Multiple early factors anticipate post-acute COVID-19 sequelae.

Post-acute sequelae of COVID-19 (PASC) represent an emerging global crisis. However, quantifiable risk factors for PASC and their biological associations are poorly resolved. We executed a deep multi-omic, longitudinal investigation of 309 COVID-19 patients from initial diagnosis to convalescence (2-3 months later), integrated with clinical data and patient-reported symptoms. We resolved four PASC-anticipating risk factors at the time of initial COVID-19 diagnosis: type 2 diabetes, SARS-CoV-2 RNAemia, Epstein-Barr virus viremia, and specific auto-antibodies. In patients with gastrointestinal PASC, SARS-CoV-2-specific and CMV-specific CD8+ T cells exhibited unique dynamics during recovery from COVID-19. Analysis of symptom-associated immunological signatures revealed coordinated immunity polarization into four endotypes, exhibiting divergent acute severity and PASC. We find that immunological associations between PASC factors diminish over time, leading to distinct convalescent immune states. Detectability of most PASC factors at COVID-19 diagnosis emphasizes the importance of early disease measurements for understanding emergent chronic conditions and suggests PASC treatment strategies.

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

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.

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

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.

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

Over the past eight hundred thousand years, glacial-interglacial cycles oscillated with a period of one hundred thousand years ('100k world'1). 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 world2-6. Between about 2.8 and 1.2 million years ago, glacial cycles were smaller in magnitude and shorter in duration ('40k world'7). 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 world8-10, 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 δDice, 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.

Applying single-cell highly multiplexed secretome proteomics to characterize immunotherapeutic products and predict clinical responses.

Genetically and phenotypically identical immune cell populations can be highly heterogenous in terms of their immune functions and protein secretion profiles. The microfluidic chip-based single-cell highly multiplexed secretome proteomics enables characterization of cellular heterogeneity of immune responses at different cellular and molecular layers. Increasing evidence has demonstrated that polyfunctional T cells that simultaneously produce 2+ proteins per cell at the single-cell level are key effector cells that contribute to the development of potent and durable cellular immunity against pathogens and cancers. The functional proteomic technology offers a wide spectrum of cellular function assessment and can uniquely define highly polyfunctional cell subsets with cytokine signatures from live individual cells. This high-dimensional single-cell analysis provides deep dissection into functional heterogeneity and helps identify predictive biomarkers and potential correlates that are crucial for immunotherapeutic product design optimization and personalized immunotherapy development to achieve better clinical outcomes.

Experiences of mothers and significant others in accessing comprehensive healthcare in the first 1000 days of life post-conception during COVID-19 in rural Uganda.

BACKGROUND: COVID-19 presented an unprecedented global public health challenge because of its rapid and relentless spread, and many countries instituted lockdowns to prevent the spread of infection. Although this strategy may have been appropriate to reduce infection, it presented unintended difficulties in rural Uganda, especially in maternal and born newborn care. For example, some services were suspended, meaning the nearest health facility was at a considerable distance. This study explored the experiences of mothers and their significant others of comprehensive care in the first 1000 days of life post-conception during the COVID-19 pandemic in Bunghokho-Mutoto sub-county, Mbale District, Uganda. METHODS: A qualitative exploratory descriptive design was used with data collected in semi-structured interviews. Mothers (pregnant or with a child under 2 years) and their significant others were purposively recruited for this study. The sample size (N = 14) was determined by data saturation. DATA: were analysed using thematic analysis. RESULTS: One theme emerged "Increasing barriers to healthcare", which encompassed six sub-themes: accessing healthcare, distressing situations, living in fear, making forced choices, navigating the gatekeepers, and 'coping with increased poverty. CONCLUSION: This study found that the COVID-19 pandemic increased barriers to accessing healthcare services in the region. Participants' narratives emphasised the lack of access to expert care and the shortage of skilled health workers, especially midwives.

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

Antibody-mediated deposition of complement membrane attack complexes (MACs) on IFN-γ-primed human endothelial cells (ECs) triggers autocrine/paracrine IL-1ß-mediated EC activation and IL-15 transpresentation to alloreactive effector memory T cells (TEM ), 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 TEM , assessed as secreted proteins. IFN-γ and TNF-α remain the predominant cytokines made by alloreactive TEM , but a few CD4+ TEM also made IL-4 while more CD8+ TEM 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.

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

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-2Rgcnull (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-2Rgcnull (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 ß-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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

Evidence for phospholipid self-organisation in concentrated ammonia-water environments.

Titan, the largest moon of Saturn is thought to have the potential to support primordial life. The surface of Titan contains bodies of liquid hydrocarbons, and modelling suggests that an ammonia-water ocean resides deep beneath the surface, both of which have been speculated to support primordial chemistry. Here we present the first evidence that both preformed and self-organised phospholipid vesicles remain stable and can maintain concentration gradients in ammonia-water environments; a fundamental requirement for primordial chemistry and biology to originate. We further reveal the remarkable stability of a diether phospholipid, such as those found in extremophilic bacteria, under these conditions and demonstrate that electron microscopy and tomography are useful tools to investigate macromolecular structure under diverse physico-chemical environments.

Single-Cell CD4 and CD8 T-Cell Secretome Profiling Reveals Temporal and Niche Differences in Acute Myeloid Leukemia Following Immune Checkpoint Blockade Therapy.

Acute myeloid leukemia (AML) is a heterogeneous malignancy of the blood primarily treated with intensive chemotherapy. The allogeneic T-cell antileukemic activity via donor lymphocyte infusions and stem cell transplantation suggests a potential role for checkpoint blockade therapy in AML. While clinical trials employing these treatments have fallen short of expected results, a deeper exploration into the functional states of T cells in AML could bridge this knowledge gap. In this study, we analyzed the polyfunctional activity of T cells in a cohort of patients with relapsed/refractory (RelRef) AML treated on the clinical trial (ClinicalTrials.gov identifier: NCT02397720) of combination therapy using azacitidine and nivolumab (Aza/Nivo). We utilized the single-cell polyfunctional multiplexed immune assay IsoPlexis to evaluate the CD4 and CD8 T cells in peripheral blood and bone marrow samples collected before and after immunotherapy. This revealed at a pseudobulk level that the CD4 T cells exhibited higher functional activity post-immunotherapy (post-IO), suggesting that CD4-directed therapies may play a role in RelRef AML. Additional single-cell analysis revealed significant differences in baseline polyfunctionality in bone marrows of responders as compared with nonresponders for both CD4 and CD8 T cells. Overall, this study highlights the impact of polyfunctional assessment in understanding CD4 and CD8 dynamics in contexts of therapy in AML. SIGNIFICANCE: We found T-cell polyfunctionality differs between local and systemic microenvironments. Enhanced variability in proteomic profiles of bone marrow CD4 T cells post-IO suggests their pivotal role in AML treatment response. Single-cell analysis identified novel CD4 and CD8 T-cell functional groups linked to immunotherapy response within the bone marrow.

Laparoscopic Repair of Bochdalek Hernia: A Rare Presentation of Abdominal Pain in the Elderly.

Bochdalek hernias (BHs) are rare, and the presentation, diagnosis, and management of them can be complex. We present a 70-year-old man presenting with left flank pain who underwent a successful laparoscopic repair of BH with mesh placement.

A Phase I/II Trial of HER2 Vaccine-Primed Autologous T-Cell Infusions in Patients with Treatment Refractory HER2-Overexpressing Breast Cancer.

PURPOSE: High levels of type I T cells are needed for tumor eradication. We evaluated whether the HER2-specific vaccine-primed T cells are readily expanded ex vivo to achieve levels needed for therapeutic infusion. PATIENTS AND METHODS: Phase I/II nonrandomized trial of escalating doses of ex vivo-expanded HER2-specific T cells after in vivo priming with a multiple peptide-based HER2 intracellular domain (ICD) vaccine. Vaccines were given weekly for a total of three immunizations. Two weeks after the third vaccine, patients underwent leukapheresis for T-cell expansion, then received three escalating cell doses over 7- to 10-day intervals. Booster vaccines were administered after the T-cell infusions. The primary objective was safety. The secondary objectives included extent and persistence of HER2-specific T cells, development of epitope spreading, and clinical response. Patients received a CT scan prior to enrollment and 1 month after the last T-cell infusion. RESULTS: Nineteen patients received T-cell infusions. Treatment was well tolerated. One month after the last T-cell infusion, 82% of patients had significantly augmented T cells to at least one of the immunizing epitopes and 81% of patients demonstrated enhanced intramolecular epitope spreading compared with baseline (P < 0.05). There were no complete responses, one partial response (6%), and eight patients with stable disease (47%), for a disease control rate of 53%. The median survival for those with progressive disease was 20.5 months and for responders (PR+SD) was 45.0 months. CONCLUSIONS: Adoptive transfer of HER2 vaccine-primed T cells was feasible, was associated with minimal toxicity, and resulted in an increased overall survival in responding patients. See related commentary by Crosby et al., p. 3256.

Camel nanobody-based B7-H3 CAR-T cells show high efficacy against large solid tumours.

Rational design of chimeric antigen receptor T (CAR-T) cells based on the recognition of antigenic epitopes capable of evoking the most potent CAR activation is an important objective in optimizing immune therapy. In solid tumors, the B7-H3 transmembrane protein is an emerging target that harbours two distinct epitope motifs, IgC and IgV, in its ectodomain. Here, we generate dromedary camel nanobodies targeting B7-H3 and demonstrate that CAR-T cells, based on the nanobodies recognizing the IgC but not IgV domain, had potent antitumour activity against large tumors in female mice. These CAR-T cells are characterized by highly activated T cell signaling and significant tumor infiltration. Single-cell transcriptome RNA sequencing coupled with functional T-cell proteomics analysis uncovers the top-upregulated genes that might be critical for the persistence of polyfunctional CAR-T cells in mice. Our results highlight the importance of the specific target antigen epitope in governing optimal CAR-T activity and provide a nanobody-based B7-H3 CAR-T product for use in solid tumor therapy.

The IgG4 hinge with CD28 transmembrane domain improves VHH-based CAR T cells targeting a membrane-distal epitope of GPC1 in pancreatic cancer.

Heterogeneous antigen expression is a key barrier influencing the activity of chimeric antigen receptor (CAR) T cells in solid tumors. Here, we develop CAR T cells targeting glypican-1 (GPC1), an oncofetal antigen expressed in pancreatic cancer. We report the generation of dromedary camel VHH nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We find that a structurally rigid IgG4H and CD28TM domain brings the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells reveals specific genes (e.g., HMGB1) associated with high T-cell polyfunctionality. This study demonstrates the potential of VHH-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes.