Publications by authors named "Sean Mackay"

50 Publications

Longitudinal profiling of respiratory and systemic immune responses reveals myeloid cell-driven lung inflammation in severe COVID-19.

Immunity 2021 Mar 11. Epub 2021 Mar 11.

Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA; Department of Surgery, Columbia University Irving Medical Center, New York, NY 10032, USA. Electronic address:

Immune response dynamics in coronavirus disease 2019 (COVID-19) and their severe manifestations have largely been studied in circulation. Here, we examined the relationship between immune processes in the respiratory tract and circulation through longitudinal phenotypic, transcriptomic, and cytokine profiling of paired airway and blood samples from patients with severe COVID-19 relative to heathy controls. In COVID-19 airways, T cells exhibited activated, tissue-resident, and protective profiles; higher T cell frequencies correlated with survival and younger age. Myeloid cells in COVID-19 airways featured hyperinflammatory signatures, and higher frequencies of these cells correlated with mortality and older age. In COVID-19 blood, aberrant CD163 monocytes predominated over conventional monocytes, and were found in corresponding airway samples and in damaged alveoli. High levels of myeloid chemoattractants in airways suggest recruitment of these cells through a CCL2-CCR2 chemokine axis. Our findings provide insights into immune processes driving COVID-19 lung pathology with therapeutic implications for targeting inflammation in the respiratory tract.
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http://dx.doi.org/10.1016/j.immuni.2021.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7951561PMC
March 2021

Complement-activated human endothelial cells stimulate increased polyfunctionality in alloreactive T cells.

Am J Transplant 2021 Jan 8. Epub 2021 Jan 8.

Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut.

Antibody-mediated deposition of complement membrane attack complexes (MACs) on IFN-γ-primed human endothelial cells (ECs) triggers autocrine/paracrine IL-1β-mediated EC activation and IL-15 transpresentation to alloreactive effector memory T cells (T ), changes that enable ECs to increase T cell proliferation and cytokine release. Here, we report the use of single-cell microchip 32-plex proteomics to more deeply assess the functionality of the activated T cells and dependence upon EC-derived signals. Compared to control ECs, MAC-activated human ECs increase both the frequency and degree of polyfunctionality among both CD4 and CD8 -proliferated T , assessed as secreted proteins. IFN-γ and TNF-α remain the predominant cytokines made by alloreactive T , but a few CD4 T also made IL-4 while more CD8 T made perforin and granzyme B. Increased polyfunctionality was attenuated by treatment of the MAC-activated ECs with anti-IL-15 blocking antibody more effectively than IL-1 receptor blockade. The increased polyfunctionality of T cells resulting from interactions with MAC-activated ECs may further link binding of donor-specific antibody to T cell-mediated allograft pathologies.
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http://dx.doi.org/10.1111/ajt.16485DOI Listing
January 2021

Multi-Omics Resolves a Sharp Disease-State Shift between Mild and Moderate COVID-19.

Cell 2020 12 28;183(6):1479-1495.e20. Epub 2020 Oct 28.

Institute for Systems Biology, Seattle, WA 98109, USA; Department of Bioengineering, University of Washington, Seattle, WA 98105, USA. Electronic address:

We present an integrated analysis of the clinical measurements, immune cells, and plasma multi-omics of 139 COVID-19 patients representing all levels of disease severity, from serial blood draws collected during the first week of infection following diagnosis. We identify a major shift between mild and moderate disease, at which point elevated inflammatory signaling is accompanied by the loss of specific classes of metabolites and metabolic processes. Within this stressed plasma environment at moderate disease, multiple unusual immune cell phenotypes emerge and amplify with increasing disease severity. We condensed over 120,000 immune features into a single axis to capture how different immune cell classes coordinate in response to SARS-CoV-2. This immune-response axis independently aligns with the major plasma composition changes, with clinical metrics of blood clotting, and with the sharp transition between mild and moderate disease. This study suggests that moderate disease may provide the most effective setting for therapeutic intervention.
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http://dx.doi.org/10.1016/j.cell.2020.10.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598382PMC
December 2020

Analysis of respiratory and systemic immune responses in COVID-19 reveals mechanisms of disease pathogenesis.

medRxiv 2020 Oct 18. Epub 2020 Oct 18.

Immune responses to respiratory viruses like SARS-CoV-2 originate and function in the lung, yet assessments of human immunity are often limited to blood. Here, we conducted longitudinal, high-dimensional profiling of paired airway and blood samples from patients with severe COVID-19, revealing immune processes in the respiratory tract linked to disease pathogenesis. Survival from severe disease was associated with increased CD4 T cells and decreased monocyte/macrophage frequencies in the airway, but not in blood. Airway T cells and macrophages exhibited tissue-resident phenotypes and activation signatures, including high level expression and secretion of monocyte chemoattractants CCL2 and CCL3 by airway macrophages. By contrast, monocytes in blood expressed the CCL2-receptor CCR2 and aberrant CD163 and immature phenotypes. Extensive accumulation of CD163 monocyte/macrophages within alveolar spaces in COVID-19 lung autopsies suggested recruitment from circulation. Our findings provide evidence that COVID-19 pathogenesis is driven by respiratory immunity, and rationale for site-specific treatment and prevention strategies.
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http://dx.doi.org/10.1101/2020.10.15.20208041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587837PMC
October 2020

Targeted Co-delivery of Tumor Antigen and α-Galactosylceramide to CD141 Dendritic Cells Induces a Potent Tumor Antigen-Specific Human CD8 T Cell Response in Human Immune System Mice.

Front Immunol 2020 18;11:2043. Epub 2020 Aug 18.

Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY, United States.

Active co-delivery of tumor antigens (Ag) and α-galactosylceramide (α-GalCer), a potent agonist for invariant Natural Killer T (NKT) cells, to cross-priming CD8α dendritic cells (DCs) was previously shown to promote strong anti-tumor responses in mice. Here, we designed a nanoparticle-based vaccine able to target human CD141 (BDCA3) DCs - the equivalent of murine CD8α DCs - and deliver both tumor Ag (Melan A) and α-GalCer. This nanovaccine was inoculated into humanized mice that mimic the human immune system (HIS) and possess functional NKT cells and CD8 T cells, called HIS-CD8/NKT mice. We found that multiple immunizations of HIS-CD8/NKT mice with the nanovaccine resulted in the activation and/or expansion of human CD141 DCs and NKT cells and ultimately elicited a potent Melan-A-specific CD8 T cell response, as determined by tetramer staining and ELISpot assay. Single-cell proteomics further detailed the highly polyfunctional CD8 T cells induced by the nanovaccine and revealed their predictive potential for vaccine potency. This finding demonstrates for the first time the unique ability of human NKT cells to license cross-priming DCs and adds a new dimension to the current strategy of cancer vaccine development.
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http://dx.doi.org/10.3389/fimmu.2020.02043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461784PMC
August 2020

Changes in Peripheral and Local Tumor Immunity after Neoadjuvant Chemotherapy Reshape Clinical Outcomes in Patients with Breast Cancer.

Clin Cancer Res 2020 Nov 21;26(21):5668-5681. Epub 2020 Aug 21.

Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee.

Purpose: The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME).

Experimental Design: We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1-high (PD-1) CD8 peripheral T cells and examination of a cytolytic gene signature in whole blood.

Results: In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden.

Conclusions: We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642197PMC
November 2020

Multiomic Immunophenotyping of COVID-19 Patients Reveals Early Infection Trajectories.

bioRxiv 2020 Jul 31. Epub 2020 Jul 31.

Host immune responses play central roles in controlling SARS-CoV2 infection, yet remain incompletely characterized and understood. Here, we present a comprehensive immune response map spanning 454 proteins and 847 metabolites in plasma integrated with single-cell multi-omic assays of PBMCs in which whole transcriptome, 192 surface proteins, and T and B cell receptor sequence were co-analyzed within the context of clinical measures from 50 COVID19 patient samples. Our study reveals novel cellular subpopulations, such as proliferative exhausted CD8 and CD4 T cells, and cytotoxic CD4 T cells, that may be features of severe COVID-19 infection. We condensed over 1 million immune features into a single immune response axis that independently aligns with many clinical features and is also strongly associated with disease severity. Our study represents an important resource towards understanding the heterogeneous immune responses of COVID-19 patients and may provide key information for informing therapeutic development.
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http://dx.doi.org/10.1101/2020.07.27.224063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402042PMC
July 2020

Acute encephalopathy with elevated CSF inflammatory markers as the initial presentation of COVID-19.

Res Sq 2020 May 12. Epub 2020 May 12.

Background: COVID-19 is caused by the severe acute respiratory syndrome virus SARS-CoV-2. It is widely recognized as a respiratory pathogen, but neurologic complications can be the presenting manifestation in a subset of infected patients.

Case Presentation: We describe a 78-year old immunocompromised woman who presented with altered mental status after witnessed seizure-like activity at home. She was found to have SARS-CoV-2 infection and associated neuroinflammation. In this case, we undertake the first detailed analysis of cerebrospinal fluid (CSF) cytokines during COVID-19 infection and find a unique pattern of inflammation in CSF, but no evidence of viral neuroinvasion.

Conclusion: Our findings suggest that neurologic symptoms such as encephalopathy and seizures may be the initial presentation of COVID-19. Central nervous system inflammation may associate with neurologic manifestations of disease.
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http://dx.doi.org/10.21203/rs.3.rs-28583/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336693PMC
May 2020

Acute encephalopathy with elevated CSF inflammatory markers as the initial presentation of COVID-19.

BMC Neurol 2020 Jun 18;20(1):248. Epub 2020 Jun 18.

Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT, 06510, USA.

Background: COVID-19 is caused by the severe acute respiratory syndrome virus SARS-CoV-2. It is widely recognized as a respiratory pathogen, but neurologic complications can be the presenting manifestation in a subset of infected patients.

Case Presentation: We describe a 78-year old immunocompromised woman who presented with altered mental status after witnessed seizure-like activity at home. She was found to have SARS-CoV-2 infection and associated neuroinflammation. In this case, we undertake the first detailed analysis of cerebrospinal fluid (CSF) cytokines during COVID-19 infection and find a unique pattern of inflammation in CSF, but no evidence of viral neuroinvasion.

Conclusion: Our findings suggest that neurologic symptoms such as encephalopathy and seizures may be the initial presentation of COVID-19. Central nervous system inflammation may associate with neurologic manifestations of disease.
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http://dx.doi.org/10.1186/s12883-020-01812-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301053PMC
June 2020

Advanced Cell Mapping Visualizations for Single Cell Functional Proteomics Enabling Patient Stratification.

Proteomics 2020 07;20(13):e1900270

IsoPlexis Corporation, Branford, CT, 06405, USA.

Highly multiplexed single-cell functional proteomics has emerged as one of the next-generation toolkits for a deeper understanding of functional heterogeneity in cell. Different from the conventional population-based bulk and single-cell RNA-Seq assays, the microchip-based proteomics at the single-cell resolution enables a unique identification of highly polyfunctional cell subsets that co-secrete many proteins from live single cells and most importantly correlate with patient response to a therapy. The 32-plex IsoCode chip technology has defined a polyfunctional strength index (PSI) of pre-infusion anti-CD19 chimeric antigen receptor (CAR)-T products, that is significantly associated with patient response to the CAR-T cell therapy. To complement the clinical relevance of the PSI, a comprehensive visualization toolkit of 3D uniform manifold approximation and projection (UMAP) and t-distributed stochastic neighbor embedding (t-SNE) in a proteomic analysis pipeline is developed, providing more advanced analytical algorithms for more intuitive data visualizations. The UMAP and t-SNE of anti-CD19 CAR-T products reveal distinct cytokine profiles between nonresponders and responders and demonstrate a marked upregulation of antitumor-associated cytokine signatures in CAR-T cells from responding patients. Using this powerful while user-friendly analytical tool, the multi-dimensional single-cell data can be dissected from complex immune responses and uncover underlying mechanisms, which can promote correlative biomarker discovery, improved bioprocessing, and personalized treatment development.
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http://dx.doi.org/10.1002/pmic.201900270DOI Listing
July 2020

Persistent Polyfunctional Chimeric Antigen Receptor T Cells That Target Glypican 3 Eliminate Orthotopic Hepatocellular Carcinomas in Mice.

Gastroenterology 2020 06 12;158(8):2250-2265.e20. Epub 2020 Feb 12.

Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. Electronic address:

Background And Aims: Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody-binding properties.

Methods: We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NOD/SCID/IL-2Rgc (NSG) mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (mock control), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NOD/SCID/IL-2Rgc (NSG) mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice.

Results: Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active β-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T-cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice.

Conclusions: In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC.
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http://dx.doi.org/10.1053/j.gastro.2020.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282931PMC
June 2020

Persistence of adoptively transferred T cells with a kinetically engineered IL-2 receptor agonist.

Nat Commun 2020 01 31;11(1):660. Epub 2020 Jan 31.

Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.

Interleukin-2 (IL-2) is a component of most protocols of adoptive cell transfer (ACT) therapy for cancer, but is limited by short exposure and high toxicities. NKTR-214 is a kinetically-engineered IL-2 receptor βγ (IL-2Rβγ)-biased agonist consisting of IL-2 conjugated to multiple releasable polyethylene glycol chains resulting in sustained signaling through IL-2Rβγ. We report that ACT supported by NKTR-214 increases the proliferation, homing and persistence of anti-tumor T cells compared to ACT with IL-2, resulting in superior antitumor activity in a B16-F10 murine melanoma model. The use of NKTR-214 increases the number of polyfunctional T cells in murine spleens and tumors compared to IL-2, and enhances the polyfunctionality of T and NK cells in the peripheral blood of patients receiving NKTR-214 in a phase 1 trial. In conclusion, NKTR-214 may have the potential to improve the antitumor activity of ACT in humans through increased in vivo expansion and polyfunctionality of the adoptively transferred T cells.
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http://dx.doi.org/10.1038/s41467-019-12901-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994533PMC
January 2020

Rational design of a trimeric APRIL-based CAR-binding domain enables efficient targeting of multiple myeloma.

Blood Adv 2019 11;3(21):3248-3260

Cellular Immunotherapy Program, Cancer Center, Massachusetts General Hospital, Boston, MA.

Chimeric antigen receptor (CAR) T cells (CARTs) have shown tremendous potential for the treatment of certain B-cell malignancies, including patients with relapsed/refractory multiple myeloma (MM). Targeting the B-cell maturation antigen (BCMA) has produced the most promising results for CART therapy of MM to date, but not all remissions are sustained. Emergence of BCMA escape variants has been reported under the selective pressure of monospecific anti-BCMA CART treatment. Thus, there is a clinical need for continuous improvement of CART therapies for MM. Here, we show that a novel trimeric APRIL (a proliferation-inducing ligand)-based CAR efficiently targets both BCMA+ and BCMA- MM. Modeled after the natural ligand-receptor pair, APRIL-based CARs allow for bispecific targeting of the MM-associated antigens BCMA and transmembrane activator and CAML interactor (TACI). However, natural ligands as CAR antigen-binding domains may require further engineering to promote optimal binding and multimerization to adequately trigger T-cell activation. We found that using a trimeric rather than a monomeric APRIL format as the antigen-binding domain enhanced binding to BCMA and TACI and CART activity against MM in vitro and in vivo. Dual-specific, trimeric APRIL-based CAR are a promising therapeutic approach for MM with potential for preventing and treating BCMA escape.
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http://dx.doi.org/10.1182/bloodadvances.2019000703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855119PMC
November 2019

Two-million-year-old snapshots of atmospheric gases from Antarctic ice.

Nature 2019 10 30;574(7780):663-666. Epub 2019 Oct 30.

Department of Geosciences, Princeton University, Princeton, NJ, USA.

Over the past eight hundred thousand years, glacial-interglacial cycles oscillated with a period of one hundred thousand years ('100k world'). Ice core and ocean sediment data have shown that atmospheric carbon dioxide, Antarctic temperature, deep ocean temperature, and global ice volume correlated strongly with each other in the 100k world. Between about 2.8 and 1.2 million years ago, glacial cycles were smaller in magnitude and shorter in duration ('40k world'). Proxy data from deep-sea sediments suggest that the variability of atmospheric carbon dioxide in the 40k world was also lower than in the 100k world, but we do not have direct observations of atmospheric greenhouse gases from this period. Here we report the recovery of stratigraphically discontinuous ice more than two million years old from the Allan Hills Blue Ice Area, East Antarctica. Concentrations of carbon dioxide and methane in ice core samples older than two million years have been altered by respiration, but some younger samples are pristine. The recovered ice cores extend direct observations of atmospheric carbon dioxide, methane, and Antarctic temperature (based on the deuterium/hydrogen isotope ratio δD, a proxy for regional temperature) into the 40k world. All climate properties before eight hundred thousand years ago fall within the envelope of observations from continuous deep Antarctic ice cores that characterize the 100k world. However, the lowest measured carbon dioxide and methane concentrations and Antarctic temperature in the 40k world are well above glacial values from the past eight hundred thousand years. Our results confirm that the amplitudes of glacial-interglacial variations in atmospheric greenhouse gases and Antarctic climate were reduced in the 40k world, and that the transition from the 40k to the 100k world was accompanied by a decline in minimum carbon dioxide concentrations during glacial maxima.
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http://dx.doi.org/10.1038/s41586-019-1692-3DOI Listing
October 2019

Reduction Mammaplasty: What Cup Size Will I Be?

Plast Reconstr Surg Glob Open 2019 Jun 14;7(6):e2273. Epub 2019 Jun 14.

Plastic Surgery Unit, Eastern Health, Melbourne, VIC, Australia.

Background: Predicting cup size after reduction mammaplasty is a challenge well recognized by plastic surgeons. This study presents a method whereby the weight of tissue to be excised can be predicted on the basis of the initial and desired cup size.

Methods: Breast density was calculated from resection specimens. Cup volumes of a specific range of bra style were then measured by filling the bra cups with modeling clay on a mannequin and the volume measured via water displacement. These data were then correlated to breast tissue volume and weight.

Results: The average breast tissue density calculated was 0.98 g/ml (SD = 0.05). Bra cup volume measurements showed a steady progression according to both cup and band sizes. A table was constructed to predict the weight of tissue required for excision to achieve the desired change in cup size.

Conclusion: These results can assist plastic surgeons in predicting the amount of breast tissue to excise to achieve a given cup size. A secondary use of these results is a breast volume guide for implant planning.
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http://dx.doi.org/10.1097/GOX.0000000000002273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635214PMC
June 2019

Single-Cell Multiplexed Proteomics on the IsoLight Resolves Cellular Functional Heterogeneity to Reveal Clinical Responses of Cancer Patients to Immunotherapies.

Methods Mol Biol 2020 ;2055:413-431

IsoPlexis, Branford, CT, USA.

Cancer immunotherapies, in particular adoptive T cell therapy and immune-checkpoint blockade therapy have demonstrated a remarkable success in the treatment of cancer. However, due to heterogeneous functionality and complex immune response of immune cells, it remains challenging to identify predictive biomarkers which have the potential to correlate with efficacy and adverse effects of immunotherapies and help selecting patients who might benefit from the therapy, developing more personalized therapeutics as well as reducing clinical trial cost. The single-cell IsoCode chip in conjunction with fluorescent ELISA-based assay enables a simultaneous detection up to 40+ proteins secreted from live single immune cells, providing a large portion of the assayable functions for each immune cell type, and thus precise assessment of multifunctional, or polyfunctional, heterogeneity of each immune cell type.This unique functional detection capability provides a powerful solution to unmet needs in immunotherapy patient profiling today. Recently, the single-cell metric termed polyfunctional strength index (PSI™) by IsoCode chip computed from preinfusion anti-CD19 chimeric antigen receptor (CAR)-T cell products has demonstrated a significant association with clinical response and cytokine release syndrome (CRS) of cancer patient to the therapy after cell product infusion. This chapter elucidates IsoPlexis single-cell highly multiplexed proteomic platform and provides technical details for characterizing cell products and various cell subsets from peripheral blood, bone marrow, or tumor tissues using this assay.
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http://dx.doi.org/10.1007/978-1-4939-9773-2_19DOI Listing
November 2020

BRCA1 mRNA expression modifies the effect of T cell activation score on patient survival in breast cancer.

BMC Cancer 2019 Apr 25;19(1):387. Epub 2019 Apr 25.

Department of Biostatistics, Yale School of Public Health, School of Medicine, Center for Biomedical Data Science, Yale Cancer Center, Yale University, 60 College Street, New Haven, CT, 06520-8034, USA.

Background: Effector CD8 T cell activation and its cytotoxic function to eradicate tumor cells depend on the T cell recognition of tumor neoantigens, and are positively associated with improved survival in breast cancer. Tumor suppressor BRCA1 and cell cycle regulator CCND1 play a critical role in maintaining genome integrity and tumorigenesis, respectively. However, it is still unclear how BRCA1 and CCND1 expression levels affect the effect of T cell activation on breast cancer patient survival.

Methods: The interactions between T cell activation status and either BRCA1 or CCND1 expression were evaluated using Kaplan-Meier survival curves and multivariate Cox regression models in a public dataset with 1088 breast cancer patients.

Results: Among the patients with low BRCA1 or CCND1 expression, the Activation group showed better overall survival than the Exhaustion group. Adjusted hazards ratios were 0.43 (95% CI: 0.20-0.93) in patients with a low BRCA1 level, and 0.39 (95% CI: 0.19-0.81) in patients with a low CCND1 level, respectively. There was a significant trend in both subgroups (p-trend = 0.011 in the low BRCA1 group, and p-trend = 0.009 in the low CCND1 group). In contrast, there is no significant association in patients with either high BRCA1 or high CCND1 levels. There is a significant interaction between T cell activation status and BRCA1 level (p = 0.009), but not between T cell activation status and CCND1 level (p = 0.135).

Conclusions: BRCA1 expression modified the effect of T cell activation status on patient survival in breast cancer, suggesting that the existence of neoantigens and the enhancement of neoantigen presentation in combination with immune checkpoint blockade may have synergistic effects on patient outcome.
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http://dx.doi.org/10.1186/s12885-019-5595-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482542PMC
April 2019

Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL.

Blood 2018 08 12;132(8):804-814. Epub 2018 Jun 12.

Kite, a Gilead Company, Santa Monica, CA.

After treatment with chimeric antigen receptor (CAR) T cells, interleukin-15 (IL-15) elevation and CAR T-cell expansion are associated with non-Hodgkin lymphoma (NHL) outcomes. However, the association of preinfusion CAR product T-cell functionality with clinical outcomes has not been reported. A single-cell analysis of the preinfusion CD19 CAR product from patients with NHL demonstrated that CAR products contain polyfunctional T-cell subsets capable of deploying multiple immune programs represented by cytokines and chemokines, including interferon-γ, IL-17A, IL-8, and macrophage inflammatory protein 1α. A prespecified T-cell polyfunctionality strength index (PSI) applied to preinfusion CAR product was significantly associated with clinical response, and PSI combined with CAR T-cell expansion or pretreatment serum IL-15 levels conferred additional significance. Within the total product cell population, associations with clinical outcomes were greater with polyfunctional CD4 T cells compared with CD8 cells. Grade ≥3 cytokine release syndrome was associated with polyfunctional T cells, and both grade ≥3 neurologic toxicity and antitumor efficacy were associated with polyfunctional IL-17A-producing T cells. The findings in this exploratory study show that a preinfusion CAR product T-cell subset with a definable polyfunctional profile has a major association with clinical outcomes of CAR T-cell therapy. This trial was registered at www.clinicaltrials.gov as #NCT00924326.
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http://dx.doi.org/10.1182/blood-2018-01-828343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107882PMC
August 2018

Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response.

J Immunother Cancer 2017 11 21;5(1):85. Epub 2017 Nov 21.

IsoPlexis Corporation, 35 NE Industrial Rd, Branford, CT, 06405, USA.

Background: It remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies.

Methods: We employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods.

Results: We demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors.

Conclusions: Single-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high dimensional data requires new visualization methods to further define precise polyfunctional response differences in these products. The presented biomarker capture and analysis system provides a more sensitive and comprehensive functional assessment of CAR-T pre-infusion products and may provide insights into the safety and efficacy of CAR-T cell therapy.
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http://dx.doi.org/10.1186/s40425-017-0293-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697351PMC
November 2017

Why Should We Use Online Research Methods? Four Doctoral Health Student Perspectives.

Qual Health Res 2018 08 26;28(10):1650-1657. Epub 2017 Jul 26.

1 Liverpool John Moores University, Liverpool, United Kingdom.

The growth of the Internet has led to an increase in researchers utilizing online methods. Online communities such as forums, blogs, and video platforms are particularly useful for research involving populations that are Internet savvy, seldom heard or discussing sensitive or illicit behavior. Drawing upon the experiences of four doctoral health students who are using online methods, this article discusses the value and benefits of conducting online research as well as the limitations and difficulties encountered. Consideration is given to the methodological and ethical implications of online research. Our own research leads us to reflect on participants' perceptions of what is public, preserving anonymity and protecting participants from harm.
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http://dx.doi.org/10.1177/1049732317721698DOI Listing
August 2018

CD8+ T-cell mediated anti-malaria protection induced by malaria vaccines; assessment of hepatic CD8+ T cells by SCBC assay.

Hum Vaccin Immunother 2017 07;13(7):1625-1629

a IsoPlexis , Branford , CT , USA.

Malaria is a severe infectious disease with relatively high mortality, thus having been a scourge of humanity. There are a few candidate malaria vaccines that have shown a protective efficacy in humans against malaria. One of the candidate human malaria vaccines, which is based on human malaria sporozoites and called PfSPZ Vaccine, has been shown to protect a significant proportion of vaccine recipients from getting malaria. PfSPZ Vaccine elicits a potent response of hepatic CD8+ T cells that are specific for malaria antigens in non-human primates. To further characterize hepatic CD8+ T cells induced by the sporozoite-based malaria vaccine in a mouse model, we have used a cutting-edge Single-cell Barcode (SCBC) assay, a recently emerged approach/method for investigating the nature of T-cells responses during infection or cancer. Using the SCBC technology, we have identified a population of hepatic CD8+ T cells that are polyfunctional at a single cell level only in a group of vaccinated mice upon malaria challenge. The cytokines/chemokines secreted by these polyfunctional CD8+ T-cell subsets include MIP-1α, RANTES, IFN-γ, and/or IL-17A, which have shown to be associated with protective T-cell responses against certain pathogens. Therefore, a successful induction of such polyfunctional hepatic CD8+ T cells may be a key to the development of effective human malaria vaccine. In addition, the SCBC technology could provide a new level of diagnostic that will allow for a more accurate determination of vaccine efficacy.
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http://dx.doi.org/10.1080/21645515.2017.1304333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5512776PMC
July 2017

Obliquity-paced climate change recorded in Antarctic debris-covered glaciers.

Nat Commun 2017 02 10;8:14194. Epub 2017 Feb 10.

Department of Earth and Environment, Boston University, Boston, Massachusetts 02215, USA.

The degree to which debris-covered glaciers record past environmental conditions is debated. Here we describe a novel palaeoclimate archive derived from the surface morphology and internal debris within cold-based debris-covered glaciers in Antarctica. Results show that subtle changes in mass balance impart major changes in the concentration of englacial debris and corresponding surface topography, and that over the past ∼220 ka, at least, the changes are related to obliquity-paced solar radiation, manifest as variations in total summer energy. Our findings emphasize solar radiation as a significant driver of mass balance changes in high-latitude mountain systems, and demonstrate that debris-covered glaciers are among the most sensitive recorders of obliquity-paced climate variability in interior Antarctica, in contrast to most other Antarctic archives that favour eccentricity-paced forcing over the same time period. Furthermore, our results open the possibility that similar-appearing debris-covered glaciers on Mars may likewise hold clues to environmental change.
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http://dx.doi.org/10.1038/ncomms14194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5309835PMC
February 2017

Interfacing with Neural Activity via Femtosecond Laser Stimulation of Drug-Encapsulating Liposomal Nanostructures.

eNeuro 2016 Nov-Dec;3(6). Epub 2016 Nov 16.

Neurobiology Research Unit, Okinawa Institute of Science and Technology Graduate University , Onna-son 904-0412, Okinawa, Japan.

External control over rapid and precise release of chemicals in the brain potentially provides a powerful interface with neural activity. Optical manipulation techniques, such as optogenetics and caged compounds, enable remote control of neural activity and behavior with fine spatiotemporal resolution. However, these methods are limited to chemicals that are naturally present in the brain or chemically suitable for caging. Here, we demonstrate the ability to interface with neural functioning via a wide range of neurochemicals released by stimulating loaded liposomal nanostructures with femtosecond lasers. Using a commercial two-photon microscope, we released inhibitory or excitatory neurochemicals to evoke subthreshold and suprathreshold changes in membrane potential in a live mouse brain slice. The responses were repeatable and could be controlled by adjusting laser stimulation characteristics. We also demonstrate the release of a wider range of chemicals-which previously were impossible to release by optogenetics or uncaging-including synthetic analogs of naturally occurring neurochemicals. In particular, we demonstrate the release of a synthetic receptor-specific agonist that exerts physiological effects on long-term synaptic plasticity. Further, we show that the loaded liposomal nanostructures remain functional for weeks in a live mouse. In conclusion, we demonstrate new techniques capable of interfacing with live neurons, and extendable to applications.
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http://dx.doi.org/10.1523/ENEURO.0107-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5110951PMC
October 2017

Post-oesophagectomy mortality: the centralization debate revisited.

ANZ J Surg 2016 Mar;86(3):116-7

Department of Upper GI Surgery, Box Hill Hospital, Melbourne, Victoria, Australia.

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http://dx.doi.org/10.1111/ans.13357DOI Listing
March 2016

Dieulafoy lesion of the gallbladder presenting with bleeding and a pseudo-mirizzi syndrome: A case report and review of the literature.

Int J Surg Case Rep 2016 6;21:12-5. Epub 2016 Feb 6.

Eastern Health Surgical Research Group, Monash University, Eastern Health Clinical School, 5 Arnold St, Box Hill, Melbourne, Victoria 3128, Australia. Electronic address:

Introduction: Gastrointestinal bleeding can have significant morbidity and mortality. Pathological processes that cause it are diverse, and timely investigation and management are vital. Dieulafoy lesions are a rare cause of gastrointestinal bleeding and here we describe a case of a gallbladder dieulafoy lesion causing gastrointestinal bleeding.

Presentation Of Case: Recently discharged from hospital following an open anterior resection and loop ileostomy for diverticular disease, an 84-year-old female re-presented with lower abdominal pain associated with jaundice and lymphocytosis. Imaging demonstrated two possible rectal stump collections (treated with antibiotics), and heterogeneous material in the gallbladder. The patient deteriorated, developing melena, coffee ground vomitus and right upper quadrant pain. Investigation sourced the bleeding to the gallbladder that resolved following cholecystectomy, and histopathology was consistent with a dieulafoy lesion. The patient made a full recovery.

Discussion: Dieulafoy lesions have rarely been reported in the gallbladder, and as such can be an occult source of massive gastrointestinal bleeding. It should be considered where gastrointestinal bleeding accompanies jaundice and abdominal pain.

Conclusion: This case highlights that dieulafoy lesions can occur in the gallbladder. Massive gastrointestinal bleeding can occur within the gallbladder, and a gallbladder dieulafoy lesion should be considered as a potential cause of such, especially when a source has not been identified on endoscopy. It also demonstrates the effectiveness of cholecystectomy as a definitive management strategy.
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http://dx.doi.org/10.1016/j.ijscr.2016.01.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802134PMC
April 2016

Signaled drug delivery and transport across the blood-brain barrier.

J Liposome Res 2016 Sep 16;26(3):233-45. Epub 2015 Nov 16.

f New Zealand's National School of Pharmacy, University of Otago , Dunedin , New Zealand.

We use a mathematical model to describe the delivery of a drug to a specific region of the brain. The drug is carried by liposomes that can release their cargo by application of focused ultrasound (US). Thereupon, the drug is absorbed through the endothelial cells that line the brain capillaries and form the physiologically important blood-brain barrier (BBB). We present a compartmental model of a capillary that is able to capture the complex binding and transport processes the drug undergoes in the blood plasma and at the BBB. We apply this model to the delivery of levodopa (L-dopa, used to treat Parkinson's disease) and doxorubicin (an anticancer agent). The goal is to optimize the delivery of drug while at the same time minimizing possible side effects of the US.
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http://dx.doi.org/10.3109/08982104.2015.1102277DOI Listing
September 2016

Endoscopic sphincterotomy with sphincteroplasty for the management of choledocholithiasis: a single-centre experience.

ANZ J Surg 2017 Sep 17;87(9):695-699. Epub 2015 Mar 17.

Department of Upper Gastrointestinal Surgery, Eastern Health, Box Hill Hospital, Melbourne, Victoria, Australia.

Background: Balloon dilatation of the ampulla at endoscopic retrograde cholangiopancreatography (ERCP) is increasingly utilized in the management of large bile duct stones. The aim of this study was to review and compare the outcomes of using endoscopic sphincterotomy with endoscopic balloon dilatation (sphincteroplasty) in a combined approach as a single-stage (immediate) or a two-stage procedure (delayed).

Methods: A retrospective review of medical records for all patients undergoing ERCP and balloon dilatation for choledocholithiasis between January 2010 and December 2012 was undertaken. Outcomes measured included patient demographics, stone size, degree of dilatation performed, success of stone extraction, number of procedures required for duct clearance and procedure-related complications.

Results: One hundred and thirty-six ERCPs were performed with balloon sphincteroplasty. One hundred and four had a previous sphincterotomy with a delayed balloon dilatation and 32 had sphincterotomy with immediate dilatation. The overall clearance rate of the common bile duct for immediate and delayed groups was 93% (28/30) and 93% (81/87), respectively. Bile duct clearance after the first procedure was achieved in 70% (21/30) of patients in the immediate group and 74% (64/87) in the delayed group. There were six complications in the delayed group and four in the immediate group. The most frequently used balloon size was 10 mm for both groups with mean sizes of 10.34 (2.93) and 11.73 (2.87) in the immediate and delayed groups, respectively.

Conclusion: Our study suggests that use of a combined approach is safe and effective and may provide benefits over using endoscopic balloon dilatation or endoscopic sphincterotomy alone in the treatment of choledocholithiasis.
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http://dx.doi.org/10.1111/ans.13058DOI Listing
September 2017

Quality of patient health information on the Internet: reviewing a complex and evolving landscape.

Australas Med J 2014 31;7(1):24-8. Epub 2014 Jan 31.

Eastern Health Surgical Research Group, Melbourne, Victoria, Australia ; Monash University, Melbourne, Victoria, Australia.

Background: The popularity of the Internet has enabled unprecedented access to health information. As a largely unregulated source, there is potential for inconsistency in the quality of information that reaches the patient.

Aims: To review the literature relating to the quality indicators of health information for patients on the Internet.

Method: A search of English language literature was conducted using PubMed, Google Scholar and EMBASE databases.

Results: Many articles have been published which assess the quality of information relating to specific medical conditions. Indicators of quality have been defined in an attempt to predict higher quality health information on the Internet. Quality evaluation tools are scoring systems based on indicators of quality. Established tools such as the HONcode may help patients navigate to more reliable information. Google and Wikipedia are important emerging sources of patient health information.

Conclusion: The Internet is crucial for modern dissemination of health information, but it is clear that quality varies significantly between sources. Quality indicators for web-information have been developed but there is no agreed standard yet. We envisage that reliable rating tools, effective search engine ranking and progress in crowd-edited websites will enhance patient access to health information on the Internet.
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http://dx.doi.org/10.4066/AMJ.2014.1900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3920473PMC
March 2014

CareTrack: assessing the appropriateness of health care delivery in Australia.

Authors:
Sean Mackay

Med J Aust 2012 Nov;197(10):548; author reply 549-50

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http://dx.doi.org/10.5694/mja12.11186DOI Listing
November 2012