall brain expansion

Characterization of highly frequent epitope-specific CD45RA(+)/CCR7(+/- )T lymphocyte responses against p53-binding domains of the human polyomavirus BK large tumor antigen in HLA-A*0201+ BKV-seropositive donors

Human polyomavirus BK (BKV) has been implicated in oncogenic transformation. Its ability to replicate is determined by the binding of its large tumor antigen (LTag) to products of tumor-suppressor genes regulating cell cycle, as specifically p53. We investigated CD8+ T immune responses to BKV LTag portions involved in p53 binding in HLA-A*0201+ BKV LTag experienced individuals. Peptides selected from either p53-binding region (LTag(351–450 )and LTag(533–626)) by current algorithms and capacity to bind HLA-A*0201 molecule were used to stimulate CD8+ T responses, as assessed by IFN-γ gene expression ex vivo and detected by cytotoxicity assays following in vitro culture. We observed epitope-specific immune responses in all HLA-A*0201+ BKV LTag experienced individuals tested. At least one epitope, LTag(579–587); LLLIWFRPV, was naturally processed in non professional antigen presenting cells and induced cytotoxic responses with CTL precursor frequencies in the order of 1/20'000. Antigen specific CD8+ T cells were only detectable in the CD45RA+ subset, in both CCR7+ and CCR7- subpopulations. These data indicate that widespread cellular immune responses against epitopes within BKV LTag-p53 binding regions exist and question their roles in immunosurveillance against tumors possibly associated with BKV infection

Type D personality is associated with increased metabolic syndrome prevalence and an unhealthy lifestyle in a cross-sectional Dutch community sample

<p>Abstract</p> <p>Background</p> <p>People with Type D-Distressed-personality have a general tendency towards increased negative affectivity (NA), while at the same time inhibiting these emotions in social situations (SI). Type D personality is associated with an increased risk of adverse outcomes in patients with cardiovascular disease. Whether Type D personality is a cardiovascular risk factor in healthy populations remains to be investigated. In the present study, the relations between Type D personality and classical cardiovascular risk factors, i.e. metabolic syndrome and lifestyle were investigated in a Dutch community sample.</p> <p>Methods</p> <p>In a cross-sectional study 1592 participants were included, aged 20-80 years. Metabolic syndrome was defined by self-report, following the International Diabetes Federation-IDF-guidelines including an increased waist circumference, dyslipidemia, hypertension, and diabetes. In addition lifestyle factors smoking, alcohol use, exercise and dietary habits were examined. Metabolic syndrome prevalence was stratified by Type D personality (a high score on both NA and SI), lifestyle and confounders age, gender, having a partner, higher education level, cardiac history, family history of cardiovascular disease.</p> <p>Results</p> <p>Metabolic syndrome was more prevalent in persons with a Type D personality (13% vs. 6%). Persons with Type D personality made poorer lifestyle choices, adhered less to the physical activity norm (OR = 1.5, 95%CI = 1.1-2.0, <it>p </it>= .02), had a less varied diet (OR = 0.50, 95%CI = 0.40-0.70, <it>p </it>< .0005), and were less likely to restrict their fat intake (OR = 0.70, 95%CI = 0.50-0.90, <it>p </it>= .01). Type D personality was related to a twofold increased risk of metabolic syndrome (OR = 2.2, 95%CI = 1.2-4.0, <it>p </it>= .011), independent of lifestyle factors and confounders.</p> <p>Conclusions</p> <p>Type D personality is related to an increased prevalence of metabolic syndrome and unhealthy lifestyle, which suggests both behavioral and biological vulnerability for development of cardiovascular disorders and diabetes.</p

Evaluating comorbidities in total hip and knee arthroplasty: available instruments

Each year millions of patients are treated for joint pain with total joint arthroplasty, and the numbers are expected to rise. Comorbid disease is known to influence the outcome of total joint arthroplasty, and its documentation is therefore of utmost importance in clinical evaluation of the individual patient as well as in research. In this paper, we examine the various methods for obtaining and assessing comorbidity information for patients undergoing joint replacement. Multiple instruments are reliable and validated for this purpose, such as the Charlson Index, Index of Coexistent Disease, and the Functional Comorbidity Index. In orthopedic studies, the Charnley classification and the American Society of Anesthesiologists physical function score (ASA) are widely used. We recommend that a well-documented comorbidity index that incorporates some aspect of mental health is used along with other appropriate instruments to objectively assess the preoperative status of the patient