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1.
Proc Natl Acad Sci U S A ; 120(19): e2300706120, 2023 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-37126700

RESUMO

Although viral hepatocellular carcinoma (HCC) is declining, nonviral HCC, which often is the end stage of nonalcoholic or alcoholic steatohepatitis (NASH, ASH), is on an upward trajectory. Immune checkpoint inhibitors (ICIs) that block the T cell inhibitory receptor PD-1 were approved for treatment of all HCC types. However, only a minority of HCC patients show a robust and sustained response to PD-1 blockade, calling for improved understanding of factors that negatively impact response rate and duration and the discovery of new adjuvant treatments that enhance ICI responsiveness. Using a mouse model of NASH-driven HCC, we identified peritumoral fibrosis as a potential obstacle to T cell-mediated tumor regression and postulated that antifibrotic medications may increase ICI responsiveness. We now show that the angiotensin II receptor inhibitor losartan, a commonly prescribed and safe antihypertensive drug, reduced liver and peritumoral fibrosis and substantially enhanced anti-PD-1-induced tumor regression. Although losartan did not potentiate T cell reinvigoration, it substantially enhanced HCC infiltration by effector CD8+ T cells compared to PD-1 blockade alone. The beneficial effects of losartan correlated with blunted TGF-ß receptor signaling, reduced collagen deposition, and depletion of immunosuppressive fibroblasts.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Humanos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Linfócitos T CD8-Positivos , Losartan , Cirrose Hepática/patologia
2.
PLoS Pathog ; 19(8): e1011452, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37549185

RESUMO

Recent HIV-1 vaccine development has centered on "near native" soluble envelope glycoprotein (Env) trimers that are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their effects in this context are unclear. To address this question, we used virus-like particle (VLP) produced in 293T cells. Uncleaved (UNC) trimers were laterally unstable upon gentle lysis from membranes. However, gp120/gp41 processing improved lateral stability. Due to inefficient gp120/gp41 processing, UNC is incorporated into VLPs. A linker between gp120 and gp41 neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed post-expression gp120/gp41 processing, concomitantly increasing trimer stability. Gp41 N-helix mutations I559P and NT1-5 imparted lateral trimer stability, but also reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ human plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, and also reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common side effects of these mutations. In some cases, this may be due to increased rigidity which limits access to glycan processing enzymes. In contrast, viral gp120 did not show glycan skipping. A second, minor species of high mannose gp160 was unaffected by any mutations and instead bypasses normal folding and glycan maturation. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and greatly increased the proportion of high mannose gp160. Remarkably, monoclonal antibodies were unable to bind to this high mannose gp160 in native protein gels. Overall, our findings suggest caution in leveraging stabilizing mutations in nucleic acid-based immunogens to ensure they impart valuable membrane trimer phenotypes for vaccine use.


Assuntos
Proteína gp41 do Envelope de HIV , HIV-1 , Humanos , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , Glicosilação , Manose/metabolismo , Mutação , Glicoproteínas/metabolismo , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Anticorpos Anti-HIV
3.
J Biol Chem ; 299(10): 105192, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37625589

RESUMO

Point mutations in leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease and augment LRRK2's kinase activity. However, cellular pathways that endogenously enhance LRRK2 kinase function have not been identified. While overexpressed Rab29 draws LRRK2 to Golgi membranes to increase LRRK2 kinase activity, there is little evidence that endogenous Rab29 performs this function under physiological conditions. Here, we identify Rab38 as a novel physiologic regulator of LRRK2 in melanocytes. In mouse melanocytes, which express high levels of Rab38, Rab32, and Rab29, knockdown (or CRISPR knockout) of Rab38, but not Rab32 or Rab29, decreases phosphorylation of multiple LRRK2 substrates, including Rab10 and Rab12, by both endogenous LRRK2 and exogenous Parkinson's disease-mutant LRRK2. In B16-F10 mouse melanoma cells, Rab38 drives LRRK2 membrane association and overexpressed kinase-active LRRK2 shows striking pericentriolar recruitment, which is dependent on the presence of endogenous Rab38 but not Rab32 or Rab29. Consistently, knockdown or mutation of BLOC-3, the guanine nucleotide exchange factor for Rab38 and Rab32, inhibits Rab38's regulation of LRRK2. Deletion or mutation of LRRK2's Rab38-binding site in the N-terminal armadillo domain decreases LRRK2 membrane association, pericentriolar recruitment, and ability to phosphorylate Rab10. In sum, our data identify Rab38 as a physiologic regulator of LRRK2 function and lend support to a model in which LRRK2 plays a central role in Rab GTPase coordination of vesicular trafficking.


Assuntos
Membranas Intracelulares , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Melanócitos , Proteínas rab de Ligação ao GTP , Animais , Camundongos , Complexo de Golgi/enzimologia , Complexo de Golgi/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Melanócitos/metabolismo , Mutação , Doença de Parkinson/metabolismo , Fosforilação , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Expressão Gênica , Domínios Proteicos , Ligação Proteica , Membranas Intracelulares/metabolismo
4.
Clin Gastroenterol Hepatol ; 21(7): 1802-1809.e6, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36967102

RESUMO

BACKGROUND & AIMS: Early detection of pancreatic cancer (PaC) can drastically improve survival rates. Approximately 25% of subjects with PaC have type 2 diabetes diagnosed within 3 years prior to the PaC diagnosis, suggesting that subjects with type 2 diabetes are at high risk of occult PaC. We have developed an early-detection PaC test, based on changes in 5-hydroxymethylcytosine (5hmC) signals in cell-free DNA from plasma. METHODS: Blood was collected from 132 subjects with PaC and 528 noncancer subjects to generate epigenomic and genomic feature sets yielding a predictive PaC signal algorithm. The algorithm was validated in a blinded cohort composed of 102 subjects with PaC, 2048 noncancer subjects, and 1524 subjects with non-PaCs. RESULTS: 5hmC differential profiling and additional genomic features enabled the development of a machine learning algorithm capable of distinguishing subjects with PaC from noncancer subjects with high specificity and sensitivity. The algorithm was validated with a sensitivity for early-stage (stage I/II) PaC of 68.3% (95% confidence interval [CI], 51.9%-81.9%) and an overall specificity of 96.9% (95% CI, 96.1%-97.7%). CONCLUSIONS: The PaC detection test showed robust early-stage detection of PaC signal in the studied cohorts with varying type 2 diabetes status. This assay merits further clinical validation for the early detection of PaC in high-risk individuals.


Assuntos
Ácidos Nucleicos Livres , Diabetes Mellitus Tipo 2 , Neoplasias Pancreáticas , Humanos , Diabetes Mellitus Tipo 2/diagnóstico , Epigenômica , Detecção Precoce de Câncer , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética
5.
J Immunol ; 204(5): 1075-1083, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32071090

RESUMO

The mammalian intestine is a complex environment that is constantly exposed to Ags derived from food, microbiota, and metabolites. Intestinal dendritic cells (DC) have the responsibility of establishing oral tolerance against these Ags while initiating immune responses against mucosal pathogens. We now know that DC are a heterogeneous population of innate immune cells composed of classical and monocyte-derived DC, Langerhans cells, and plasmacytoid DC. In the intestine, DC are found in organized lymphoid tissues, such as the mesenteric lymph nodes and Peyer's patches, as well as in the lamina propria. In this Brief Review, we review recent work that describes a division of labor between and collaboration among gut DC subsets in the context of intestinal homeostasis and inflammation. Understanding relationships between DC subtypes and their biological functions will rationalize oral vaccine design and will provide insights into treatments that quiet pathological intestinal inflammation.


Assuntos
Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Células de Langerhans/imunologia , Nódulos Linfáticos Agregados/imunologia , Animais , Humanos , Inflamação/imunologia , Inflamação/patologia , Mucosa Intestinal/patologia , Células de Langerhans/patologia , Linfonodos/imunologia , Linfonodos/patologia , Mesentério/imunologia , Mesentério/patologia , Nódulos Linfáticos Agregados/patologia
6.
Biotechnol Bioeng ; 116(2): 427-443, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30450542

RESUMO

Human mesenchymal stem cells (hMSCs) are under intense study for applications of cell and gene therapeutics because of their unique immunomodulatory and regenerative properties. Safe and efficient genetic modification of hMSCs could increase their clinical potential by allowing functional expression of therapeutic transgenes or control over behavior and differentiation. Viral gene delivery is efficient, but suffers from safety issues, while nonviral methods are safe, but highly inefficient, especially in hMSCs. Our lab previously demonstrated that priming cells before delivery of DNA complexes with dexamethasone (DEX), an anti-inflammatory glucocorticoid drug, significantly increases hMSC transfection success. This work systematically investigates the mechanisms of hMSC transfection and DEX-mediated enhancement of transfection. Our results show that hMSC transfection and its enhancement by DEX are decreased by inhibiting classical intracellular transport and nuclear import pathways, but DEX transfection priming does not increase cellular or nuclear internalization of plasmid DNA (pDNA). We also show that hMSC transgene expression is largely affected by pDNA promoter and enhancer sequence changes, but DEX-mediated enhancement of transfection is unaffected by any pDNA sequence changes. Furthermore, DEX-mediated transfection enhancement is not the result of increased transgene messenger RNA transcription or stability. However, DEX-priming increases total protein synthesis by preventing hMSC apoptosis induced by transfection, resulting in increased translation of transgenic protein. DEX may also promote further enhancement of transgenic reporter enzyme activity by other downstream mechanisms. Mechanistic studies of nonviral gene delivery will inform future rationally designed technologies for safe and efficient genetic modification of clinically relevant cell types.


Assuntos
Dexametasona/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Transfecção/métodos , Transformação Genética , Células Cultivadas , Expressão Gênica , Humanos
7.
J Cardiothorac Vasc Anesth ; 33(10): 2814-2825, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31060943

RESUMO

Peripartum cardiomyopathy is a rare form of acute heart failure but the major cause of all deaths in pregnant patients with heart failure. Improved survival rates in recent years, however, emphasize the importance of early recognition and initiation of heart failure treatment. This article, therefore, attempts to raise awareness among cardiac and obstetric anesthesiologists as well as intensivists of this often fatal diagnosis. This review summarizes theories of the pathophysiology and outcome of peripartum cardiomyopathy. Based on the most recent literature, it further outlines diagnostic criteria and treatment options including medical management, mechanical circulatory support devices, and heart transplantation. Earlier recognition of this rare condition and a new generation of mechanical circulatory devices has contributed to the improved outcome. More frequently, patients in cardiogenic shock who fail medical management are successfully bridged to recovery on extracorporeal circulatory devices or survive with a long-lasting implantable ventricular assist device. The outcome of transplanted patients with peripartum cardiomyopathy, however, is worse compared to other recipients of heart transplants and warrants further investigation in the future.


Assuntos
Cardiomiopatias/terapia , Oxigenação por Membrana Extracorpórea/métodos , Insuficiência Cardíaca/terapia , Período Periparto , Complicações Cardiovasculares na Gravidez/terapia , Doença Aguda , Cardiomiopatias/diagnóstico , Cardiomiopatias/fisiopatologia , Feminino , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/fisiopatologia , Humanos , Período Periparto/fisiologia , Gravidez , Complicações Cardiovasculares na Gravidez/diagnóstico , Complicações Cardiovasculares na Gravidez/fisiopatologia , Resultado do Tratamento
8.
World J Surg ; 42(7): 1939-1948, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29143088

RESUMO

BACKGROUND: Patients with anemia frequently undergo surgery, as it is unclear at what threshold clinicians should consider delaying surgery for preoperative anemia optimization. The primary objective of this study was to determine whether there is an association of varying degrees of anemia and transfusion with 30-day mortality. METHODS: This is a retrospective cohort study using the American College of Surgeons National Surgical Quality Improvement Program database from 2011 to 2013. Cohorts were analyzed based on preoperative hematocrit range-patients with: (1) no anemia, (2) hematocrit ≥33% and <36% in females or <39% in males, (3) hematocrit ≥30% and <33%, (4) hematocrit ≥27% and <30%, (5) hematocrit ≥24% and <27%, and (6) hematocrit ≥21% and less than 24%. Multivariable logistic regression was used to analyze the association of anemia and transfusion with 30-day in-hospital mortality. RESULTS: The odds for 30-day mortality increased incrementally as the hematocrit ranges decreased, in which preoperative hematocrit between 21 and 24% had the highest odds for this outcome (odds ratio [OR] 6.50, p < 0.0001) compared to the reference group (no anemia). The use of transfusion increased the odds of mortality even further (OR 5.57, p < 0.0001). Among patients that received an intra-/postoperative transfusion, preoperative anemia was not predictive of mortality. CONCLUSIONS: Healthcare providers making preoperative clinical decisions for patients undergoing elective surgery should consider the degree of preoperative anemia and likelihood of perioperative transfusion.


Assuntos
Transfusão de Sangue , Procedimentos Cirúrgicos Eletivos/mortalidade , Hematócrito , Idoso , Anemia/mortalidade , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/mortalidade , Cuidados Pré-Operatórios , Estudos Retrospectivos , Fatores de Risco
9.
Crit Care Nurs Q ; 41(4): 413-425, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30153186

RESUMO

Intra-hospital transport (IHT) of intensive care unit (ICU) patients is associated with a 30% to 60% incidence of adverse events (AEs). This prospective observational study collected data from 200 patient transports from a 24-bed cardiovascular intensive care unit (ICU) between July 2017 and December 2017. Phase 1 of the study focused on identifying and correcting deficiencies in nurses' knowledge regarding IHT. Phase 2 observed the occurrence and type of AEs during the IHT of ICU patients with and without physician accompaniment. The preeducation mean nursing knowledge score was 30.8 ± 10.2 (scale 0-100), and postcurriculum test mean score was 80 ± 20.2 (p < .001). In a series of 200 ICU transports, the incidence of AEs was 21.5% (n = 43). In patients who were unstable prior to transport, there was no difference in complications with or without a physician present (p = 0.40, χ = 0.696, odds ratio = 0.643, 95% confidence interval: 0.245-1.96). Patient needs during transport were met with preexisting orders or treatment orders received telephonically. Nurses' knowledge of transport standards improved significantly with education. Physician presence did not affect outcomes. The interventions needed to respond to complications did not require physician presence. In this cohort, there was no statistically significant benefit from physician attendance in transport.


Assuntos
Doenças Cardiovasculares/terapia , Cuidados Críticos/normas , Estado Terminal/terapia , Transporte de Pacientes , Enfermagem de Cuidados Críticos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
10.
J Cardiothorac Vasc Anesth ; 31(4): 1361-1369, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28455097

RESUMO

OBJECTIVE: Perioperative risk factors and the clinical impact of acute kidney injury (AKI) and failure after lung transplantation are not well described. The incidences of AKI and acute renal failure (ARF), potential perioperative contributors to their development, and postdischarge healthcare needs were evaluated. DESIGN: Retrospective. SETTING: University hospital. PARTICIPANTS: Patients undergoing lung transplantation between January 1, 2011 and December 31, 2015. MEASURED DATA: The incidences of AKI and ARF, as defined using the Risk, Injury, Failure, Loss, End-Stage Renal Disease criteria, were measured. Perioperative events were analyzed to identify risk factors for renal compromise. A comparison of ventilator days, intensive care unit (ICU) and hospital lengths of stay (LOS), 1-year readmissions, and emergency department visits was performed among AKI, ARF, and uninjured patients. MEASUREMENTS AND MAIN RESULTS: Ninety-seven patients underwent lung transplantation; 22 patients developed AKI and 35 patients developed ARF. Patients with ARF had significantly longer ICU LOS (12 days v 4 days, p < 0.001); ventilator days (4.5 days v 1 day, p < 0.001); and hospital LOS (22.5 days v 14 days, p < 0.001) compared with uninjured patients. Patients with AKI also had significantly longer ICU and hospital LOS. Patients with ARF had significantly more emergency department visits and hospital readmissions (2 v 1 readmissions, p = 0.002) compared with uninjured patients. A univariable analysis suggested that prolonged surgical time, intraoperative vasopressor use, and cardiopulmonary bypass use were associated with the highest increased risk for AKI. Intraoperative vasopressor use and cardiopulmonary bypass mean arterial pressure <60 mmHg were identified as independent risk factors by multivariable analysis for AKI. CONCLUSION: The severity of AKI was associated with an increase in the use of healthcare resources after surgery and discharge. Certain risk factors appeared modifiable and may reduce the incidence of AKI and ARF.


Assuntos
Injúria Renal Aguda/economia , Custos de Cuidados de Saúde , Recursos em Saúde/economia , Recursos em Saúde/estatística & dados numéricos , Transplante de Pulmão/economia , Complicações Pós-Operatórias/economia , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/etiologia , Adulto , Feminino , Custos de Cuidados de Saúde/tendências , Humanos , Transplante de Pulmão/efeitos adversos , Transplante de Pulmão/tendências , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/etiologia , Estudos Retrospectivos , Índice de Gravidade de Doença
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