RESUMO
Although combination antiretroviral therapy (ART) blocks HIV replication, it is not curative because infected CD4+ T cells that carry intact, infectious proviruses persist. Understanding the behavior of clones of infected T cells is important for understanding the stability of the reservoir; however, the stabilities of clones of infected T cells in persons on long-term ART are not well defined. We determined the relative stabilities of clones of infected and uninfected CD4+ T cells over time intervals of one to four years in three individuals who had been on ART for 9-19 years. The largest clones of uninfected T cells were larger than the largest clones of infected T cells. Clones of infected CD4+ T cells were more stable than clones of uninfected CD4+ T cells of a similar size. Individual clones of CD4+ T cells carrying intact, infectious proviruses can expand, contract, or remain stable over time.
Assuntos
Infecções por HIV , HIV-1 , Linfócitos T CD4-Positivos , Células Clonais , DNA Viral , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , Provírus/genéticaRESUMO
Effective strategies to eliminate human immunodeficiency virus type 1 (HIV-1) reservoirs are likely to require more thorough characterizations of proviruses that persist on antiretroviral therapy (ART). The rarity of infected CD4+ T-cells and related technical challenges have limited the characterization of integrated proviruses. Current approaches using next-generation sequencing can be inefficient and limited sequencing depth can make it difficult to link proviral sequences to their respective integration sites. Here, we report on an efficient method by which HIV-1 proviruses and their sites of integration are amplified and sequenced. Across five HIV-1-positive individuals on clinically effective ART, a median of 41.2% (n = 88 of 209) of amplifications yielded near-full-length proviruses and their 5'-host-virus junctions containing a median of 430 bp (range, 18 to 1,363 bp) of flanking host sequence. Unexpectedly, 29.5% (n = 26 of 88) of the sequenced proviruses had structural asymmetries between the 5' and 3' long terminal repeats (LTRs), commonly in the form of major 3' deletions. Sequence-intact proviruses were detected in 3 of 5 donors, and infected CD4+ T-cell clones were detected in 4 of 5 donors. The accuracy of the method was validated by amplifying and sequencing full-length proviruses and flanking host sequences directly from peripheral blood mononuclear cell DNA. The individual proviral sequencing assay (IPSA) described here can provide an accurate, in-depth, and longitudinal characterization of HIV-1 proviruses that persist on ART, which is important for targeting proviruses for elimination and assessing the impact of interventions designed to eradicate HIV-1. IMPORTANCE The integration of human immunodeficiency virus type 1 (HIV-1) into chromosomal DNA establishes the long-term persistence of HIV-1 as proviruses despite effective antiretroviral therapy (ART). Characterizing proviruses is difficult because of their rarity in individuals on long-term suppressive ART, their highly polymorphic sequences and genetic structures, and the need for efficient amplification and sequencing of the provirus and its integration site. Here, we describe a novel, integrated, two-step method (individual proviral sequencing assay [IPSA]) that amplifies the host-virus junction and the full-length provirus except for the last 69 bp of the 3' long terminal repeat (LTR). Using this method, we identified the integration sites of proviruses, including those that are sequence intact and replication competent or defective. Importantly, this new method identified previously unreported asymmetries between LTRs that have implications for how proviruses are detected and quantified. The IPSA method reported is unaffected by LTR asymmetries, permitting a more accurate and comprehensive characterization of the proviral landscape.
Assuntos
HIV-1 , Provírus , Sequências Repetidas Terminais , Infecções por HIV/virologia , HIV-1/genética , HIV-1/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucócitos Mononucleares/virologia , Provírus/genética , Provírus/metabolismo , Sequências Repetidas Terminais/genéticaRESUMO
Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was no way to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for "viral reconstruction" to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality.
Assuntos
HIV-1/genética , Integração Viral/genética , Replicação Viral/genética , Antirretrovirais/uso terapêutico , Sequência de Bases , Linhagem Celular , DNA Viral/genética , Farmacorresistência Viral , Infecções por HIV/virologia , Humanos , Leucócitos Mononucleares/virologia , Linfonodos/virologia , Mutação , Provírus/genética , Integração Viral/fisiologiaRESUMO
In adults starting antiretroviral therapy (ART) during acute infection, 2% of proviruses that persist on ART are genetically intact by sequence analysis. In contrast, a recent report in children treated early failed to detect sequence-intact proviruses. In another cohort of children treated early, we sought to detect and characterize proviral sequences after 6 to 9 years on suppressive ART. Peripheral blood mononuclear cells (PBMC) from perinatally infected children from the Children with HIV Early antiRetroviral (CHER) study were analyzed. Nearly full-length proviral amplification and sequencing (NFL-PAS) were performed at one time point after 6 to 9 years on ART. Amplicons with large internal deletions were excluded (<9 kb). All amplicons of ≥9 kb were sequenced and analyzed through a bioinformatic pipeline to detect indels, frameshifts, or hypermutations that would render them defective. In eight children who started ART at a median age of 5.4 months (range, 2.0 to 11.1 months), 733 single NFL-PAS amplicons were generated. Of these, 534 (72.9%) had large internal deletions, 174 (23.7%) had hypermutations, 15 (1.4%) had small internal deletions, 3 (1.0%) had deletions in the packaging signal/major splice donor site, and 7 (1.0%) were sequence intact. These 7 intact sequences were from three children who initiated ART after 2.3 months of age, one of whom had two identical intact sequences, suggestive of a cell clone harboring a replication-competent provirus. No intact proviruses were detected in four children who initiated ART before 2.3 months of age. Rare, intact proviruses can be detected in children who initiate ART after 2.3 months of age and are probably, as in adults, maintained by clonal expansion of cells infected before ART initiation.IMPORTANCE There are limited data about the proviral landscape in children exhibiting long-term suppression after early treatment, particularly in Sub-Saharan Africa where HIV-1 subtype C predominates. Investigating the sequence-intact reservoir could provide insight on the mechanisms by which intact proviruses persist and inform ongoing cure efforts. Through nearly full-length proviral amplification and sequencing (NFL-PAS), we generated 733 NFL-PAS amplicons from eight children. We showed that rare, genetically intact proviruses could be detected in children who initiated ART after 2.3 months of age. The frequency of intact proviruses was lower (P < 0.05) than that reported for HIV subtype B-infected adults treated during early HIV infection. We show that cells harboring genetically intact HIV proviruses are rare in children exhibiting long-term suppression after early treatment and may require the processing of a large number of cells to assess reservoir size. This points to the need for efficient methods to accurately quantify latent reservoirs, particularly in pediatric studies where sample availability is limited.
Assuntos
Infecções por HIV/genética , HIV-1/genética , Provírus/genética , Antirretrovirais/uso terapêutico , Terapia Antirretroviral de Alta Atividade/métodos , Linfócitos T CD4-Positivos/virologia , Criança , Estudos de Coortes , DNA Viral/sangue , Feminino , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Leucócitos Mononucleares/virologia , Masculino , Análise de Sequência de DNA/métodos , África do Sul , Carga Viral/genética , Carga Viral/métodosRESUMO
BACKGROUND: Most literature regarding traumatic Le Fort or maxillary fractures exists in the adult population, with limited information regarding the epidemiology and management of pediatric fractures. The purpose of this study was to evaluate fracture mechanism, surgical management, and associated injuries in pediatric patients with Le Fort fractures. METHODS: A retrospective chart analysis of all pediatric patients age ≤18 years diagnosed with facial fractures at a single level 1 trauma center over a 10-year period (January 2006-December 2015) was performed. Demographics, fracture location, mechanism of injury, and hospital course were abstracted as well as associated injuries and need for operative management. RESULTS: A total of 1274 patients met inclusion criteria. Sixty-nine (5.4%) presented with Le Fort fractures. Factors associated with Le Fort fractures included motor vehicle collisions (Pâ<â0.001), increased age (Pâ<â0.001), and traumatic brain injury (Pâ<â0.04). Patients with Le Fort fractures were more likely to need intensive care unit admission (Pâ<â0.001), surgical management (Pâ<â0.001), transfusions (Pâ<â0.001), secondary fixation surgery (Pâ<â0.001), and have a longer length of stay (Pâ<â0.001). Multivariate showed increased odds for increased age (OR 1.1; 95%CI 1.04-1.17) and concomitant orbit fractures (OR 8.33; 95%CI 4.08-19.34). Decreased odds were associated for all mechanisms of injury other than motor vehicle collisions (Other blunt trauma: OR 0.36; 95%CI 0.2-0.6. Penetrating trauma: OR 0.13; 95%CI 0.01-0.6). CONCLUSION: Maxillary or Le Fort fractures represent a small portion of pediatric facial fractures but require high rates of operative management. The high velocity required to create this fracture type is associated with significant traumatic comorbidities, which can complicate the hospital course.
Assuntos
Fraturas Maxilares , Fraturas Orbitárias , Fraturas Cranianas , Acidentes de Trânsito , Adolescente , Adulto , Criança , Humanos , Fraturas Maxilares/epidemiologia , Fraturas Maxilares/cirurgia , Fraturas Orbitárias/epidemiologia , Fraturas Orbitárias/cirurgia , Estudos Retrospectivos , Fraturas Cranianas/epidemiologia , Fraturas Cranianas/cirurgiaRESUMO
In epilepsy, the GABA and glutamate balance may be disrupted and a transient decrease in extracellular calcium occurs before and during a seizure. Flow Cytometry based fluorescence activated particle sorting experiments quantified synaptosomes from human neocortical tissue, from both epileptic and non-epileptic patients (27.7% vs. 36.9% GABAergic synaptosomes, respectively). Transporter-mediated release of GABA in human and rat neocortical synaptosomes was measured using the superfusion technique for the measurement of endogenous GABA. GABA release was evoked by either a sodium channel activator or a sodium/potassium-ATPase inhibitor when exocytosis was possible or prevented, and when the sodium/calcium exchanger was active or inhibited. The transporter-mediated release of GABA is because of elevated intracellular sodium. A reduction in the extracellular calcium increased this release (in both non-epileptic and epileptic, except Rasmussen encephalitis, synaptosomes). The inverse was seen during calcium doubling. In humans, GABA release was not affected by exocytosis inhibition, that is, it was solely transporter-mediated. However, in rat synaptosomes, an increase in GABA release at zero calcium was only exhibited when the exocytosis was prevented. The absence of calcium amplified the sodium/calcium exchanger activity, leading to elevated intracellular sodium, which, together with the stimulation-evoked intracellular sodium increment, enhanced GABA transporter reversal. Sodium/calcium exchange inhibitors diminished GABA release. Thus, an important seizure-induced extracellular calcium reduction might trigger a transporter- and sodium/calcium exchanger-related anti-seizure mechanism by augmenting transporter-mediated GABA release, a mechanism absent in rats. Uniquely, the additional increase in GABA release because of calcium-withdrawal dwindled during the course of illness in Rasmussen encephalitis. Seizures cause high Na(+) influx through action potentials. A transient decrease in [Ca(2+)]e (seizure condition) increases GABA transporter (GAT)-mediated GABA release because of elevated [Na(+)]i. This amplifies the Sodium-Calcium-Exchanger (NCX) activity, further increasing [Na(+)]i and GABA release. The reduction in [Ca(2+)]e triggers a GAT-NCX related anti-seizure mechanism by augmenting GAT-mediated GABA release. This mechanism, obvious in humans, is absent in rats.
Assuntos
Cálcio/metabolismo , Neocórtex/metabolismo , Neocórtex/patologia , Convulsões/patologia , Sódio/metabolismo , Sinaptossomos/metabolismo , Adolescente , Adulto , Idoso , Compostos de Anilina/farmacologia , Animais , Criança , Pré-Escolar , Inibidores Enzimáticos/farmacologia , Feminino , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Neurotoxinas/farmacologia , Ouabaína/farmacologia , Éteres Fenílicos/farmacologia , Ratos , Ratos Wistar , Sinaptossomos/efeitos dos fármacos , Toxina Tetânica/farmacologia , Tioureia/análogos & derivados , Tioureia/farmacologia , Trítio/metabolismo , Veratridina/farmacologia , Adulto JovemRESUMO
Leptin is an anorexigenic hormone that acts via its receptor (LepR) to regulate the hypothalamic arcuate nucleus circuitry to mediate energy homeostasis and feeding behavior. Moreover, leptin decreases the reward value of natural and artificial rewards, and low levels of circulating leptin have been implicated in several mood disorders linking leptin to the mesolimbic system. Therefore, the purpose of this study was to assess whether and to what extent an acute intranasal application of leptin is able to modulate monoamine neurotransmitters in the nucleus accumbens (NAc). Microdialysis experiments were carried out in freely moving Wistar rats and in LepR-deficient Zucker rats (LepRfa/fa). Samples were analysed for the levels of dopamine (DA), serotonin (5-HT), and their metabolites using high-performance liquid chromatography with electrochemical detection. We show that in Wistar rats, nasal application of leptin dose-dependently increased extracellular DA and 5-HT levels in the NAc. By contrast, in the LepRfa/fa rats, nasal application of 0.12 mg/kg leptin failed to increase levels of either DA or 5-HT, but their metabolites (DOPAC and HIAA, respectively) were significantly decreased. In addition, leptin interaction with the melanocortin system was tested. Nasal co-administration of leptin and the melanocortin receptor antagonist, SHU9119, completely abolished the leptin-induced increase of both DA and 5-HT outflow in the NAc. These results indicate a marked leptin effect on the basal ganglia-related reward system involving melanocortin receptors.
Assuntos
Dopamina/metabolismo , Leptina/administração & dosagem , Neurotransmissores/administração & dosagem , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Serotonina/metabolismo , Administração Intranasal , Animais , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Leptina/genética , Leptina/metabolismo , Masculino , Microdiálise , Ratos Transgênicos , Ratos Wistar , Ratos Zucker , Receptores de Melanocortina/antagonistas & inibidores , Receptores de Melanocortina/metabolismoRESUMO
Glutamate is thought to be the most important excitatory neurotransmitter in the CNS, while glutamine predominantly serves as a precursor and metabolite in the glutamate-glutamine cycle. To verify the interaction between intrinsic extracellular glutamate, y-aminobutyric acid (GABA) levels and glial glutamine outflow in human tissue, fresh brain slices from human frontal cortex were incubated in superfusion chambers in vitro. Human neocortical tissue was obtained during surgical treatment of subcortical brain tumors. For superfusion experiments, the white matter was separated and discarded from the gray matter, which finally contained all six neocortical layers. Outflows of endogenous glutamate, GABA and glutamine were established after a 40-min washout period and amounts were simultaneously quantified after two-phase derivatization by high-performance liquid chromatography with electrochemical detection. Under basal conditions, amounts of glutamate could be found 20-fold in comparison to the inhibitory neurotransmitter GABA, whereas this excitatory predominance markedly declined after veratridine-induced activation. The basal glutamate:glutamine ratio of extracellular levels was approximately 1:2. Blockade or activation of the voltage-gated sodium channel by tetrodotoxin or veratridine significantly modulated glutamate levels, but the glutamate:glutamine ratio was nearly constant with 1:2. When the EAAT blocker TBOA was employed, glutamine remained nearly unchanged whereas glutamate significantly enhanced. These results led us to suggest that glutamine release through glial SN1 is related to EAAT activity that can be modulated by intrinsic extracellular glutamate in human cortical slices.
Assuntos
Córtex Cerebral/citologia , Líquido Extracelular/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/fisiologia , Adulto , Idoso , Ácido Aspártico/farmacologia , Cromatografia Líquida de Alta Pressão , Líquido Extracelular/efeitos dos fármacos , Feminino , Glutamina/metabolismo , Humanos , Técnicas In Vitro , Masculino , Metionina/análogos & derivados , Metionina/farmacologia , Pessoa de Meia-Idade , Neurônios/efeitos dos fármacos , Tetrodotoxina/farmacologia , Fatores de Tempo , Veratridina/farmacologia , Ácido gama-Aminobutírico/metabolismoRESUMO
Baclofen is a commonly prescribed muscle relaxant that functions to reduce spasticity associated with a variety of musculoskeletal and neurological conditions. Patients with decreased renal function, however, are at an increased risk of suffering from baclofen withdrawal symptoms. We discuss the case of a 77-year-old male who presented to the emergency room with altered mental status and was admitted for acute metabolic encephalopathy found to be from abrupt cessation of baclofen. The presence of kidney disease in this patient further increased his susceptibility to the withdrawal symptoms that began after cessation of the normal medication regimen. This case further illustrates the importance of both comprehensive history and a high index of suspicion when it comes to patient presentation of baclofen withdrawal.
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Variations in the arch of the aorta and aortic valves among fetal, cadaveric, and post-mortem specimens present a spectrum of anatomical configurations, posing challenges in establishing a standard norm. While some variations hold surgical significance, many bear little functional consequence but provide insights into embryological origins. The aortic arch exhibits diverse branching patterns, including common trunks and different orders, relevant for endovascular surgeries. Meanwhile, malformations in the aortic valve, affecting the aorta, may lead to ischemia and cerebral infarction, warranting understanding of coexisting arch and valve anomalies to predict complications like aortic dissection. Studies in the Indian population mirror global variations, underscoring the need to explore embryological, clinical, and surgical implications for safer vascular surgeries involving the aortic arch and valves. The study's objectives included examining branching patterns, diameters, and distances between arch branches and exploring aortic valve variations. Employing a cross-sectional design, the study was conducted across Anatomy, Forensic Medicine, and Obstetrics and Gynecology departments. A sample of 100, comprising cadavers, fetuses, and postmortem specimens, were gathered. Specimens ranged from 14 weeks of intrauterine life to 85 years, with intact thoracic cages as inclusion criteria. Methodology involved dissection, specimen fixation, and macroscopic examination for variations and morphological parameters. Results showed aortic diameter increase with age, with significant gender differences. A statistically significant association between arch variations and anomalous valves was observed, suggesting mutual predictability. Individuals with valve anomalies should undergo comprehensive cardiology evaluation to avert complications like aortic dissection during endovascular surgeries. While atheromatous plaques were prevalent in younger groups, their frequency rose with age, necessitating vigilant vascular monitoring. Careful handling during surgeries is paramount, given potential adverse outcomes resulting from variations. Overall, the study underscores the importance of comprehensive anatomical understanding in clinical contexts, guiding effective management strategies and ensuring patient safety in vascular surgeries.
Assuntos
Variação Anatômica , Aorta Torácica , Valva Aórtica , Cadáver , Humanos , Feminino , Masculino , Valva Aórtica/anatomia & histologia , Valva Aórtica/anormalidades , Aorta Torácica/anatomia & histologia , Aorta Torácica/embriologia , Idoso , Adulto , Pessoa de Meia-Idade , Idoso de 80 Anos ou mais , Adolescente , Adulto Jovem , Criança , Lactente , Pré-Escolar , Feto/anatomia & histologia , Recém-Nascido , Estudos TransversaisRESUMO
BACKGROUND: Prior reports indicate that modulation of the endocannabinoid system (ECS) may have a protective benefit for Covid-19 patients. However, associations between cannabis use (CU) or CU not in remission (active cannabis use (ACU)), and Covid-19-related outcomes among hospitalized patients is unknown. METHODS: In this multicenter retrospective observational cohort analysis of adults (≥ 18 years-old) identified from 2020 National Inpatient Sample database, we utilize multivariable regression analyses and propensity score matching analysis (PSM) to analyze trends and outcomes among Covid-19-related hospitalizations with CU and without CU (N-CU) for primary outcome of interest: Covid-19-related mortality; and secondary outcomes: Covid-19-related hospitalization, mechanical ventilation (MV), and acute pulmonary embolism (PE) compared to all-cause admissions; for CU vs N-CU; and for ACU vs N-ACU. RESULTS: There were 1,698,560 Covid-19-related hospitalizations which were associated with higher mortality (13.44% vs 2.53%, p ≤ 0.001) and worse secondary outcomes generally. Among all-cause hospitalizations, 1.56% of CU and 6.29% of N-CU were hospitalized with Covid-19 (p ≤ 0.001). ACU was associated with lower odds of MV, PE, and death among the Covid-19 population. On PSM, ACU(N(unweighted) = 2,382) was associated with 83.97% lower odds of death compared to others(N(unweighted) = 282,085) (2.77% vs 3.95%, respectively; aOR:0.16, [0.10-0.25], p ≤ 0.001). CONCLUSIONS: These findings suggest that the ECS may represent a viable target for modulation of Covid-19. Additional studies are needed to further explore these findings.
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Glioblastoma (GBM) is a highly lethal type of cancer. GBM recurrence following chemoradiation is typically attributed to the regrowth of invasive and resistant cells. Therefore, there is a pressing need to gain a deeper understanding of the mechanisms underlying GBM resistance to chemoradiation and its ability to infiltrate. Using a combination of transcriptomic, proteomic, and phosphoproteomic analyses, longitudinal imaging, organotypic cultures, functional assays, animal studies, and clinical data analyses, we demonstrate that chemoradiation and brain vasculature induce cell transition to a functional state named VC-Resist (vessel co-opting and resistant cell state). This cell state is midway along the transcriptomic axis between proneural and mesenchymal GBM cells and is closer to the AC/MES1-like state. VC-Resist GBM cells are highly vessel co-opting, allowing significant infiltration into the surrounding brain tissue and homing to the perivascular niche, which in turn induces even more VC-Resist transition. The molecular and functional characteristics of this FGFR1-YAP1-dependent GBM cell state, including resistance to DNA damage, enrichment in the G2M phase, and induction of senescence/stemness pathways, contribute to its enhanced resistance to chemoradiation. These findings demonstrate how vessel co-option, perivascular niche, and GBM cell plasticity jointly drive resistance to therapy during GBM recurrence.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Camundongos , Quimiorradioterapia/métodos , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Tolerância a Radiação , Proteínas de Sinalização YAP/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , ProteômicaRESUMO
Single-cell RNA-sequencing (scRNA-seq) is becoming a ubiquitous method in profiling the cellular transcriptomes of both malignant and non-malignant cells from the human brain. Here, we present a protocol to isolate viable tumor cells from human ex vivo glioblastoma cultures for single-cell transcriptomic analysis. We describe steps including surgical tissue collection, sectioning, culturing, primary tumor cells inoculation, growth tracking, fluorescence-based cell sorting, and population-enriched scRNA-seq. This comprehensive methodology empowers in-depth understanding of brain tumor biology at the single-cell level. For complete details on the use and execution of this protocol, please refer to Ravi et al.1.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/genética , Perfilação da Expressão Gênica , Neoplasias Encefálicas/genética , Encéfalo , Transcriptoma/genéticaRESUMO
Spatial resolution of the T cell repertoire is essential for deciphering cancer-associated immune dysfunction. Current spatially resolved transcriptomic technologies are unable to directly annotate T cell receptors (TCR). We present spatially resolved T cell receptor sequencing (SPTCR-seq), which integrates optimized target enrichment and long-read sequencing for highly sensitive TCR sequencing. The SPTCR computational pipeline achieves yield and coverage per TCR comparable to alternative single-cell TCR technologies. Our comparison of PCR-based and SPTCR-seq methods underscores SPTCR-seq's superior ability to reconstruct the entire TCR architecture, including V, D, J regions and the complementarity-determining region 3 (CDR3). Employing SPTCR-seq, we assess local T cell diversity and clonal expansion across spatially discrete niches. Exploration of the reciprocal interaction of the tumor microenvironmental and T cells discloses the critical involvement of NK and B cells in T cell exhaustion. Integrating spatially resolved omics and TCR sequencing provides as a robust tool for exploring T cell dysfunction in cancers and beyond.
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Receptores de Antígenos de Linfócitos T , Linfócitos T , Receptores de Antígenos de Linfócitos T/genética , Regiões Determinantes de Complementaridade/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Perfilação da Expressão Gênica , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
BACKGROUND: In glioblastoma (GBM), the effects of altered glycocalyx are largely unexplored. The terminal moiety of cell coating glycans, sialic acid, is of paramount importance for cell-cell contacts. However, sialic acid turnover in gliomas and its impact on tumor networks remain unknown. METHODS: We streamlined an experimental setup using organotypic human brain slice cultures as a framework for exploring brain glycobiology, including metabolic labeling of sialic acid moieties and quantification of glycocalyx changes. By live, 2-photon and high-resolution microscopy we have examined morphological and functional effects of altered sialic acid metabolism in GBM. By calcium imaging we investigated the effects of the altered glycocalyx on a functional level of GBM networks. RESULTS: The visualization and quantitative analysis of newly synthesized sialic acids revealed a high rate of de novo sialylation in GBM cells. Sialyltrasferases and sialidases were highly expressed in GBM, indicating that significant turnover of sialic acids is involved in GBM pathology. Inhibition of either sialic acid biosynthesis or desialylation affected the pattern of tumor growth and lead to the alterations in the connectivity of glioblastoma cells network. CONCLUSIONS: Our results indicate that sialic acid is essential for the establishment of GBM tumor and its cellular network. They highlight the importance of sialic acid for glioblastoma pathology and suggest that dynamics of sialylation have the potential to be targeted therapeutically.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/patologia , Ácido N-Acetilneuramínico/metabolismo , Ácidos Siálicos/metabolismo , Transdução de Sinais , Linhagem Celular TumoralRESUMO
PURPOSE: The association between bariatric surgery and IBD-related inpatient outcomes is not well characterized. We report, analyze, and compare inpatient trends and outcomes among encounters with a history of bariatric surgery (Hx-MBS) compared to those receiving bariatric surgery during index admission (PR-MBS) admitted from 2009 to 2020. METHODS: Retrospective cohort design: the 2009-2020 National Inpatient Sample (NIS) databases were used to identify hospital encounters with patients aged ≥ 18 years with a history of MBS (Hx-MBS) or with procedure coding indicating MBS procedure (PR-MBS) according to International Classification of Diseases, Ninth (ICD-9-CM/ ICD-9-PCS) or Tenth Revision (ICD-10-CM/ICD-10-PCS) Clinical Modification/Procedure Coding System during index admission (ICD-9-CM: V4586; ICD-10-CM: Z9884; ICD-9-PR: 4382, 4389; ICD-10-PR: 0DB64Z3, 0DB63ZZ). Pearson χ2 analysis, analysis of variance, multivariable regression analyses, and propensity matching on independent variables were conducted to analyze significant associations between variables and for primary outcome inflammatory bowel disease-related admission, and secondary outcomes: diagnosis of nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, or chronic mesenteric ischemia during admission. RESULTS: We identified 3,365,784 (76.20%) Hx-MBS hospitalizations and 1,050,900 hospitalizations with PR-MBS (23.80%). Propensity score matching analysis demonstrated significantly higher odds of inflammatory bowel disease, and chronic mesenteric ischemia for Hx-MBS compared to PR-MBS, and significantly lower odds of nonalcoholic steatohepatitis and nonalcoholic fatty liver disease for Hx-MBS compared to PR-MBS. CONCLUSION: In our study, Hx-MBS was associated with significantly increased odds of inflammatory bowel disease and other GI pathologies compared to matched controls. The mechanism by which this occurs is unclear. Additional studies are needed to examine these findings.
Assuntos
Cirurgia Bariátrica , Doenças Inflamatórias Intestinais , Isquemia Mesentérica , Hepatopatia Gordurosa não Alcoólica , Obesidade Mórbida , Humanos , Estudos Retrospectivos , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Hepatopatia Gordurosa não Alcoólica/cirurgia , Hepatopatia Gordurosa não Alcoólica/complicações , Obesidade Mórbida/cirurgia , Doenças Inflamatórias Intestinais/epidemiologia , Doenças Inflamatórias Intestinais/cirurgia , Doenças Inflamatórias Intestinais/complicações , Cirurgia Bariátrica/métodos , GastrectomiaRESUMO
The United States witnessed a nearly 4-fold increase in personal health care expenditures between 1980 and 2010. Despite innovations and obvious benefits to health, participants enrolled in clinical trials still do not accurately represent the racial and ethnic composition of patients nationally or globally. This lack of diversity in cohorts limits the generalizability and significance of results among all populations and has deep repercussions for patient equity. To advance diversity in clinical trials, robust evidence for the most effective strategies for recruitment of diverse participants is needed. A major limitation of previous literature on clinical trial diversity is the lack of control or comparator groups for different strategies. To date, interventions have focused primarily on (1) community-based interventions, (2) institutional practices, and (3) digital health systems. This review article outlines prior intervention strategies across these 3 categories and considers health policy and ethical incentives for substantiation before US Food and Drug Administration approval. There are no current studies that comprehensively compare these interventions against one another. The American Heart Association Strategically Focused Research Network on the Science of Diversity in Clinical Trials represents a multicenter, collaborative network between Stanford School of Medicine and Morehouse School of Medicine created to understand the barriers to diversity in clinical trials by contemporaneous head-to-head interventional strategies accessing digital, institutional, and community-based recruitment strategies to produce informed recruitment strategies targeted to improve underrepresented patient representation in clinical trials.
Assuntos
American Heart Association , Instalações de Saúde , Estados Unidos , Humanos , Política de Saúde , Assistência Médica , Diversidade Cultural , Estudos Multicêntricos como AssuntoRESUMO
Microglia appear activated in the vicinity of amyloid beta (Aß) plaques, but whether microglia contribute to Aß propagation into unaffected brain regions remains unknown. Using transplantation of wild-type (WT) neurons, we show that Aß enters WT grafts, and that this is accompanied by microglia infiltration. Manipulation of microglia function reduced Aß deposition within grafts. Furthermore, in vivo imaging identified microglia as carriers of Aß pathology in previously unaffected tissue. Our data thus argue for a hitherto unexplored mechanism of Aß propagation.
Assuntos
Peptídeos beta-Amiloides , Microglia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Humanos , Microglia/metabolismo , Neurônios/metabolismo , Placa Amiloide/patologiaRESUMO
Despite recent advances in cancer immunotherapy, certain tumor types, such as Glioblastomas, are highly resistant due to their tumor microenvironment disabling the anti-tumor immune response. Here we show, by applying an in-silico multidimensional model integrating spatially resolved and single-cell gene expression data of 45,615 immune cells from 12 tumor samples, that a subset of Interleukin-10-releasing HMOX1+ myeloid cells, spatially localizing to mesenchymal-like tumor regions, drive T-cell exhaustion and thus contribute to the immunosuppressive tumor microenvironment. These findings are validated using a human ex-vivo neocortical glioblastoma model inoculated with patient derived peripheral T-cells to simulate the immune compartment. This model recapitulates the dysfunctional transformation of tumor infiltrating T-cells. Inhibition of the JAK/STAT pathway rescues T-cell functionality both in our model and in-vivo, providing further evidence of IL-10 release being an important driving force of tumor immune escape. Our results thus show that integrative modelling of single cell and spatial transcriptomics data is a valuable tool to interrogate the tumor immune microenvironment and might contribute to the development of successful immunotherapies.
Assuntos
Neoplasias Encefálicas/imunologia , Glioblastoma/imunologia , Interleucina-10/metabolismo , Células Mieloides/metabolismo , Linfócitos T/imunologia , Adulto , Idoso , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Voluntários Saudáveis , Heme Oxigenase-1/metabolismo , Humanos , Imunoterapia/métodos , Inibidores de Janus Quinases/farmacologia , Inibidores de Janus Quinases/uso terapêutico , Janus Quinases/antagonistas & inibidores , Janus Quinases/metabolismo , Masculino , Pessoa de Meia-Idade , Neocórtex/citologia , Neocórtex/imunologia , Neocórtex/patologia , Cultura Primária de Células , RNA-Seq , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Análise de Célula Única , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Técnicas de Cultura de Tecidos , Evasão Tumoral , Microambiente Tumoral/imunologiaRESUMO
Lactate is a central metabolite in brain physiology but also contributes to tumor development. Glioblastoma (GB) is the most common and malignant primary brain tumor in adults, recognized by angiogenic and invasive growth, in addition to its altered metabolism. We show herein that lactate fuels GB anaplerosis by replenishing the tricarboxylic acid (TCA) cycle in absence of glucose. Lactate dehydrogenases (LDHA and LDHB), which we found spatially expressed in GB tissues, catalyze the interconversion of pyruvate and lactate. However, ablation of both LDH isoforms, but not only one, led to a reduction in tumor growth and an increase in mouse survival. Comparative transcriptomics and metabolomics revealed metabolic rewiring involving high oxidative phosphorylation (OXPHOS) in the LDHA/B KO group which sensitized tumors to cranial irradiation, thus improving mouse survival. When mice were treated with the antiepileptic drug stiripentol, which targets LDH activity, tumor growth decreased. Our findings unveil the complex metabolic network in which both LDHA and LDHB are integrated and show that the combined inhibition of LDHA and LDHB strongly sensitizes GB to therapy.