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The ability for cells to localize and activate peripheral membrane binding proteins is critical for signal transduction. Ubiquitously important in these signaling processes are phosphatidylinositol phosphate (PIP) lipids, which are dynamically phosphorylated by PIP lipid kinases on intracellular membranes. Functioning primarily at the plasma membrane, phosphatidylinositol-4-phosphate 5-kinases (PIP5K) catalyzes the phosphorylation of PI(4)P to generate most of the PI(4,5)P2 lipids found in eukaryotic plasma membrane. Recently, we determined that PIP5K displays a positive feedback loop based on membrane-mediated dimerization and cooperative binding to its product, PI(4,5)P2. Here, we examine how two motifs contribute to PI(4,5)P2 recognition to control membrane association and catalysis of PIP5K. Using a combination of single molecule TIRF microscopy and kinetic analysis of PI(4)P lipid phosphorylation, we map the sequence of steps that allow PIP5K to cooperatively engage PI(4,5)P2. We find that the specificity loop regulates the rate of PIP5K membrane association and helps orient the kinase to more effectively bind PI(4,5)P2 lipids. After correctly orienting on the membrane, PIP5K transitions to binding PI(4,5)P2 lipids near the active site through a motif previously referred to as the substrate or PIP binding motif (PIPBM). The PIPBM has broad specificity for anionic lipids and serves a role in regulating membrane association in vitro and in vivo. Overall, our data supports a two-step membrane binding model where the specificity loop and PIPBM act in concert to help PIP5K orient and productively engage anionic lipids to drive the positive feedback during PI(4,5)P2 production.
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An influenza vaccine approach that overcomes the problem of viral sequence diversity and provides long-lived heterosubtypic protection is urgently needed to protect against pandemic influenza viruses. Here, to determine if lung-resident effector memory T cells induced by cytomegalovirus (CMV)-vectored vaccines expressing conserved internal influenza antigens could protect against lethal influenza challenge, we immunize Mauritian cynomolgus macaques (MCM) with cynomolgus CMV (CyCMV) vaccines expressing H1N1 1918 influenza M1, NP, and PB1 antigens (CyCMV/Flu), and challenge with heterologous, aerosolized avian H5N1 influenza. All six unvaccinated MCM died by seven days post infection with acute respiratory distress, while 54.5% (6/11) CyCMV/Flu-vaccinated MCM survived. Survival correlates with the magnitude of lung-resident influenza-specific CD4 + T cells prior to challenge. These data demonstrate that CD4 + T cells targeting conserved internal influenza proteins can protect against highly pathogenic heterologous influenza challenge and support further exploration of effector memory T cell-based vaccines for universal influenza vaccine development.
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Linfócitos T CD4-Positivos , Citomegalovirus , Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza , Macaca fascicularis , Animais , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Citomegalovirus/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Virus da Influenza A Subtipo H5N1/imunologia , Pulmão/imunologia , Pulmão/virologia , Pulmão/patologia , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Masculino , Feminino , Células T de Memória/imunologia , Memória Imunológica/imunologia , VacinaçãoRESUMO
Methods for describing and reporting the clinical and histologic characteristics of cutaneous tissue samples from patients with hidradenitis suppurativa (HS) are not currently standardized, limiting clinicians' and scientists' ability to uniformly record, report, and communicate about the characteristics of tissue used in translational experiments. A recently published consensus statement outlined morphological definitions of typical HS lesions, but no consensus has been reached regarding clinical characterization and examination of HS tissue samples. In this study, we aimed to establish a protocol for reporting histopathologic and clinical characteristics of HS tissue specimens. This study was conducted from May 2023 to August 2023. Experts in clinical care, dermatopathology, and translational research were recruited, and a modified Delphi technique was used to develop a protocol for histologic reporting and clinical characterization of submitted tissue specimens from patients with HS. A total of 27 experts participated (14 dermatologists, 3 fellowship-trained dermatopathologists, 3 plastic surgeons, 3 general surgeons, and 4 research scientists) in creating and reviewing protocols for the clinical and histopathological examination of HS tissue specimens. The protocols were formatted as a synoptic report and will help to consistently classify specimens in biobanks on the basis of histologic features and more accurately report and select samples used in translational research projects.
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The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E-restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E-restricted CD8+ T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E-restricted CD8+ T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E+ prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8+ T cell-based immunotherapies.
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Antígenos de Neoplasias , Linfócitos T CD8-Positivos , Antígenos HLA-E , Animais , Humanos , Masculino , Fosfatase Ácida , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Citomegalovirus/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Macaca mulatta , MesotelinaRESUMO
The γ-aminobutyric acid (GABA) type A receptor (GABAAR), a GABA activated pentameric chloride channel, mediates fast inhibitory neurotransmission in the brain. The lipid environment is critical for GABAAR function. How lipids regulate the channel in the cell membrane is not fully understood. Here we employed super resolution imaging of lipids to demonstrate that the agonist GABA induces a rapid and reversible membrane translocation of GABAAR to phosphatidylinositol 4,5-bisphosphate (PIP2) clusters in mouse primary cortical neurons. This translocation relies on nanoscopic separation of PIP2 clusters and lipid rafts (cholesterol-dependent ganglioside clusters). In a resting state, the GABAAR associates with lipid rafts and this colocalization is enhanced by uptake of astrocytic secretions. These astrocytic secretions enhance endocytosis and delay desensitization. Our findings suggest intercellular signaling from astrocytes regulates GABAAR location based on lipid uptake in neurons. The findings have implications for treating mood disorders associated with altered neural excitability.
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Phosphoinositide 3-kinase (PI3K) beta (PI3Kß) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kß prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kß localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kß when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kß membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kß prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GßGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kß to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GßGγ or pY/Rac1(GTP), PI3Kß activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kß is synergistically activated by pY/GßGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.
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Classe I de Fosfatidilinositol 3-Quinases , Proteínas rac1 de Ligação ao GTP , Proteínas rho de Ligação ao GTP , Humanos , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Microscopia de Fluorescência , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/química , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/química , Transdução de Sinais , Classe I de Fosfatidilinositol 3-Quinases/química , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
PURPOSE: Resection of sacral neoplasms such as chordoma and chondrosarcoma with subsequent reconstruction of large soft tissue defects is a complex multidisciplinary process. Radiotherapy and prior abdominal surgery play a role in reconstructive planning; however, there is no consensus on how to maximize outcomes. In this study, we present our institution's experience with the reconstructive surgical management of this unique patient population. METHODS: We conducted a retrospective review of patients who underwent reconstruction after resection of primary or recurrent pelvic chordoma or chondrosarcoma between 2002 and 2019. Surgical details, hospital stay, and postoperative outcomes were assessed. Patients were divided into 3 groups for comparison based on reconstruction technique: gluteal-based flaps, vertical rectus abdominus myocutaneous (VRAM) flaps, and locoregional fasciocutaneous flaps. RESULTS: Twenty-eight patients (17 males, 11 females), with mean age of 62 years (range, 34-86 years), were reviewed. Twenty-two patients (78.6%) received gluteal-based flaps, 3 patients (10.7%) received VRAM flaps, and 3 patients (10.7%) were reconstructed with locoregional fasciocutaneous flaps. Patients in the VRAM group were significantly more likely to have undergone total sacrectomy (P < 0.01) in a 2-stage operation (P < 0.01) compared with patients in the other 2 groups. Patients in the VRAM group also had a significantly greater average number of reoperations (2 ± 3.5, P = 0.04) and length of stay (29.7 ± 20.4 days, P = 0.01) compared with the 2 other groups. The overall minor and major wound complication rates were 17.9% and 42.9%, respectively, with 17.9% of patients experiencing at least 1 infection or seroma. There was no association between prior abdominal surgery, surgical stages, or radiation therapy and an increased risk of wound complications. CONCLUSIONS: Vertical rectus abdominus myocutaneous flaps are a more suitable option for patients with larger defects after total sacrectomy via 2-staged anteroposterior resections, whereas gluteal myocutaneous flaps are effective options for posterior-only resections. For patients with small- to moderate-sized defects, local fasciocutaneous flaps are a less invasive and effective option. Paraspinous flaps may be used in combination with other techniques to provide additional bulk and coverage for especially long postresection wounds. Furthermore, mesh is a useful adjunct for any reconstruction aimed at protecting against intra-abdominal complications.
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Cordoma , Procedimentos de Cirurgia Plástica , Sacro , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Estudos Retrospectivos , Procedimentos de Cirurgia Plástica/métodos , Idoso , Adulto , Idoso de 80 Anos ou mais , Cordoma/cirurgia , Sacro/cirurgia , Condrossarcoma/cirurgia , Retalhos Cirúrgicos , São Francisco , Neoplasias da Coluna Vertebral/cirurgiaRESUMO
BACKGROUND: Incisional negative pressure wound therapy (iNPWT) is an adjunctive treatment that uses constant negative pressure suction to facilitate healing. The utility of this treatment modality on vascular operations for critical limb-threatening ischemia (CLTI) has yet to be elucidated. This study compares the incidence of postoperative wound complications between the Prevena Incision Management System, a type of iNPWT, and standard wound dressings for vascular patients who also underwent plastic surgery closure of groin incisions for CLTI. METHOD: We performed a retrospective cohort study of 40 patients with CLTI who underwent 53 open vascular surgeries with subsequent sartorius muscle flap closure. Patient demographics, intraoperative details, and wound complications were measured from 2015 to 2018 at the University of California San Francisco. Two cohorts were generated based on the modality of postoperative wound management and compared on wound healing outcomes. RESULTS: Of the 53 groin incisions, 29 were managed with standard dressings, and 24 received iNPWT. Patient demographics, comorbidities, and operative characteristics were similar between the 2 groups. Patients who received iNPWT had a significantly lower rate of infection (8.33% vs 31.0%, P = 0.04) and dehiscence (0% vs 41.3%, P < 0.01). Furthermore, the iNPWT group had a significantly lower rate of reoperation (0% vs 17.2%, P = 0.03) for wound complications within 30 days compared with the control group and a moderately reduced rate of readmission (4.17% vs 20.7%, P = 0.08). CONCLUSIONS: Rates of infection, reoperation, and dehiscence were significantly reduced in patients whose groin incisions were managed with iNPWT compared with standard wound care. Readmission rates were also decreased, but this difference was not statistically significant. Our results suggest that implementing iNPWT for the management of groin incisions, particularly in patients undergoing vascular operations for CLTI, may significantly improve clinical outcomes.
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Virilha , Isquemia , Tratamento de Ferimentos com Pressão Negativa , Cicatrização , Humanos , Tratamento de Ferimentos com Pressão Negativa/métodos , Masculino , Estudos Retrospectivos , Feminino , Virilha/cirurgia , Isquemia/cirurgia , Isquemia/etiologia , Idoso , Pessoa de Meia-Idade , Procedimentos Cirúrgicos Vasculares/métodos , Estudos de Coortes , Complicações Pós-Operatórias/epidemiologiaRESUMO
In September 2021, 14 smallmouth bass (SMB; Micropterus dolomieu) with skin lesions were collected from Green Bay waters of Lake Michigan and submitted for diagnostic evaluation. All the skin samples tested positive for largemouth bass virus (LMBV) by conventional PCR. The complete genome of the LMBV (99,328 bp) isolated from a homogenized skin sample was determined using an Illumina MiSeq sequencer. A maximum likelihood (ML) phylogenetic analysis based on the 21 core iridovirus genes supported the LMBV isolated from SMB (LMBV-WVL21117) as a member of the species Santee-Cooper ranavirus. Pairwise nucleotide comparison of the major capsid protein (MCP) gene showed that LMBV-WVL21117 is identical to other LMBV reported from the United States and nearly identical to doctor fish virus and guppy virus 6 (99.2%) from Southeast Asia, as well as LMBV isolates from China and Thailand (99.1%). In addition, ML phylogenetic analysis based on the MCP gene suggests three genotypes of LMBV separated by region: genotype one from the United States, genotype two from Southeast Asia, and genotype three from China and Thailand. Additional research is needed to understand the prevalence and genetic diversity of LMBV strains circulating in wild and managed fish populations from different regions.
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Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Genoma Viral , Filogenia , Ranavirus , Animais , Ranavirus/genética , Ranavirus/isolamento & purificação , Ranavirus/classificação , Bass/virologia , Infecções por Vírus de DNA/virologia , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/virologia , Proteínas do Capsídeo/genética , Genótipo , Lagos/virologiaRESUMO
PURPOSE: The benefits of paraspinous flaps in adult complex spine surgery patients are established in the literature; however, their use in pediatric patients has not been well described. This study compares clinical outcomes with and without paraspinous muscle flap closure in pediatric patients who have undergone spine surgery. METHODS: We conducted a retrospective review of all pediatric spine surgeries at the University of California, San Francisco from 2011 to 2022. Patients were divided into 2 cohorts based on whether the plastic surgery service closed or did not close the wound with paraspinous muscle flaps. We matched patients by age, American Society of Anesthesiology classification, prior spinal surgical history, and diagnosis. Surgical outcomes were compared between the 2 cohorts. RESULTS: We identified 226 pediatric patients who underwent at least one spinal surgery, 14 of whom received paraspinous flap closure by plastic surgery. They were matched in a 1:4 ratio with controls (n = 56) that did not have plastic surgery closure. The most common indication for plastic surgery involvement was perceived complexity of disease by the spine surgeon with concern for inadequate healthy tissue coverage (78.6%), followed by infection (21.4%). Postoperative complications were similar between the two groups. The plastic surgery cohort had a higher rate of patients who were underweight (57.1% vs 14.3%, P < 0.01) and had positive preoperative wound cultures (28.6% vs 8.9%, P = 0.05), as well as a higher rate of postoperative antibiotic usage (78.6 vs 17.9%, P < 0.01). There was no difference in recorded postoperative outcomes. CONCLUSIONS: Spine surgeons requested paraspinous flap closure for patients with more complex disease, preoperative infections, history of chemotherapy, or if they were underweight. Patients with paraspinous flap coverage did not have increased postoperative complications despite their elevated risk profile. Our findings suggest that paraspinous muscle flaps should be considered in high-risk pediatric patients who undergo spine surgery.
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Músculos Paraespinais , Procedimentos de Cirurgia Plástica , Retalhos Cirúrgicos , Humanos , Estudos Retrospectivos , Feminino , Masculino , Criança , Adolescente , Procedimentos de Cirurgia Plástica/métodos , Retalhos Cirúrgicos/transplante , Pré-Escolar , Complicações Pós-Operatórias/epidemiologia , Resultado do Tratamento , Doenças da Coluna Vertebral/cirurgiaRESUMO
Human cytomegalovirus (HCMV) encodes four viral Fc-gamma receptors (vFcγRs) that counteract antibody-mediated activation in vitro , but their role in infection and pathogenesis is unknown. To examine the in vivo function of vFcγRs in animal hosts closely related to humans, we identified and characterized vFcγRs encoded by rhesus CMV (RhCMV). We demonstrate that Rh05, Rh152/151 and Rh173 represent the complete set of RhCMV vFcγRs, each displaying functional similarities to their respective HCMV orthologs with respect to antagonizing host FcγR activation in vitro . When RhCMV-naïve rhesus macaques were infected with vFcγR-deleted RhCMV, peak plasma viremia levels and anti-RhCMV antibody responses were comparable to wildtype infections. However, the duration of plasma viremia was significantly shortened in immunocompetent, but not in CD4+ T cell-depleted animals. Since vFcγRs were not required for superinfection, we conclude that vFcγRs delay control by virus-specific adaptive immune responses, particularly antibodies, during primary infection.
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Background: RhCMV/SIV vaccines protect â¼59% of vaccinated rhesus macaques against repeated limiting-dose intra-rectal exposure with highly pathogenic SIVmac239M, but the exact mechanism responsible for the vaccine efficacy is not known. It is becoming evident that complex interactions exist between gut microbiota and the host immune system. Here we aimed to investigate if the rhesus gut microbiome impacts RhCMV/SIV vaccine-induced protection. Methods: Three groups of 15 rhesus macaques naturally pre-exposed to RhCMV were vaccinated with RhCMV/SIV vaccines. Rectal swabs were collected longitudinally both before SIV challenge (after vaccination) and post challenge and were profiled using 16S rRNA based microbiome analysis. Results: We identified â¼2,400 16S rRNA amplicon sequence variants (ASVs), representing potential bacterial species/strains. Global gut microbial profiles were strongly associated with each of the three vaccination groups, and all animals tended to maintain consistent profiles throughout the pre-challenge phase. Despite vaccination group differences, using newly developed compositional data analysis techniques we identified a common gut microbial signature predictive of vaccine protection outcome across the three vaccination groups. Part of this microbial signature persisted even after SIV challenge. We also observed a strong correlation between this microbial signature and an early signature derived from whole blood transcriptomes in the same animals. Conclusions: Our findings indicate that changes in gut microbiomes are associated with RhCMV/SIV vaccine-induced protection and early host response to vaccination in rhesus macaques.
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Rapid conversion of force into a biological signal enables living cells to respond to mechanical forces in their environment. The force is believed to initially affect the plasma membrane and then alter the behavior of membrane proteins. Phospholipase D2 (PLD2) is a mechanosensitive enzyme that is regulated by a structured membrane-lipid site comprised of cholesterol and saturated ganglioside (GM1). Here we show stretch activation of TWIK-related K+ channel (TREK-1) is mechanically evoked by PLD2 and spatial patterning involving ordered GM1 and 4,5-bisphosphate (PIP2) clusters in mammalian cells. First, mechanical force deforms the ordered lipids, which disrupts the interaction of PLD2 with the GM1 lipids and allows a complex of TREK-1 and PLD2 to associate with PIP2 clusters. The association with PIP2 activates the enzyme, which produces the second messenger phosphatidic acid (PA) that gates the channel. Co-expression of catalytically inactive PLD2 inhibits TREK-1 stretch currents in a biological membrane. Cellular uptake of cholesterol inhibits TREK-1 currents in culture and depletion of cholesterol from astrocytes releases TREK-1 from GM1 lipids in mouse brain. Depletion of the PLD2 ortholog in flies results in hypersensitivity to mechanical force. We conclude PLD2 mechanosensitivity combines with TREK-1 ion permeability to elicit a mechanically evoked response.
"Ouch!": you have just stabbed your little toe on the sharp corner of a coffee table. That painful sensation stems from nerve cells converting information about external forces into electric signals the brain can interpret. Increasingly, new evidence is suggesting that this process may be starting at fat-based structures within the membrane of these cells. The cell membrane is formed of two interconnected, flexible sheets of lipids in which embedded structures or molecules are free to move. This organisation allows the membrane to physically respond to external forces and, in turn, to set in motion chains of molecular events that help fine-tune how cells relay such information to the brain. For instance, an enzyme known as PLD2 is bound to lipid rafts precisely arranged, rigid fatty 'clumps' in the membrane that are partly formed of cholesterol. PLD2 has also been shown to physically interact with and then activate the ion channel TREK-1, a membrane-based protein that helps to prevent nerve cells from relaying pain signals. However, the exact mechanism underpinning these interactions is difficult to study due to the nature and size of the molecules involved. To address this question, Petersen et al. combined a technology called super-resolution imaging with a new approach that allowed them to observe how membrane lipids respond to pressure and fluid shear. The experiments showed that mechanical forces disrupt the careful arrangement of lipid rafts, causing PLD2 and TREK-1 to be released. They can then move through the surrounding membrane where they reach a switch that turns on TREK-1. Further work revealed that the levels of cholesterol available to mouse cells directly influenced how the clumps could form and bind to PLD2, and in turn, dialled up and down the protective signal mediated by TREK-1. Overall, the study by Petersen et al. shows that the membrane of nerve cells can contain cholesterol-based 'fat sensors' that help to detect external forces and participate in pain regulation. By dissecting these processes, it may be possible to better understand and treat conditions such as diabetes and lupus, which are associated with both pain sensitivity and elevated levels of cholesterol in tissues.
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Gangliosídeo G(M1) , Transdução de Sinais , Animais , Camundongos , Sistemas do Segundo Mensageiro , Membrana Celular , Colesterol , MamíferosRESUMO
Hidradenitis suppurativa (HS) is a chronic skin condition affecting approximately 1% of the US population. HS skin lesions are highly inflammatory and characterized by a large immune infiltrate. While B cells and plasma cells comprise a major component of this immune milieu, the biology and the contribution of these cells in HS pathogenesis are unclear. We aimed to investigate the dynamics and microenvironmental interactions of B cells within cutaneous HS lesions. Combining histological analysis, single-cell RNA sequencing, and spatial transcriptomics profiling of HS lesions, we defined the tissue microenvironment relative to B cell activity within this disease. Our findings identified tertiary lymphoid structures (TLSs) within HS lesions and described organized interactions among T cells, B cells, antigen-presenting cells, and skin stroma. We found evidence that B cells within HS TLSs actively underwent maturation, including participation in germinal center reactions and class switch recombination. Moreover, skin stroma and accumulating T cells were primed to support the formation of TLSs and facilitate B cell recruitment during HS. Our data definitively demonstrated the presence of TLSs in lesional HS skin and point to ongoing cutaneous B cell maturation through class switch recombination and affinity maturation during disease progression in this inflamed nonlymphoid tissue.
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Hidradenite Supurativa , Estruturas Linfoides Terciárias , Humanos , Hidradenite Supurativa/patologia , Estruturas Linfoides Terciárias/patologia , Pele/patologia , Linfócitos B/patologia , Linfócitos T/patologiaRESUMO
Various sorption processes affect leaching of per- and polyfluoroalkyl substances (PFAS) such as PFOA and PFOS. The objectives of this study are to (1) compare rate-limited leaching in column and lysimeter experiments, (2) investigate the relevance of sorption to air-water interfaces (AWI), and (3) examine colloid-facilitated transport as a process explaining early experimental breakthrough. A continuum model (CM) with two-domain sorption is used to simulate equilibrium and rate-limited sorption. A random walk particle tracking (PT) model was developed and applied to analyze complex leaching characteristics. Results show that sorption parameters derived from column experiments underestimate long-term PFOA leaching in lysimeter experiments due to early depletion, suggesting that transformation of precursors contributes to the observed long-term leaching in the lysimeters (approximately 0.003 µg/kg/d PFOA). Both models demonstrate that sorption to AWI is the dominant retention mechanism for PFOS in lysimeter experiments, with retardation due to AWI being 3 (CM) to 3.7 (PT) times higher than retardation due to solid phase sorption. Notably, despite a simplified conception of AWI sorption, the PT results are closer to the observations. The PT simulations demonstrate possible colloid-facilitated transport at early time; however, results using substance-specific varying transport parameters align better with the observations, which should be equal if colloid-facilitated transport without additional kinetics is the sole mechanism affecting early breakthrough. Possibly, rate-limited sorption to AWI is relevant during the early stages of the lysimeter experiment. Our findings demonstrate that rate-limited sorption is less relevant for long-term leaching under field conditions compared to transformation of precursors and that sorption to AWI can be the dominant retention mechanism on contaminated sites. Moreover, they highlight the potential of random walk particle tracking as a practical alternative to continuum models for estimating the relative contributions of various retention mechanisms.
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Fluorocarbonos , Água , Coloides , CinéticaRESUMO
Congenital cytomegalovirus (cCMV) is the leading infectious cause of neurologic defects in newborns with particularly severe sequelae in the setting of primary CMV infection in the first trimester of pregnancy. The majority of cCMV cases worldwide occur after non-primary infection in CMV-seropositive women; yet the extent to which pre-existing natural CMV-specific immunity protects against CMV reinfection or reactivation during pregnancy remains ill-defined. We previously reported on a novel nonhuman primate model of cCMV in rhesus macaques where 100% placental transmission and 83% fetal loss were seen in CD4+ T lymphocyte-depleted rhesus CMV (RhCMV)-seronegative dams after primary RhCMV infection. To investigate the protective effect of preconception maternal immunity, we performed reinfection studies in CD4+ T lymphocyte-depleted RhCMV-seropositive dams inoculated in late first / early second trimester gestation with RhCMV strains 180.92 (n = 2), or RhCMV UCD52 and FL-RhCMVΔRh13.1/SIVgag, a wild-type-like RhCMV clone with SIVgag inserted as an immunological marker, administered separately (n = 3). An early transient increase in circulating monocytes followed by boosting of the pre-existing RhCMV-specific CD8+ T lymphocyte and antibody response was observed in the reinfected dams but not in control CD4+ T lymphocyte-depleted dams. Emergence of SIV Gag-specific CD8+ T lymphocyte responses in macaques inoculated with the FL-RhCMVΔRh13.1/SIVgag virus confirmed reinfection. Placental transmission was detected in only one of five reinfected dams and there were no adverse fetal sequelae. Viral whole genome, short-read, deep sequencing analysis confirmed transmission of both reinfection RhCMV strains across the placenta with ~30% corresponding to FL-RhCMVΔRh13.1/SIVgag and ~70% to RhCMV UCD52, consistent with the mixed human CMV infections reported in infants with cCMV. Our data showing reduced placental transmission and absence of fetal loss after non-primary as opposed to primary infection in CD4+ T lymphocyte-depleted dams indicates that preconception maternal CMV-specific CD8+ T lymphocyte and/or humoral immunity can protect against cCMV infection.
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Infecções por Citomegalovirus , Citomegalovirus , Recém-Nascido , Animais , Feminino , Gravidez , Humanos , Citomegalovirus/genética , Macaca mulatta , Reinfecção , Placenta , Imunidade InataRESUMO
The plasma membrane is a well-organized structure of lipids and proteins, segmented into lipid compartments under 200 nm in size. This specific spatial patterning is crucial for the function of proteins and necessitates super-resolution imaging for its elucidation. Here, we establish that the genetically encoded enhanced green fluorescent protein (EGFP), when combined with direct optical reconstruction microscopy (dSTORM), tracks shear- and cholesterol-induced nanoscopic patterning of potassium channels overexpressed in HEK293T cells. Leveraging EGFP in dSTORM (EGFP-STORM), our findings indicate that cholesterol directs the C-terminus of TWIK-related potassium channel (TREK-1) to ceramide-enriched lipid ganglioside (GM1) clusters. In the absence of the C-terminus, the channel associates with phosphatidylinositol 4,5-bisphosphate (PIP2) cluster. Similarly, cholesterol derived from astrocytes repositions EGFP-tagged inward-rectifying potassium (Kir) channels into GM1 clusters. Without cholesterol, the channel aligns with PIP2 lipids. We deduce that cholesterol's interaction with Kir sequesters the channel, separating it from its activating lipid PIP2. Fundamentally, a genetically encoded EGFP tag should make any protein amenable to dSTORM analysis.
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While microscopy-based cellular assays, including microfluidics, have significantly advanced over the last several decades, there has not been concurrent development of widely-accessible techniques to analyze time-dependent microscopy data incorporating phenomena such as fluid flow and dynamic cell adhesion. As such, experimentalists typically rely on error-prone and time-consuming manual analysis, resulting in lost resolution and missed opportunities for innovative metrics. We present a user-adaptable toolkit packaged into the open-source, standalone Interactive Cellular assay Labeled Observation and Tracking Software (iCLOTS). We benchmark cell adhesion, single-cell tracking, velocity profile, and multiscale microfluidic-centric applications with blood samples, the prototypical biofluid specimen. Moreover, machine learning algorithms characterize previously imperceptible data groupings from numerical outputs. Free to download/use, iCLOTS addresses a need for a field stymied by a lack of analytical tools for innovative, physiologically-relevant assays of any design, democratizing use of well-validated algorithms for all end-user biomedical researchers who would benefit from advanced computational methods.
Assuntos
Inteligência Artificial , Microfluídica , Microscopia , Software , Células SanguíneasRESUMO
The lipid molecule phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] controls all aspects of plasma membrane (PM) function in animal cells, from its selective permeability to the attachment of the cytoskeleton. Although disruption of PI(4,5)P2 is associated with a wide range of diseases, it remains unclear how cells sense and maintain PI(4,5)P2 levels to support various cell functions. Here, we show that the PIP4K family of enzymes, which synthesize PI(4,5)P2 via a minor pathway, also function as sensors of tonic PI(4,5)P2 levels. PIP4Ks are recruited to the PM by elevated PI(4,5)P2 levels, where they inhibit the major PI(4,5)P2-synthesizing PIP5Ks. Perturbation of this simple homeostatic mechanism reveals differential sensitivity of PI(4,5)P2-dependent signaling to elevated PI(4,5)P2 levels. These findings reveal that a subset of PI(4,5)P2-driven functions might drive disease associated with disrupted PI(4,5)P2 homeostasis.
Assuntos
Fosfatidilinositol 4,5-Difosfato , Transdução de Sinais , Animais , Fosfatidilinositol 4,5-Difosfato/metabolismo , Transdução de Sinais/fisiologia , Membrana Celular/metabolismo , Fosfatidilinositóis/metabolismo , HomeostaseRESUMO
Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neonatal neurological impairment but essential virological determinants of transplacental CMV transmission remain unclear. The pentameric complex (PC), composed of five subunits, glycoproteins H (gH), gL, UL128, UL130, and UL131A, is essential for efficient entry into non-fibroblast cells in vitro . Based on this role in cell tropism, the PC is considered a possible target for CMV vaccines and immunotherapies to prevent cCMV. To determine the role of the PC in transplacental CMV transmission in a non-human primate model of cCMV, we constructed a PC-deficient rhesus CMV (RhCMV) by deleting the homologues of the HCMV PC subunits UL128 and UL130 and compared congenital transmission to PC-intact RhCMV in CD4+ T cell-depleted or immunocompetent RhCMV-seronegative, pregnant rhesus macaques (RM). Surprisingly, we found that the transplacental transmission rate was similar for PC-intact and PC-deleted RhCMV based on viral genomic DNA detection in amniotic fluid. Moreover, PC-deleted and PC-intact RhCMV acute infection led to similar peak maternal plasma viremia. However, there was less viral shedding in maternal urine and saliva and less viral dissemination in fetal tissues in the PC-deleted group. As expected, dams inoculated with PC-deleted RhCMV demonstrated lower plasma IgG binding to PC-intact RhCMV virions and soluble PC, as well as reduced neutralization of PC-dependent entry of the PC-intact RhCMV isolate UCD52 into epithelial cells. In contrast, binding to gH expressed on the cell surface and neutralization of entry into fibroblasts by the PC-intact RhCMV was higher for dams infected with PC-deleted RhCMV compared to those infected with PC-intact RhCMV. Our data demonstrates that the PC is dispensable for transplacental CMV infection in our non-human primate model. One Sentence Summary: Congenital CMV transmission frequency in seronegative rhesus macaques is not affected by the deletion of the viral pentameric complex.