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The stiffness of the extracellular matrix induces differential tension within integrin-based adhesions, triggering differential mechanoresponses. However, it has been unclear if the stiffness-dependent differential tension is induced solely by myosin activity. Here, we report that in the absence of myosin contractility, 3T3 fibroblasts still transmit stiffness-dependent differential levels of traction. This myosin-independent differential traction is regulated by polymerizing actin assisted by actin nucleators Arp2/3 and formin where formin has a stronger contribution than Arp2/3 to both traction and actin flow. Intriguingly, despite only slight changes in F-actin flow speed observed in cells with the combined inhibition of Arp2/3 and myosin compared to cells with sole myosin inhibition, they show a 4-times reduction in traction than cells with myosin-only inhibition. Our analyses indicate that traditional models based on rigid F-actin are inadequate for capturing such dramatic force reduction with similar actin flow. Instead, incorporating the F-actin network's viscoelastic properties is crucial. Our new model including the F-actin viscoelasticity reveals that Arp2/3 and formin enhance stiffness sensitivity by mechanically reinforcing the F-actin network, thereby facilitating more effective transmission of flow-induced forces. This model is validated by cell stiffness measurement with atomic force microscopy and experimental observation of model-predicted stiffness-dependent actin flow fluctuation.
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Introduction: Nontuberculous mycobacteria (NTM) mediated infections are important to consider in cases with neuroinflammatory presentations. We aimed to characterize cases of NTM with neurological manifestations at the National Institutes of Health (NIH) Clinical Center and review the relevant literature. Materials and methods: Between January 1995 and December 2020, six cases were identified. Records were reviewed for demographic, clinical, and radiological characteristics. A MEDLINE search found previously reported cases. Data were extracted, followed by statistical analysis to compare two groups [cases with slow-growing mycobacteria (SGM) vs. those with rapidly growing mycobacteria (RGM)] and evaluate for predictors of survival. NIH cases were evaluated for clinical and radiological characteristics. Cases from the literature were reviewed to determine the differences between SGM and RGM cases and to identify predictors of survival. Results: Six cases from NIH were identified (age 41 ± 13, 83% male). Five cases were caused by SGM [Mycobacterium avium complex (MAC) n = 4; Mycobacterium haemophilum n = 1] and one due to RGM (Mycobacterium abscessus). Underlying immune disorders were identified only in the SGM cases [genetic (n = 2), HIV (n = 1), sarcoidosis (n = 1), and anti-interferon-gamma antibodies (n = 1)]. All cases were diagnosed using tissue analysis. A literature review found 81 reports on 125 cases (SGM n = 85, RGM n = 38, non-identified n = 2). No immune disorder was reported in 26 cases (21%). Within SGM cases, the most common underlying disease was HIV infection (n = 55, 65%), and seizures and focal lesions were more common. In RGM cases, the most common underlying condition was neurosurgical intervention or implants (55%), and headaches and meningeal signs were common. Tissue-based diagnosis was used more for SGM than RGM (39% vs. 13%, p = 0.04). Survival rates were similar in both groups (48% SGM and 55% in RGM). Factors associated with better survival were a solitary CNS lesion (OR 5.9, p = 0.01) and a diagnosis made by CSF sampling only (OR 9.9, p = 0.04). Discussion: NTM infections cause diverse neurological manifestations, with some distinctions between SGM and RGM infections. Tissue sampling may be necessary to establish the diagnosis, and an effort should be made to identify an underlying immune disorder.
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Integrated preclinical multimodal imaging systems, such as X-ray computed tomography (CT) combined with positron emission tomography (PET) or magnetic resonance imaging (MRI) combined with PET, are widely available and typically provide robustly co-registered volumes. However, separate devices are often needed to combine a standalone MRI with an existing PET-CT or to incorporate additional data from optical tomography or high-resolution X-ray microtomography. This necessitates image co-registration, which involves complex aspects such as multimodal mouse bed design, fiducial marker inclusion, image reconstruction, and software-based image fusion. Fiducial markers often pose problems for in vivo data due to dynamic range issues, limitations on the imaging field of view, difficulties in marker placement, or marker signal loss over time (e.g., from drying or decay). These challenges must be understood and addressed by each research group requiring image co-registration, resulting in repeated efforts, as the relevant details are rarely described in existing publications. This protocol outlines a general workflow that overcomes these issues. Although a differential transformation is initially created using fiducial markers or visual structures, such markers are not required in production scans. The requirements for the volume data and the metadata generated by the reconstruction software are detailed. The discussion covers achieving and verifying requirements separately for each modality. A phantom-based approach is described to generate a differential transformation between the coordinate systems of two imaging modalities. This method showcases how to co-register production scans without fiducial markers. Each step is illustrated using available software, with recommendations for commercially available phantoms. The feasibility of this approach with different combinations of imaging modalities installed at various sites is showcased.
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Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tomografia Computadorizada por Raios X , Animais , Camundongos , Tomografia Computadorizada por Raios X/métodos , Tomografia por Emissão de Pósitrons/métodos , Marcadores Fiduciais , Software , Imageamento por Ressonância Magnética/métodos , Imagens de FantasmasRESUMO
Introduction: Allergic reactions are mediated by human IgE antibodies that bind to and cross-link allergen molecules. The sites on allergens that are recognized by IgE antibodies have been difficult to investigate because of the paucity of IgE antibodies in a human serum. Here, we report the production of unique human IgE monoclonal antibodies to major inhaled allergens and food allergens that can be produced at scale in perpetuity. Materials and methods: The IgE antibodies were derived from peripheral blood mononuclear cells of symptomatic allergic patients, mostly children aged 3-18 years, using hybridoma fusion technology. Total IgE and allergen-specific IgE was measured by ImmunoCAP. Their specificity was confirmed through ELISA and immunoblotting. Allergenic potency measurements were determined by ImmunoCAP inhibition. Biological activity was determined in vitro by comparing ß-hexosaminidase release from a humanized rat basophilic cell line. Results: Human IgE monoclonal antibodies (n = 33) were derived from 17 allergic patients with symptoms of allergic rhinitis, asthma, atopic dermatitis, food allergy, eosinophilic esophagitis, or red meat allergy. The antibodies were specific for five inhaled allergens, nine food allergens, and alpha-gal and had high levels of IgE (53,450-1,702,500â kU/L) with ratios of specific IgE to total IgE ranging from <0.01 to 1.39. Sigmoidal allergen binding curves were obtained through ELISA, with low limits of detection (<1â kU/L). Allergen specificity was confirmed through immunoblotting. Pairs of IgE monoclonal antibodies to Ara h 6 were identified that cross-linked after allergen stimulation and induced release of significant levels of ß-hexosaminidase (35%-80%) from a humanized rat basophilic cell line. Conclusions: Human IgE monoclonal antibodies are unique antibody molecules with potential applications in allergy diagnosis, allergen standardization, and identification of allergenic epitopes for the development of allergy therapeutics. The IgE antibody probes will enable the unequivocal localization and validation of allergenic epitopes.
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[This corrects the article DOI: 10.3389/fimmu.2023.1155613.].
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Complexo AIDS Demência , Fármacos Anti-HIV , Infecções por HIV , Humanos , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Complexo AIDS Demência/diagnóstico por imagem , Complexo AIDS Demência/tratamento farmacológico , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta AtividadeRESUMO
Ulcerative colitis (UC) is a type of inflammatory bowel disease characterized by inflammation, ulcers and irritation of the mucosal lining. Oral drug delivery in UC encounters challenges because of multifaceted barriers. Dexamethasone-loaded galactosylated-PLGA/Eudragit S100/pullulan nanocargoes (Dexa-GP/ES/Pu NCs) have been developed with a dual stimuli-sensitive coating responsive to both colonic pH and microbiota, and an underneath galactosylated-PLGA core (GP). The galactose ligand of the GP preferentially binds to the macrophage galactose type-lectin-C (MGL-2) surface receptor. Therefore, both stimuli and ligand-mediated targeting facilitate nanocargoes to deliver Dexa specifically to the colon with enhanced macrophage uptake. Modified emulsion method coupled with a solvent evaporation coating technique was employed to prepare Dexa-GP/ES/Pu NCs. The nanocargoes were tested using in vitro, ex vivo techniques and dextran sodium sulfate (DSS) induced UC model. Prepared nanocargoes had desired physicochemical properties, drug release, cell uptake and cellular viability. Investigations using a DSS-colitis model showed high localization and mitigation of colitis with downregulation of NF-ĸB and COX-2, and restoration of clinical, histopathological, biochemical indices, antioxidant balance, microbial alterations, FTIR spectra, and epithelial junctions' integrity. Thus, Dexa-GP/ES/Pu NCs found to be biocompatible nanocargoes capable of delivering drugs to the inflamed colon with unique targeting properties for prolonged duration.
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Curcumin (CUR) has potent anticancer activities, and its bioformulations, including biodegradable polymers, are increasingly able to improve CUR's solubility, stability, and delivery to cancer cells. In this study, copolymers comprising poly (L-lactide)-poly (ethylene glycol)-poly (L-lactide) (PLA-PEG-PLA) and poly (ethylene glycol)-poly (L-lactide)-poly (ethylene glycol) (PEG-PLA-PEG) were designed and synthesized to assess and compare their CUR-delivery capacity and inhibitory potency on MCF-7 breast cancer cells. Molecular dynamics simulations and free energy analysis indicated that PLA-PEG-PLA has a higher propensity to interact with the cell membrane and more negative free energy, suggesting it is the better carrier for cell membrane penetration. To characterize the copolymer synthesis, Fourier transform-infrared (FT-IR) and proton nuclear magnetic resonance (1H-NMR) were employed, copolymer size was measured using dynamic light scattering (DLS), and their surface charge was determined by zeta potential analysis. Characterization indicated that the ring-opening polymerization (ROP) reaction was optimal for synthesizing high-quality polymers. Microspheres comprising the copolymers were then synthesized successfully. Of the two formulations, PLA-PEG-PLA experimentally exhibited better results, with an initial burst release of 17.5%, followed by a slow, constant release of the encapsulated drug up to 80%. PLA-PEG-PLA-CUR showed a significant increase in cell death in MCF-7 cancer cells (IC50 = 23.01 ± 0.85 µM) based on the MTT assay. These data were consistent with gene expression studies of Bax, Bcl2, and hTERT, which showed that PLA-PEG-PLA-CUR induced apoptosis more efficiently in these cells. Through the integration of nano-informatics and in vitro approaches, our study determined that PLA-PEG-PLA-CUR is an optimal system for delivering curcumin to inhibit cancer cells.
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Reservoirs of HIV maintained in anatomic compartments during antiretroviral therapy prevent HIV eradication. However, mechanisms driving their persistence and interventions to control them remain elusive. Here we report the presence of an inducible HIV reservoir within antigen-specific CD4+T cells in the central nervous system of a 59-year-old male with progressive multifocal leukoencephalopathy immune reconstitution inflammatory syndrome (PML-IRIS). HIV production during PML-IRIS was suppressed by modulating inflammation with corticosteroids; selection of HIV drug resistance caused subsequent breakthrough viremia. Therefore, inflammation can influence the composition, distribution and induction of HIV reservoirs, warranting it as a key consideration for developing effective HIV remission strategies.
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Infecções por HIV , Síndrome Inflamatória da Reconstituição Imune , Leucoencefalopatia Multifocal Progressiva , Masculino , Humanos , Pessoa de Meia-Idade , Síndrome Inflamatória da Reconstituição Imune/tratamento farmacológico , Síndrome Inflamatória da Reconstituição Imune/etiologia , Leucoencefalopatia Multifocal Progressiva/tratamento farmacológico , Leucoencefalopatia Multifocal Progressiva/etiologia , Encéfalo , Sistema Nervoso CentralRESUMO
Background: Toxoplasmic encephalitis (TE) is a life-threatening complication of people with human immunodeficiency virus (PWH) with severe immunodeficiency, especially those with a CD4+ T-cell count <100 cells/µL. Following a clinical response to anti-Toxoplasma therapy, and immune reconstitution after initiation of combination antiretroviral therapy (ART), anti-Toxoplasma therapy can be discontinued with a low risk of relapse. Methods: To better understand the evolution of magnetic resonance imaging (MRI)-defined TE lesions in PWH receiving ART, we undertook a retrospective study of PWH initially seen at the National Institutes of Health between 2001 and 2012, who had at least 2 serial MRI scans. Lesion size and change over time were calculated and correlated with clinical parameters. Results: Among 24 PWH with TE and serial MRI scans, only 4 had complete clearance of lesions at the last MRI (follow-up, 0.09-5.8 years). Of 10 PWH off all anti-Toxoplasma therapy (median, 3.2 years after TE diagnosis), 6 had persistent MRI enhancement. In contrast, all 5 PWH seen in a pre-ART era study who were followed for >6 months had complete clearance of lesions. TE lesion area at diagnosis was associated with the absolute change in area (P < .0001). Conclusions: Contrast enhancement can persist even when TE has been successfully treated and anti-Toxoplasma therapy has been stopped, highlighting the need to consider diagnostic alternatives in successfully treated patients with immune reconstitution presenting with new neurologic symptoms.
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Background: Human Immunoglobulin E monoclonal antibodies (hIgE mAb) are unique tools for investigating IgE responses. Here, the biological activity of hIgE mAb, derived from immortalized B cells harvested from the blood of allergic donors, targeting three allergens (Der p 2, Fel d 1 and Ara h 2) was investigated. Methods: Three Der p 2-, three Fel d 1- and five Ara h 2-specific hIgE mAb produced by human B cell hybridomas, were combined in pairs and used to passively sensitize humanized rat basophilic leukemia cells and compared with sensitization using serum pools. Sensitized cells were stimulated with corresponding allergens (recombinant or purified), allergen extracts or structural homologs, having 40-88% sequence similarity, and compared for mediator (ß-hexosaminidase) release. Results: One, two and eight pairs of Der p 2-, Fel d 1- and Ara h 2-specific hIgE mAb, respectively, produced significant mediator release (>50%). A minimum hIgE mAb concentration of 15-30 kU/L and a minimum antigen concentration between 0.01-0.1 µg/mL were sufficient to induce a pronounced mediator release. Individual sensitization with one Ara h 2-specific hIgE mAb was able to induce crosslinking independently of a second specific hIgE mAb. Der p 2- and Ara h 2-specific mAb showed a high allergen specificity when compared to homologs. Mediator release from cells sensitized with hIgE mAb was comparable to serum sensitization. Conclusion: The biological activity of hIgE mAb reported here provides the foundation for novel methods of standardization and quality control of allergen products and for mechanistic studies of IgE-mediated allergic diseases, using hIgE mAb.
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Basófilos , Imunoglobulina E , Animais , Humanos , Ratos , Alérgenos , Anticorpos Monoclonais , ParaproteínasRESUMO
BACKGROUND AND OBJECTIVES: SARS-CoV-2 infection has been associated with a syndrome of long-term neurologic sequelae that is poorly characterized. We aimed to describe and characterize in-depth features of neurologic postacute sequelae of SARS-CoV-2 infection (neuro-PASC). METHODS: Between October 2020 and April 2021, 12 participants were seen at the NIH Clinical Center under an observational study to characterize ongoing neurologic abnormalities after SARS-CoV-2 infection. Autonomic function and CSF immunophenotypic analysis were compared with healthy volunteers (HVs) without prior SARS-CoV-2 infection tested using the same methodology. RESULTS: Participants were mostly female (83%), with a mean age of 45 ± 11 years. The median time of evaluation was 9 months after COVID-19 (range 3-12 months), and most (11/12, 92%) had a history of only a mild infection. The most common neuro-PASC symptoms were cognitive difficulties and fatigue, and there was evidence for mild cognitive impairment in half of the patients (MoCA score <26). The majority (83%) had a very disabling disease, with Karnofsky Performance Status ≤80. Smell testing demonstrated different degrees of microsmia in 8 participants (66%). Brain MRI scans were normal, except 1 patient with bilateral olfactory bulb hypoplasia that was likely congenital. CSF analysis showed evidence of unique intrathecal oligoclonal bands in 3 cases (25%). Immunophenotyping of CSF compared with HVs showed that patients with neuro-PASC had lower frequencies of effector memory phenotype both for CD4+ T cells (p < 0.0001) and for CD8+ T cells (p = 0.002), an increased frequency of antibody-secreting B cells (p = 0.009), and increased frequency of cells expressing immune checkpoint molecules. On autonomic testing, there was evidence for decreased baroreflex-cardiovagal gain (p = 0.009) and an increased peripheral resistance during tilt-table testing (p < 0.0001) compared with HVs, without excessive plasma catecholamine responses. DISCUSSION: CSF immune dysregulation and neurocirculatory abnormalities after SARS-CoV-2 infection in the setting of disabling neuro-PASC call for further evaluation to confirm these changes and explore immunomodulatory treatments in the context of clinical trials.
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Linfócitos T CD8-Positivos , COVID-19 , Feminino , Masculino , Humanos , COVID-19/complicações , SARS-CoV-2 , Encéfalo , CatecolaminasRESUMO
BACKGROUND AND OBJECTIVES: Neurologic outcomes in people with HIV (PWH) on long-duration antiretroviral therapy (ART) are not fully understood, and the underlying pathophysiology is unclear. To address this, we established a cohort of such individuals and compared them with HIV-negative controls using a novel matching technique. Both groups underwent extensive cognitive testing, evaluation for psychiatric measures, and MRI and CSF analyses. METHODS: Participants underwent comprehensive neuropsychological testing and completed standardized questionnaires measuring depressive symptoms, perceptions of own functioning, and activities of daily living as part of an observational study. Brain MRI and lumbar puncture were optional. Coarsened Exact Matching was used to reduce between-group differences in age and sex, and weighted linear/logistic regression models were used to assess the effect of HIV on outcomes. RESULTS: Data were analyzed from 155 PWH on ART for at least 15 years and 100 HIV-negative controls. Compared with controls, PWH scored lower in the domains of attention/working memory (PWH least square mean [LSM] = 50.4 vs controls LSM = 53.1, p = 0.008) and motor function (44.6 vs 47.7, p = 0.009) and a test of information processing speed (symbol search 30.3 vs 32.2, p = 0.003). They were more likely to self-report a higher number of cognitive difficulties in everyday life (p = 0.011). PWH also reported more depressive symptoms, general anxiety, and use of psychiatric medications (all with p < 0.05). PWH had reduced proportions of subcortical gray matter on MRI (ß = -0.001, p < 0.001), and CSF showed elevated levels of neurofilament light chain (664 vs 529 pg/mL, p = 0.01) and tumor necrosis factor α (0.229 vs 0.156 ng/mL, p = 0.0008). DISCUSSION: PWH, despite effective ART for over a decade, displayed neurocognitive deficits and mood abnormalities. MRI and CSF analyses revealed reduced brain volume and signs of ongoing neuronal injury and neuroinflammation. As the already large proportion of virologically controlled PWH continues to grow, longitudinal studies should be conducted to elucidate the implications of cognitive, psychiatric, MRI, and CSF abnormalities in this group.
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Disfunção Cognitiva , Infecções por HIV , Humanos , Atividades Cotidianas , Infecções por HIV/tratamento farmacológico , Cognição , Memória de Curto PrazoRESUMO
Chronic, treated HIV and the neurologic effects, both clinically and virologically, is a field with significant potential for research that will have impacts for patients and virologists alike as we seek to better understand HIV reservoirs with the ultimate aim of curing them.
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Complexo AIDS Demência , Humanos , Doença CrônicaRESUMO
PURPOSE OF REVIEW: Progressive multifocal leukoencephalopathy (PML) is a severe opportunistic infection that remains an important cause of morbidity and mortality in people living with HIV (PLWH). Immune checkpoint molecules are negative regulators of the immune response that have been targeted as a strategy to bolster anti-viral immunity in PML, with varied outcomes reported. While initiation and optimization of antiretroviral therapy remains the standard of care in HIV-related PML, the specific opportunities and risks for checkpoint blockade in these cases should be explored. RECENT FINDINGS: As of April 15, 2022, only 5 of the 53 total published cases of PML treated with checkpoint blockade had underlying HIV infection; four of these had a favorable outcome. The risk of promoting immune reconstitution inflammatory syndrome is a major concern and underscores the importance of patient selection and monitoring. Checkpoint blockade warrants further exploration as a potentially promising option for treatment escalation in HIV-related PML.
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Infecções por HIV , Síndrome Inflamatória da Reconstituição Imune , Vírus JC , Leucoencefalopatia Multifocal Progressiva , Humanos , Leucoencefalopatia Multifocal Progressiva/tratamento farmacológico , Leucoencefalopatia Multifocal Progressiva/etiologia , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Síndrome Inflamatória da Reconstituição Imune/tratamento farmacológico , Síndrome Inflamatória da Reconstituição Imune/complicações , Antivirais/uso terapêuticoRESUMO
BACKGROUND: Retinal measurements correlate with disease progression in patients with multiple sclerosis; however, whether they associate with neurologic disease in people with controlled HIV is unknown. Using spectral domain optical coherence tomography, we evaluated retinal differences between people with HIV and HIV-negative controls and investigated clinical correlates of retinal thinning. METHODS: People with HIV on antiretroviral therapy for at least 1 year and HIV-negative controls recruited from the same communities underwent spectral domain optical coherence tomography, ophthalmic examination, brain MRI, and neuropsychological testing. Retinal nerve fiber layer (RNFL) and ganglion cell inner plexiform layer (GC-IPL) thicknesses were compared between groups using analysis of covariance with relevant clinical variables as covariates. Linear regression was used to explore associations of HIV history variables, cognitive domain scores, and MRI volume measurements within the HIV group. RESULTS: The HIV group (n = 69), with long-duration HIV infection (median time from diagnosis 19 years) and outstanding viral control have thinner retinal layers than HIV-negative controls (n = 28), after adjusting for covariates (GC-IPL: P = 0.002; RNFL: P = 0.024). The effect of HIV on GC-IPL thickness was stronger in women than in men (Women: P = 0.011; Men: P = 0.126). GC-IPL thickness is associated with information processing speed in the HIV group (P = 0.007, semipartial r = 0.309). No associations were found with retinal thinning and MRI volumes or HIV factors. CONCLUSIONS: People with HIV on antiretroviral therapy have thinning of the RNFL and GC-IPL of the retina, and women particularly are affected to a greater degree. This retinal thinning was associated with worse performance on tests of information processing speed.
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Infecções por HIV , Fibras Nervosas , Progressão da Doença , Feminino , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Humanos , Masculino , Células Ganglionares da Retina , Tomografia de Coerência Óptica/métodosRESUMO
Background: HIV persistence during antiretroviral therapy (ART) is the principal obstacle to cure. Lymphoid tissue is a compartment for HIV, but mechanisms of persistence during ART and viral rebound when ART is interrupted are inadequately understood. Metabolic activity in lymphoid tissue of patients on long-term ART is relatively low, and increases when ART is stopped. Increases in metabolic activity can be detected by 18F-fluorodeoxyglucose Positron Emission Tomography (FDG-PET) and may represent sites of HIV replication or immune activation in response to HIV replication. Methods: FDG-PET imaging will be used to identify areas of high and low metabolic uptake in lymphoid tissue of individuals undergoing long-term ART. Baseline tissue samples will be collected. Participants will then be randomized 1:1 to continue or interrupt ART via analytic treatment interruption (ATI). Image-guided biopsy will be repeated 10 days after ATI initiation. After ART restart criteria are met, image-guided biopsy will be repeated once viral suppression is re-achieved. Participants who continued ART will have a second FDG-PET and biopsies 12-16 weeks after the first. Genetic characteristics of HIV populations in areas of high and low FDG uptake will be assesed. Optional assessments of non-lymphoid anatomic compartments may be performed to evaluate HIV populations in distinct anatomic compartments. Anticipated results: We anticipate that PET standardized uptake values (SUV) will correlate with HIV viral RNA in biopsies of those regions and that lymph nodes with high SUV will have more viral RNA than those with low SUV within a patient. Individuals who undergo ATI are expected to have diverse viral populations upon viral rebound in lymphoid tissue. HIV populations in tissues may initially be phylogenetically diverse after ATI, with emergence of dominant viral species (clone) over time in plasma. Dominant viral species may represent the same HIV population seen before ATI. Discussion: This study will allow us to explore utility of PET for identification of HIV infected cells and determine whether high FDG uptake respresents areas of HIV replication, immune activation or both. We will also characterize HIV infected cell populations in different anatomic locations. The protocol will represent a platform to investigate persistence and agents that may target HIV populations. Study protocol registration: Identifier: NCT05419024.
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The isolation of chitin utilizing ionic liquid 1-ethyl-3-methylimidazolium acetate has been determined to result in polymer contaminated with proteins. For the first time, the proteins in chitin extracted with ionic liquid have been quantified; the protein content was found to vary from 1.3 to 1.9% of the total weight. These proteins were identified and include allergenic proteins such as tropomyosin. In order to avoid 'traditional' hydroxide-based deproteinization of chitin, which could reduce the molecular weight of the final product, alternative deproteinization strategies were attempted. Testing of the previously reported deproteinization method using aqueous K3PO4 resulted in protein reduction by factors varying from 2 to 10, but resulted in significant phosphate salt contamination of the final product. Contrarily, the incorporation of GRAS (Generally Recognized as Safe) compound Polysorbate 80 into the polymer washing step provided the polymer of comparable purity with no contaminants. This study presents new options for the deproteinization of chitin that can replace traditional approaches with methods that are environmentally friendly and can produce high purity polymer.
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Quitina , Líquidos Iônicos , Peso Molecular , Polímeros , ProteínasRESUMO
T cell immunity to natural SARS-CoV-2 infection may be more robust and longer lived than Ab responses. Accurate assessment of T cell responses is critical for understanding the magnitude and longevity of immunity across patient cohorts, and against emerging variants. By establishing a simple, accurate, and rapid whole blood test, natural and vaccine-induced SARS-CoV-2 immunity was determined. Cytokine release in whole blood stimulated with peptides specific for SARS-CoV-2 was measured in donors with previous PCR-confirmed infection, suspected infection, or with no exposure history (n = 128), as well as in donors before and after vaccination (n = 32). Longitudinal assessment of T cell responses following initial vaccination and booster vaccination was also conducted (n = 50 and n = 62, respectively). Cytokines were measured by ELISA and multiplex array. IL-2 and IFN-γ were highly elevated in PCR-confirmed donors compared with history-negative controls, with median levels â¼33-fold and â¼48-fold higher, respectively. Receiver operating curves showed IL-2 as the superior biomarker (area under the curve = 0.9950). Following vaccination, all donors demonstrated a positive IL-2 response. Median IL-2 levels increased â¼32-fold from prevaccination to postvaccination in uninfected individuals. Longitudinal assessment revealed that T cell responses were stable up to 6 mo postvaccination. No significant differences in cytokine production were observed between stimulations with Wuhan, Delta, or Omicron peptides. This rapid, whole blood-based test can be used to make comparable longitudinal assessments of vaccine-induced T cell immunity across multiple cohorts and against variants of concern, thus aiding decisions on public health policies.