Whole-blood sorting, enrichment and in situ immunolabeling of cellular subsets using acoustic microstreaming

Analyzing undiluted whole human blood is a challenge due to its complex composition of hematopoietic cellular populations, nucleic acids, metabolites, and proteins. We present a novel multi-functional microfluidic acoustic streaming platform that enables sorting, enrichment and in situ identification of cellular subsets from whole blood. This single device platform, based on lateral cavity acoustic transducers (LCAT), enables (1) the sorting of undiluted donor whole blood into its cellular subsets (platelets, RBCs, and WBCs), (2) the enrichment and retrieval of breast cancer cells (MCF-7) spiked in donor whole blood at rare cell relevant concentrations (10 mL− 1), and (3) on-chip immunofluorescent labeling for the detection of specific target cellular populations by their known marker expression patterns. Our approach thus demonstrates a compact system that integrates upstream sample processing with downstream separation/enrichment, to carry out multi-parametric cell analysis for blood-based diagnosis and liquid biopsy blood sampling

Co-delivery of human cancer-testis antigens with adjuvant in protein nanoparticles induces higher cell-mediated immune responses.

Nanoparticles have attracted considerable interest as cancer vaccine delivery vehicles for inducing sufficient CD8+ T cell-mediated immune responses to overcome the low immunogenicity of the tumor microenvironment. Our studies described here are the first to examine the effects of clinically-tested human cancer-testis (CT) peptide epitopes within a synthetic nanoparticle. Specifically, we focused on two significant clinical CT targets, the HLA-A2 restricted epitopes of NY-ESO-1 and MAGE-A3, using a viral-mimetic packaging strategy. Our data shows that simultaneous delivery of a NY-ESO-1 epitope (SLLMWITQV) and CpG using the E2 subunit assembly of pyruvate dehydrogenase (E2 nanoparticle), resulted in a 25-fold increase in specific IFN-γ secretion in HLA-A2 transgenic mice. This translated to a 15-fold increase in lytic activity toward target cancer cells expressing the antigen. Immunization with a MAGE-A3 epitope (FLWGPRALV) delivered with CpG in E2 nanoparticles yielded an increase in specific IFN-γ secretion and cell lysis by 6-fold and 9-fold, respectively. Furthermore, combined delivery of NY-ESO-1 and MAGE-A3 antigens in E2 nanoparticles yielded an additive effect that increased lytic activity towards cells bearing NY-ESO-1+ and MAGE-A3+. Our investigations demonstrate that formulation of CT antigens within a nanoparticle can significantly enhance antigen-specific cell-mediated responses, and the combination of the two antigens in a vaccine can preserve the increased individual responses that are observed for each antigen alone

Relationship between social support, quality of life, and Th2 cytokines in a biobehavioral cancer survivorship trial.

ObjectiveBenefits of social support (SS) during cancer survivorship are complex. This study examines change in SS over time in cervical cancer (CXCA) survivors who have completed definitive treatment and how changing SS impacts quality of life (QOL) and T-helper type 2 (Th2) cytokines.MethodsWe conducted a randomized trial in 204 CXCA survivors to test if psychosocial telephone counseling (PTC) could improve QOL compared to usual care (UC). Although PTC did not target SS, data were collected at baseline, 4 and 9 months post-enrollment using the Medical Outcomes Survey Social Support scale. Biospecimens were collected to investigate associations with patient-reported outcomes. Data were analyzed using multivariate linear models and stepwise regression.ResultsParticipants' mean age was 43. PTC participants experienced increasing SS compared to UC at 4 months (PTC-UC = 5.1; p = 0.055) and 9 months (PTC-UC = 6.0; p = 0.046). Higher baseline SS and increasing SS were independently associated with improved QOL at 4 and 9 months after adjusting for patient characteristics (p < 0.05). Differences between study arms were not statistically significant. Improvements in QOL at 4 months were observed with increases in emotional/informational and tangible SS. Increasing SS predicted significant longitudinal decreases in IL-4 and IL-13 at 4 months that were larger in the PTC arm (interactions p = 0.041 and p = 0.057, respectively).ConclusionImproved SS was significantly associated with improved QOL independent of patient characteristics and study arm. Decreasing Th2 cytokines with increasing SS and QOL are consistent with a biobehavioral paradigm in which modulation of the chronic stress response is associated with shifts in immune stance

Isocitrate Dehydrogenase Mutation in \u3ci\u3eVibrio anguillarum\u3c/i\u3e Results in Virulence Attenuation and Immunoprotection in Rainbow Trout (\u3ci\u3eOncorhynchus mykiss\u3c/i\u3e)

Background Vibrio anguillarum is an extracellular bacterial pathogen that is a causative agent of vibriosis in finfish and crustaceans with mortality rates ranging from 30% to 100%. Mutations in central metabolism (glycolysis and the TCA cycle) of intracellular pathogens often result in attenuated virulence due to depletion of required metabolic intermediates; however, it was not known whether mutations in central metabolism would affect virulence in an extracellular pathogen such as V. anguillarum. Results Seven central metabolism mutants were created and characterized with regard to growth in minimal and complex media, expression of virulence genes, and virulence in juvenile rainbow trout (Oncorhynchus mykiss). Only the isocitrate dehydrogenase (icd) mutant was attenuated in virulence against rainbow trout challenged by either intraperitoneal injection or immersion. Further, the icd mutant was shown to be immunoprotective against wild type V. anguillarum infection. There was no significant decrease in the expression of the three hemolysin genes detected by qRT-PCR. Additionally, only the icd mutant exhibited a significantly decreased growth yield in complex media. Growth yield was directly related to the abundance of glutamate. A strain with a restored wild type icd gene was created and shown to restore growth to a wild type cell density in complex media and pathogenicity in rainbow trout. Conclusions The data strongly suggest that a decreased growth yield, resulting from the inability to synthesize α-ketoglutarate, caused the attenuation despite normal levels of expression of virulence genes. Therefore, the ability of an extracellular pathogen to cause disease is dependent upon the availability of host-supplied nutrients for growth. Additionally, a live vaccine strain could be created from an icd deletion strain

An alternative flow cytometry strategy for peripheral blood dendritic cell enumeration in the setting of repetitive GM-CSF dosing

BACKGROUND: Enumeration of circulating peripheral blood dendritic cells (DCs) is complicated by the absence of a unique cell surface marker expressed on all DC subsets and by the use of various biological adjuvants to modulate the DC compartment, including granulocyte macrophage colony stimulating factor (GM-CSF). Common methods employ a cocktail of antibodies, typically including anti-CD14, to define a lineage negative, MHC class II positive, putative DC population. Reported flow cytometry protocols include highly variable gating strategies and DC identification criteria. Increasing appreciation of DC pleiomorphism, GM-CSF biology, and recognition of CD14 expression in some DC subsets led us to consider an alternative lineage cocktail to improve identification of the circulating DC pool. METHODS: Standard whole blood staining with appropriate fluorochrome conjugated antibodies to MHC class II and either standard CD14 containing, or an alternate CD66acde containing, lineage cocktail was performed on samples obtained from normal donors and breast cancer patients before and after administration of dose-dense, cytotoxic chemotherapy with daily GM-CSF hematopoetic growth factor support. Putative DCs were enumerated by standard flow cytometry. Data set differences were evaluated using two tailed Mann-Whitney or Wilcoxon signed rank tests. Cellular morphology was examined in cell-sorted populations from post GM-CSF samples. RESULTS: Use of either antibody cocktail defined comparably sized lineage negative, MHC class II positive populations in normal donors and at baseline in cancer patients. However, selection of lineage negative subsets with increasing MHC class II expression levels yielded larger putative DC populations identified with the alternate cocktail. Both cocktails yielded highly reproducible data. Use of the alternate cocktail: 1) yielded a putative DC population, post GM-CSF that was more homogenous and consistent with DCs, 2) resulted in less data variation across gating strategies, and 3) resulted in more uniform and concordant longitudinal data, consistent with established GM-CSF biological activity. CONCLUSION: An alternative lineage negative cocktail substituting anti-CD66 antibody for anti-CD14 is a viable option for enumerating the circulating DC population, potentially more accurately defining the circulating DC pool by including CD14 positive immature DCs, and thus, may give more reliable data, particularly in the setting of sustained GM-CSF administration

All Over the Map: Rethinking American Regions

Even as Americans keep moving all over the map in the late twentieth century, they cherish memories of the places they come from. But where do these places—these regions—come from? What makes them so real? In this groundbreaking book a distinguished group of historians explores the concept of region in America, traces changes the idea has undergone in our national experience, and examines its meaning for Americans today. Far from diminishing in importance, the authors conclude, regional differences continue to play a significant role in Americans\u27 self-image. Regional identity, in fact, has always been fed by the very forces that many people think threaten its existence today: a central government, an aggressive economy, and connections with places beyond regional boundaries. Calling into question widely held notions about how Americans came to differ from one another and explaining why those differences continue to flourish, this iconoclastic study—by scholars with differing regional ties—will refresh and redirect the centuries-old discussion over Americans\u27 conceptions of themselves.https://scholarship.richmond.edu/bookshelf/1257/thumbnail.jp

Nutritional Quality of Leaves and Unripe Fruit Consumed as Famine Foods by the Flying Foxes of Samoa

Many tropical herbivores alter their diets throughout the year in response to different levels of food availability. Fruit bats, including Pteropus samoensis Peale and Pteropus tonganus Quoy & Gaimard, are phytophagous species that may increase their consumption of foods such as unripe fruit and leaves in periods of low fruit diversity and volume. These periods include the tropical dry season or following the frequent hurricanes that batter the Samoan Archipelago. We examined the nutritional composition of leaves and immature fruits and compared the levels of organic and mineral nutrients with those of ripe fruit. We used principal components analysis (PCA) to examine patterns of variation in nutrient components of leaves, unripe fruit, and ripe fruit, as well as to compare the mean levels of nutrients. Overall, unripe fruit provided levels of nutrients comparable with those of ripe fruit of the same species for many organic and mineral components. Unripe fruit were only half as rich in iron as ripe fruit, but unripe fruit had high levels of calcium compared with ripe fruit of the same species. Leaves are often cited as a rich source of protein for fruit bats, and our results were consistent with this suggestion. Leaves were also found to be rich in zinc, manganese, and calcium. Therefore, flying foxes and other herbivores probably do not avoid unripe fruits and leaves because of their low nutrient levels. It may be that these famine foods are not normally consumed because of the presence of secondary compounds, low concentrations of palatable sugars, or a distasteful and hard pericarp on unripe fruits

Draft Genome Sequence of the New Pathogen for Bivalve Larvae \u3cem\u3eVibrio bivalvicida\u3c/em\u3e

Vibrio bivalvicida is a novel pathogen of bivalve larvae responsible for recent vibriosis outbreaks affecting shellfish hatcheries. Here, we announce the draft genome sequence of V. bivalvicida 605 and describe potential virulence factors

Draft Genome Sequence of the Emerging Bivalve Pathogen \u3cem\u3eVibrio tubiashii\u3c/em\u3e subsp. \u3cem\u3eeuropaeus\u3c/em\u3e

Vibrio tubiashii subsp. europaeus is a bivalve pathogen isolated during episodes of mortality affecting larval cultures in different shellfish hatcheries. Here, we announce the draft genome sequence of the type strain PP-638 and describe potential virulence factors, which may provide insight into the mechanism of pathogenicity

Metabolite Responsive Nanoparticle-Protein Complex

Stimuli responsive polymers are an efficient means of targeted therapy.  Compared to conventional agents, they increase bioavailability and efficacy.  In particular, polymer hydrogel nanoparticles (NPs) can be designed to respond when exposed to a specific environmental stimulus such as pH or temperature. However, targeting a specific metabolite as the trigger for stimuli response could further elevate selectivity and create a new class of bioresponsive materials.  In this work we describe an N-isopropylacrylamide (NIPAm) NP that responds to a specific metabolite characteristic of a hypoxic environment found in cancerous tumors.  NIPAm NPs were synthesized by copolymerization with an oxamate derivative, a known inhibitor of lactate dehydrogenase (LDH).  The oxamate functionalized NPs (OxNP) efficiently sequestered LDH to produce an OxNP-protein complex.  When exposed to elevated concentrations of lactic acid, a substrate of LDH and a metabolite characteristic of hypoxic tumor microenvironments, OxNP-LDH complexes swelled (65%).  The OxNP-LDH complexes were not responsive to structurally related small molecules.  This work demonstrates a proof of concept for tuning NP responsiveness by conjugation with a key protein to target a specific metabolite of disease