The rockefeller university

Purpose: All uveal melanoma and a fraction of other melanoma subtypes are driven by activation of the G-protein alpha-q (G alpha(q)) pathway. Targeting these melanomas has proven difficult despite advances in the molecular understanding of key driver signaling pathways in the disease pathogenesis. Inhibitors of G alpha(q) have shown promising preclinical results, but their therapeutic activity in distinct G alpha(q) mutational contexts and in vivo have remained elusive. Experimental Design: We used an isogenic melanocytic cellular system to systematically examine hotspot mutations in GNAQ (e.g., G48V, R183Q, Q209L) and CYSLTR2 (L129Q) found in human uveal melanoma. This cellular system and human uveal melanoma cell lines were used in vitro and in in vivo xenograft studies to assess the efficacy of G alpha(q) inhibition as a single agent and in combination with MEK inhibition. Results: We demonstrate that the G alpha(q) inhibitor YM-254890 inhibited downstream signaling and in vitro growth in all mutants. In vivo, YM-254890 slowed tumor growth but did not cause regression in human uveal melanoma xenografts. Through comprehensive transcriptome analysis, we observed that YM-254890 caused inhibition of the MAPK signaling with evidence of rebound by 24 hours and combination treatment of YM-254890 and a MEK inhibitor led to sustained MAPK inhibition. We further demonstrated that the combination caused synergistic growth inhibition in vitro and tumor shrinkage in vivo. Conclusions: These data suggest that the combination of G alpha(q) and MEK inhibition provides a promising therapeutic strategy and improved therapeutic window of broadly targeting G alpha(q) in uveal melanoma. See related commentary by Neelature Sriramareddy and Smalley, p. 1217

Objectives: Heat-sensory neurons from the dorsal root ganglia (DRG) play a pivotal role in detecting the cutaneous temperature and transmission of external signals to the brain, ensuring the maintenance of thermoregulation. However, whether these thermoreceptor neurons contribute to adaptive thermogenesis remains elusive. It is also unknown whether these neurons play a role in obesity and energy metabolism. Methods: We used genetic ablation of heat-sensing neurons expressing calcitonin gene-related peptide a (CGRPa) to assess whole-body energy expenditure, weight gain, glucose tolerance, and insulin sensitivity in normal chow and high-fat diet-fed mice. Ex vivo lipolysis and transcriptional characterization were combined with adipose tissue-clearing methods to visualize and probe the role of sensory nerves in adipose tissue. Adaptive thermogenesis was explored using infrared imaging of intrascapular brown adipose tissue (iBAT), tail, and core temperature upon various stimuli including diet, external temperature, and the cooling agent icilin. Results: In this report, we show that genetic ablation of heat-sensing CGRPa neurons promotes resistance to weight gain upon high-fat diet (HFD) feeding and increases energy expenditure in mice. Mechanistically, we found that loss of CGRPa-expressing sensory neurons was associated with reduced lipid deposition in adipose tissue, enhanced expression of fatty acid oxidation genes, higher ex vivo lipolysis in primary white adipocytes, and increased mitochondrial respiration from iBAT. Remarkably, mice lacking CGRPa sensory neurons manifested increased tail cutaneous vasoconstriction at room temperature. This exacerbated cold perception was not associated with reduced core temperature, suggesting that heat production and heat conservation mechanisms were engaged. Specific denervation of CGRPa neurons in intrascapular BAT did not contribute to the increased metabolic rate observed upon global sensory denervation. Conclusions: Taken together, these findings highlight an important role of cutaneous thermoreceptors in regulating energy metabolism by triggering counter-regulatory responses involving energy dissipation processes including lipid fuel utilization and cutaneous vasodilation. (c) 2021 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

S-palmitoylation is a reversible posttranslational lipid modification of proteins. It controls protein activity, stability, trafficking and protein-protein interactions. Recent global profiling of immune cells and targeted analysis have identified many S-palmitoylated immunity-associated proteins. Here, we review S-palmitoylated immune receptors and effectors, and their dynamic regulation at cellular membranes to generate specific and balanced immune responses. We also highlight how this understanding can drive therapeutic advances to pharmacologically modulate immune responses.

Glia in the central nervous system engulf neuron fragments to remodel synapses and recycle photoreceptor outer segments. Whether glia passively clear shed neuronal debris or actively prune neuron fragments is unknown. How pruning of single-neuron endings impacts animal behavior is also unclear. Here, we report our discovery of glia-directed neuron pruning in Caenorhabditis elegans. Adult C. elegans AMsh glia engulf sensory endings of the AFD thermosensory neuron by repurposing components of the conserved apoptotic corpse phagocytosis machinery. The phosphatidylserine (PS) flippase TAT-1/ATP8A functions with glial PS-receptor PSR-1/PSR and PAT-2/alpha-integrin to initiate engulfment. This activates glial CED-10/Rac1 GTPase through the ternary GEF complex of CED-2/CrkII, CED-5/DOCK180, CED-12/ELMO. Execution of phagocytosis uses the actin-remodeler WSP-1/nWASp. This process dynamically tracks AFD activity and is regulated by temperature, the AFD sensory input. Importantly, glial CED-10 levels regulate engulfment rates downstream of neuron activity, and engulfment-defective mutants exhibit altered AFD-ending shape and thermosensory behavior. Our findings reveal a molecular pathway underlying glia-dependent engulfment in a peripheral sense-organ and demonstrate that glia actively engulf neuron fragments, with profound consequences on neuron shape and animal sensory behavior.YY

Background Adalimumab, a tumor necrosis factor-alpha inhibitor, is a biologic used for the treatment of moderate-to-severe hidradenitis suppurativa (HS). It is well known that patients may experience loss of efficacy from its use in other conditions, and it is suggested that developing a strategy for therapeutic drug monitoring (TDM) may help secure optimal clinical outcomes. Objectives We sought to determine serum adalimumab concentrations and anti-adalimumab antibody (AAA) status in patients with moderate-to-severe HS. Methods A retrospective case series of 38 patients with suboptimal response to adalimumab 40 mg weekly was conducted at a community dermatology clinic. Adalimumab serum trough levels, AAA status, and inflammatory biomarkers were collected. Blood was drawn on identification of suboptimal response (after a minimum of 12 weeks) and was collected once prior to receiving the next scheduled dose. Kruskal-Wallis and Chi-squared tests were used for data analysis. Results A total of 38 patients had a median adalimumab trough concentration of 8.76 (interquartile range [IQR] 1.3-12.5) mu g/mL. The median duration of adalimumab therapy of all patients was 21 (IQR 12-24) months. AAAs were detected in nine patients (24%), and all had subtherapeutic serum concentrations (< 6 mu g/mL). Patients who were AAA+ had a significantly lower median adalimumab concentration than those who were AAA- (0.02 mu g/mL [range 0.02-0.81] vs. 10.14 [range 0.76-48.00]; p = 0.0006). Conclusion Patients with AAAs had significantly lower serum adalimumab levels. The current study suggests that TDM may identify underlying reasons for suboptimal response and detect patients who may benefit from dose optimization strategies.

Background: A research priority in finding a cure for HIV is to establish methods to accurately locate and quantify where and how HIV persists in people living with HIV (PLWH) receiving suppressive antiretroviral therapy (ART). Infusing copper-64 (Cu-64) radiolabelled broadly neutralising antibodies targeting HIV envelope (Env) with CT scan and positron emission tomography (PET) identified HIV Env in tissues in SIV infected non-human primates . We aimed to determine if a similar approach was effective in people living with HIV (PLWH). Methods: Unmodified 3BNC117 was compared with 3BNC117 bound to the chelator MeCOSar and Cu-64 (Cu-64-3BNC117) in vitro to assess binding and neutralization. In a clinical trial Cu-64-3BNC117 was infused into HIV uninfected (Group 1), HIV infected and viremic (viral load, VL >1000 c/mL; Group 2) and HIV infected aviremic (VL <20 c/mL; Group 3) participants using two dosing strategies: high protein (3mg/kg unlabeled 3BNC117 combined with <5mg Cu-64-3BNC117) and trace (<5mg Cu-64-3BNC117 only). All participants were screened for 3BNC117 sensitivity from virus obtained from viral outgrowth. Magnetic resonance imaging (MRI)/PET and pharmacokinetic assessments (ELISA for serum 3BNC117 concentrations and gamma counting for Cu-64) were performed 1, 24-and 48-hours post dosing. The trial (clincialtrials.gov NCT03063788) primary endpoint was comparison of PET standard uptake values (SUVs) in regions of interest (e.g lymph node groups and gastrointestinal tract). Findings: Comparison of unmodified and modified 3BNC117 in vitro demonstrated no difference in HIV binding or neutralisation. 17 individuals were enrolled of which 12 were dosed including Group 1 (n=4, 2 high protein, 2 trace dose), Group 2 (n=6, 2 high protein, 4 trace) and Group 3 (n=2, trace only). HIV+ participants had a mean CD4 of 574 cells/microL and mean age 43 years. There were no drug related adverse effects and no differences in tissue uptake in regions of interest (e.g lymph node gut, pharynx) between the 3 groups. In the high protein dosing group, serum concentrations of 3BNC117 and gamma counts were highly correlated demonstrating that Cu-64-3BNC117 remained intact in vivo. Interpretation: In PLWH on or off ART, the intervention of infusing Cu-64-3BNC117 and MRI/PET imaging over 48 hours, was unable to detect HIV-1 env expression in vivo. Future studies should investigate alternative radiolabels such as zirconium which have a longer half-life in vivo. Funding: Funded by the Alfred Foundation, The Australian Centre for HIV and Hepatitis Virology Research with additional support from the Division of AIDS, National Institute of Allergy and Infectious Disease, US National Institutes of Health (USAI126611). JHM and SRL are supported by the Australian National Health and Medical Research Council. (C) The Author(s). Published by Elsevier B.V.

The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon antiretroviral therapy (ART)-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The intact proviral DNA assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR) is a lower-throughput assay that uses limiting dilution long-distance PCR amplification followed by quantitative PCR (qPCR) and near-full-length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another, but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long-distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size. IMPORTANCE The intact proviral DNA assay (IPDA) and quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication-competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 antiretroviral therapy (ART) suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.

The germinal centre is a dynamic microenvironment in which B cells that express high-affinity antibody variants produced by somatic hypermutation are selected for clonal expansion by limiting the numbers of T follicular helper cells(1,2). Although much is known about the mechanisms that control the selection of B cells in the germinal centre, far less is understood about the clonal behaviour of the T follicular helper cells that help to regulate this process. Here we report on the dynamic behaviour of T follicular helper cell clones during the germinal centre reaction. We find that, similar to germinal centre B cells, T follicular helper cells undergo antigen-dependent selection throughout the germinal centre reaction that results in differential proliferative expansion and contraction. Increasing the amount of antigen presented in the germinal centre leads to increased division of T follicular helper cells. Competition between T follicular helper cell clones is mediated by the affinity of T cell receptors for peptide-major-histocompatibility-complex ligands. T cells that preferentially expand in the germinal centre show increased expression of genes downstream of the T cell receptor, such as those required for metabolic reprogramming, cell division and cytokine production. These dynamic changes lead to marked remodelling of the functional T follicular helper cell repertoire during the germinal centre reaction.