In vitro models of differing macrophage functions are useful since human monocyte–derived macrophages are short–lived, finite and vary from donor to donor. Published protocols using the promonocytic ...cell line THP-1 have tended to result in cells that closely resemble classically-activated macrophages, differentiated in IFNγ and LPS. However, no protocol, to date, has fully recapitulated polarization of THP-1 to the M(IL-4) or M(IL-10) macrophage phenotypes seen when human monocyte-derived macrophages are exposed to each cytokine. Here we present protocols that can be used to prepare M(IL-4) polarized THP-1 that transcribe CCL17, CCL26, CD200R and MRC1 and M(IL-10) cells which transcribe CD163, C1QA and SEPP1. We show that the inhibitory Fcγ Receptor IIb is preferentially expressed on the surface of M(IL-4) cells, altering the balance of activating to inhibitory Fcγ Receptors.
Adoption of standardized experimental conditions for macrophage polarization will make it easier to compare downstream effector functions of different macrophage polarization states, where the impact of PMA exposure is minimized and rest periods and cytokine exposure have been optimized.
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•THP-1 cells can polarized to produce both classically and alternatively–activated macrophages•THP-1 macrophages transcribe markers consistent with peripheral blood macrophages treated with IFNγ+LPS, IL-4 or IL-10•The ratio of activatory (FcγRI) to inhibitory (FcγRIIb) FcγRs is altered in different macrophage populations.
Human myeloproliferative disorders form a range of clonal haematological malignant diseases, the main members of which are polycythaemia vera, essential thrombocythaemia, and idiopathic ...myelofibrosis. The molecular pathogenesis of these disorders is unknown, but tyrosine kinases have been implicated in several related disorders. We investigated the role of the cytoplasmic tyrosine kinase JAK2 in patients with a myeloproliferative disorder.
We obtained DNA samples from patients with polycythaemia vera, essential thrombocythaemia, or idiopathic myelofibrosis. The coding exons of
JAK2 were bidirectionally sequenced from peripheral-blood granulocytes, T cells, or both. Allele-specific PCR, molecular cytogenetic studies, microsatellite PCR, Affymetrix single nucleotide polymorphism array analyses, and colony assays were undertaken on subgroups of patients.
A single point mutation (Val617Phe) was identified in JAK2 in 71 (97%) of 73 patients with polycythaemia vera, 29 (57%) of 51 with essential thrombocythaemia, and eight (50%) of 16 with idiopathic myelofibrosis. The mutation is acquired, is present in a variable proportion of granulocytes, alters a highly conserved valine present in the negative regulatory JH2 domain, and is predicted to dysregulate kinase activity. It was heterozygous in most patients, homozygous in a subset as a result of mitotic recombination, and arose in a multipotent progenitor capable of giving rise to erythroid and myeloid cells. The mutation was present in all erythropoietin-independent erythroid colonies.
A single acquired mutation of JAK2 was noted in more than half of patients with a myeloproliferative disorder. Its presence in all erythropoietin-independent erythroid colonies demonstrates a link with growth factor hypersensitivity, a key biological feature of these disorders.
Identification of the Val617Phe JAK2 mutation lays the foundation for new approaches to the diagnosis, classification, and treatment of myeloproliferative disorders.
HIV cure efforts are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in patients receiving ...antiretroviral therapy. We combine fluorescent in situ RNA hybridization with detection of HIV protein and flow cytometry, enabling detection of 0.5–1 gag-pol mRNA+/Gag protein+-infected cells per million. In the peripheral blood of untreated persons, active HIV replication correlated with viremia and occurred in CD4 T cells expressing T follicular helper cell markers and inhibitory co-receptors. In virally suppressed subjects, the approach identified latently infected cells capable of producing HIV mRNA and protein after stimulation with PMA/ionomycin and latency-reversing agents (LRAs). While ingenol-induced reactivation mirrored the effector and central/transitional memory CD4 T cell contribution to the pool of integrated HIV DNA, bryostatin-induced reactivation occurred predominantly in cells expressing effector memory markers. This indicates that CD4 T cell differentiation status differentially affects LRA effectiveness.
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•HIV RNA and protein co-expression allows ex vivo characterization of patient CD4 T cells•HIV-infected CD4s show markers of exhaustion and peripheral follicular helper cells•Translation-competent latent reservoir can be detected in most ART-treated patients•PKC agonist Bryostatin preferentially reactivates HIV from effector memory CD4s
Technological limitations hamper characterization of CD4 T cells supporting ongoing HIV infection and quantification of the latent reservoir. Baxter et al. (2016) use simultaneous detection of viral protein and mRNA to quantify and phenotype both the ongoing infection during viremia and the translation-competent inducible reservoir in virally supressed, treated patients.
An understanding of astrophysical feedback is important for constraining models of galaxy formation and for extracting cosmological information from current and future weak lensing surveys. The ...thermal Sunyaev-Zel'dovich effect, quantified via the Compton-y parameter, is a powerful tool for studying feedback, because it directly probes the pressure of the hot, ionized gas residing in dark matter halos. Cross-correlations between galaxies and maps of Compton-y obtained from cosmic microwave background surveys are sensitive to the redshift evolution of the gas pressure, and its dependence on halo mass. In this work, we use galaxies identified in year one data from the Dark Energy Survey and Compton-y maps constructed from Planck observations. We find highly significant (roughly 12σ) detections of galaxy-y cross-correlation in multiple redshift bins. By jointly fitting these measurements as well as measurements of galaxy clustering, we constrain the halo bias-weighted, gas pressure of the Universe as a function of redshift between 0.15≲z≲0.75. We compare these measurements to predictions from hydrodynamical simulations, allowing us to constrain the amount of thermal energy in the halo gas relative to that resulting from gravitational collapse.
Metasomatic reaction zones between mafic and ultramafic rocks exhumed from subduction zones provide a window into mass‐transfer processes at high pressure. However, accurate interpretation of the ...rock record requires distinguishing high‐pressure metasomatic processes from inherited oceanic signatures prior to subduction. We integrated constraints from bulk‐rock geochemical compositions and petrophysical properties, mineral chemistry, and thermodynamic modeling to understand the formation of reaction zones between juxtaposed metagabbro and serpentinite as exemplified by the Voltri Massif (Ligurian Alps, Italy). Distinct zones of variably metasomatized metagabbro are dominated by chlorite, amphibole, clinopyroxene, epidote, rutile, ilmenite, and titanite between serpentinite and eclogitic metagabbro. Whereas the precursor serpentinite and oxide gabbro formed and were likely already in contact in an oceanic setting, the reaction zones formed by diffusional Mg‐metasomatism between the two rocks from prograde to peak, to retrograde conditions in a subduction zone. Metasomatism of mafic rocks by Mg‐rich fluids that previously equilibrated with serpentinite could be widespread along the subduction interface, within the subducted slab, and the mantle wedge. Furthermore, the models predict that talc formation by Si‐metasomatism of serpentinite in subduction zones is limited by pressure‐dependent increase in the silica activity buffered by the serpentine‐talc equilibrium. Elevated activities of aqueous Ca and Al species would also favor the formation of chlorite and garnet. Accordingly, unusual conditions or processes would be required to stabilize abundant talc at high P‐T conditions. Alternatively, a different set of mineral assemblages, such as serpentine‐ or chlorite‐rich rocks, may be controlling the coupling‐decoupling transition of the plate interface.
Key Points
Fluid‐mediated mass transfer between mafic and ultramafic rocks can occur from prograde to peak, to retrograde conditions
The formation of chlorite‐rich assemblages through Mg metasomatism of mafic rocks is prevalent at high P‐T conditions
Talc formation via Si‐metasomatism of serpentinite is more limited in subduction zones than in oceanic plates
Multi-system Inflammatory Syndrome in Children (MIS-C) is a major complication of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection in pediatric patients. Weeks after an often ...mild or asymptomatic initial infection with SARS-CoV-2 children may present with a severe shock-like picture and marked inflammation. Children with MIS-C present with varying degrees of cardiovascular and hyperinflammatory symptoms. Here we perform a comprehensive analysis of the plasma proteome of more than 1400 proteins in children with SARS-CoV-2. We hypothesize that the proteome would reflect heterogeneity in hyperinflammation and vascular injury, and further identify pathogenic mediators of disease. We show that protein signatures demonstrate overlap between MIS-C, and the inflammatory syndromes macrophage activation syndrome (MAS) and thrombotic microangiopathy (TMA). We demonstrate that PLA2G2A is an important marker of MIS-C that associates with TMA. We find that IFNγ responses are dysregulated in MIS-C patients, and that IFNγ levels delineate clinical heterogeneity.
National efforts are underway to prepare the UK National Health Service (NHS) for the COVID-19 pandemic; however, the efficacy of these interventions is unknown. In view of this, a cross-sectional ...survey of front-line healthcare workers (HCWs) at two large acute NHS hospital trusts in England was undertaken to assess their confidence and perceived level of preparedness for the virus. The survey found that there has been moderate success in readying HCWs to manage COVID-19, but that more still needs to be done, particularly in relation to educating HCWs about laboratory diagnostics.
Simple, efficient and well-tolerated delivery of CRISPR genome editing systems into primary cells remains a major challenge. Here we describe an engineered Peptide-Assisted Genome Editing (PAGE) ...CRISPR-Cas system for rapid and robust editing of primary cells with minimal toxicity. The PAGE system requires only a 30-min incubation with a cell-penetrating Cas9 or Cas12a and a cell-penetrating endosomal escape peptide to achieve robust single and multiplex genome editing. Unlike electroporation-based methods, PAGE gene editing has low cellular toxicity and shows no significant transcriptional perturbation. We demonstrate rapid and efficient editing of primary cells, including human and mouse T cells, as well as human hematopoietic progenitor cells, with editing efficiencies upwards of 98%. PAGE provides a broadly generalizable platform for next-generation genome engineering in primary cells.
ABSTRACT Clusters of galaxies are expected to gravitationally lens the cosmic microwave background (CMB) and thereby generate a distinct signal in the CMB on arcminute scales. Measurements of this ...effect can be used to constrain the masses of galaxy clusters with CMB data alone. Here we present a measurement of lensing of the CMB by galaxy clusters using data from the South Pole Telescope (SPT). We develop a maximum likelihood approach to extract the CMB cluster lensing signal and validate the method on mock data. We quantify the effects on our analysis of several potential sources of systematic error and find that they generally act to reduce the best-fit cluster mass. It is estimated that this bias to lower cluster mass is roughly 0.85 in units of the statistical error bar, although this estimate should be viewed as an upper limit. We apply our maximum likelihood technique to 513 clusters selected via their Sunyaev-Zeldovich (SZ) signatures in SPT data, and rule out the null hypothesis of no lensing at 3.1 . The lensing-derived mass estimate for the full cluster sample is consistent with that inferred from the SZ flux: (68% C.L., statistical error only).
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