RESUMO
Systemic perturbations can drive a neuroimmune cascade after surgical trauma, including affecting the blood-brain barrier (BBB), activating microglia, and contributing to cognitive deficits such as delirium. Delirium superimposed on dementia (DSD) is a particularly debilitating complication that renders the brain further vulnerable to neuroinflammation and neurodegeneration, albeit these molecular mechanisms remain poorly understood. Here, we have used an orthopedic model of tibial fracture/fixation in APPSwDI/mNos2-/- AD (CVN-AD) mice to investigate relevant pathogenetic mechanisms underlying DSD. We conducted the present study in 6-month-old CVN-AD mice, an age at which we speculated amyloid-ß pathology had not saturated BBB and neuroimmune functioning. We found that URMC-099, our brain-penetrant anti-inflammatory neuroprotective drug, prevented inflammatory endothelial activation, breakdown of the BBB, synapse loss, and microglial activation in our DSD model. Taken together, our data link post-surgical endothelial activation, microglial MafB immunoreactivity, and synapse loss as key substrates for DSD, all of which can be prevented by URMC-099.
Assuntos
Delírio , Demência , Animais , Delírio/complicações , Delírio/prevenção & controle , Demência/etiologia , Demência/prevenção & controle , Hipocampo/metabolismo , Camundongos , Piridinas , Pirróis/uso terapêuticoRESUMO
Multiple sclerosis (MS) is an inflammatory, neurodegenerative disease of the CNS characterized by both grey and white matter injury. Microglial activation and a reduction in synaptic density are key features of grey matter pathology that can be modeled with MOG35-55 experimental autoimmune encephalomyelitis (EAE). Complement deposition combined with microglial engulfment has been shown during normal development and in disease as a mechanism for pruning synapses. We tested whether there is excess complement production in the EAE hippocampus and whether complement-dependent synapse loss is a source of degeneration in EAE using C1qa and C3 knockout mice. We found that C1q and C3 protein and mRNA levels were elevated in EAE mice. Genetic loss of C3 protected mice from EAE-induced synapse loss, reduced microglial activation, decreased the severity of the EAE clinical score, and protected memory/freezing behavior after contextual fear conditioning. C1qa KO mice with EAE showed little to no change on these measurements compared to WT EAE mice. Thus, pathologic expression and activation of the early complement pathway, specifically at the level of C3, contributes to hippocampal grey matter pathology in the EAE.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Doenças Neurodegenerativas , Animais , Camundongos , Camundongos Endogâmicos C57BL , SinapsesRESUMO
OBJECTIVE: Interactions between enzyme-inducing anti-seizure medications (EI-ASMs) and antiretroviral drugs (ARVs) can lead to decreased ARV levels and may increase the likelihood of viral resistance. We conducted a study to determine if co-usage of ARVs and EI-ASMs is associated with ARV-resistant human immunodeficiency virus (HIV) among people living with HIV in Zambia. METHODS: Eligible participants were ≥18 years of age and concurrently taking ASMs and ARVs for at least 1 month of the prior 6-month period. Data were obtained regarding medication and HIV history. CD4 counts, plasma viral loads (pVLs), and HIV genotype and resistance profile in participants with a pVL >1000 copies/mL were obtained. Pearson's test of independence was used to determine whether treatment with EI-ASM was associated with pVL >1000/mL copies. RESULTS: Of 50 participants, 41 (82%) were taking carbamazepine (37 on monotherapy), and all had stable regimens in the prior 6 months. Among the 13 ARV regimens used, 68% had a tenofovir/lamivudine backbone. The majority (94%) were on a stable ARV regimen for >6 months. Median CD4 nadir was 205 cells/mm3 (interquartile range [IQR] 88-389), and 60% of participants had commenced ARV treatment before advanced disease occurred. Mean CD4 count at enrollment was 464 cells/mm3 (SD 226.3). Seven participants (14%) had a CD4 count <200 cells/mm3 . Four (8%) had a pVL >1000 copies/mL; all were on carbamazepine. Three participants with elevated pVL had a CD4 count <200 cells/mm3 . None had documented adherence concerns by providers; however, two had events concerning for clinical failure. HIV genotype testing showed mutations in three participants. Carbamazepine was not found to correlate with elevated pVL (P = .58). SIGNIFICANCE: EI-ASMs are commonly used in sub-Saharan Africa. Despite concurrent use of EI-ASMs and ARVs, the majority of participants showed CD4 counts >200 cells/mm3 and were virally suppressed. Carbamazepine was not associated with an increased risk of virological failure or ARV-resistant HIV.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Anticonvulsivantes/uso terapêutico , Carbamazepina/uso terapêutico , Epilepsia/tratamento farmacológico , Infecções por HIV/tratamento farmacológico , Adulto , Instituições de Assistência Ambulatorial/estatística & dados numéricos , Fármacos Anti-HIV/efeitos adversos , Anticonvulsivantes/efeitos adversos , Contagem de Linfócito CD4 , Carbamazepina/efeitos adversos , Interações Medicamentosas , Farmacorresistência Viral , Epilepsia/complicações , Feminino , Infecções por HIV/complicações , Humanos , Masculino , Resultado do Tratamento , Carga Viral/efeitos dos fármacos , ZâmbiaRESUMO
OBJECTIVE: The present work evaluates the relationship between postoperative immune and neurovascular changes and the pathogenesis of surgery-induced delirium superimposed on dementia. BACKGROUND AND RATIONALE: Postoperative delirium is a common complication in many older adults and in patients with dementia including Alzheimer's disease (AD). The course of delirium can be particularly debilitating, while its pathophysiology remains poorly defined. HISTORICAL EVOLUTION: As of 2019, an estimated 5.8 million people of all ages have been diagnosed with AD, 97% of whom are >65 years of age. Each year, many of these patients require surgery. However, anesthesia and surgery can increase the risk for further cognitive decline. Surgery triggers neuroinflammation both in animal models and in humans, and a failure to resolve this inflammatory state may contribute to perioperative neurocognitive disorders as well as neurodegenerative pathology. UPDATED HYPOTHESIS: We propose an immunovascular hypothesis whereby dysregulated innate immunity negatively affects the blood-brain interface, which triggers delirium and thereby exacerbates AD neuropathology. EARLY EXPERIMENTAL DATA: We have developed a translational model to study delirium superimposed on dementia in APPSwDI/mNos2-/- AD mice (CVN-AD) after orthopedic surgery. At 12 months of age, CVN-AD showed distinct neuroimmune and vascular impairments after surgery, including acute microgliosis and amyloid-ß deposition. These changes correlated with attention deficits, a core feature of delirium-like behavior. FUTURE EXPERIMENTS AND VALIDATION STUDIES: Future research should determine the extent to which prevention of surgery-induced microgliosis and/or neurovascular unit dysfunction can prevent or ameliorate postoperative memory and attention deficits in animal models. Translational human studies should evaluate perioperative indices of innate immunity and neurovascular integrity and assess their potential link to perioperative neurocognitive disorders. MAJOR CHALLENGES FOR THE HYPOTHESIS: Understanding the complex relationships between delirium and dementia will require mechanistic studies aimed at evaluating the role of postoperative neuroinflammation and blood-brain barrier changes in the setting of pre-existing neurodegenerative and/or aging-related pathology. LINKAGE TO OTHER MAJOR THEORIES: Non-resolving inflammation with vascular disease that leads to cognitive impairments and dementia is increasingly important in risk stratification for AD in the aging population. The interdependence of these factors with surgery-induced neuroinflammation and cognitive dysfunction is also becoming apparent, providing a strong platform for assessing the relationship between postoperative delirium and longer term cognitive dysfunction in older adults.
Assuntos
Delírio/fisiopatologia , Demência/complicações , Inflamação , Complicações Pós-Operatórias , Animais , Barreira Hematoencefálica , Encéfalo/patologia , Transtornos Cognitivos/etiologia , Modelos Animais de Doenças , Humanos , Camundongos , Transtornos NeurocognitivosRESUMO
BACKGROUND: Patients with pre-existing neurodegenerative disease commonly experience fractures that require orthopedic surgery. Perioperative neurocognitive disorders (PND), including delirium and postoperative cognitive dysfunction, are serious complications that can result in increased 1-year mortality when superimposed on dementia. Importantly, there are no disease-modifying therapeutic options for PND. Our lab developed the "broad spectrum" mixed-lineage kinase 3 inhibitor URMC-099 to inhibit pathological innate immune responses that underlie neuroinflammation-associated cognitive dysfunction. Here, we test the hypothesis that URMC-099 can prevent surgery-induced neuroinflammation and cognitive impairment. METHODS: Orthopedic surgery was performed by fracturing the tibia of the left hindlimb with intramedullary fixation under general anesthesia and analgesia. In a pilot experiment, 9-month-old mice were treated five times with URMC-099 (10 mg/kg, i.p.), spaced 12 h apart, with three doses prior to surgery and two doses following surgery. In this experiment, microgliosis was evaluated using unbiased stereology and blood-brain barrier (BBB) permeability was assessed using immunoglobulin G (IgG) immunostaining. In follow-up experiments, 3-month-old mice were treated only three times with URMC-099 (10 mg/kg, i.p.), spaced 12 h apart, prior to orthopedic surgery. Two-photon scanning laser microscopy and CLARITY with light-sheet microscopy were used to define surgery-induced changes in microglial dynamics and morphology, respectively. Surgery-induced memory impairment was assessed using the "What-Where-When" and Memory Load Object Discrimination tasks. The acute peripheral immune response to surgery was assessed by cytokine/chemokine profiling and flow cytometry. Finally, long-term fracture healing was assessed in fracture callouses using micro-computerized tomography (microCT) and histomorphometry analyses. RESULTS: Orthopedic surgery induced BBB disruption and microglial activation, but had no effect on microglial process motility. Surgically treated mice exhibited impaired object place and identity discrimination in the "What-Where-When" and Memory Load Object Discrimination tasks. Both URMC-099 dosing paradigms prevented the neuroinflammatory sequelae that accompanied orthopedic surgery. URMC-099 prophylaxis had no effect on the mobilization of the peripheral innate immune response and fracture healing. CONCLUSIONS: These findings show that prophylactic URMC-099 treatment is sufficient to prevent surgery-induced microgliosis and cognitive impairment without affecting fracture healing. Together, these findings provide compelling evidence for the advancement of URMC-099 as a therapeutic option for PND.
Assuntos
Disfunção Cognitiva/prevenção & controle , MAP Quinase Quinase Quinases/antagonistas & inibidores , Microglia/efeitos dos fármacos , Assistência Perioperatória , Piridinas/uso terapêutico , Pirróis/uso terapêutico , Animais , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Feminino , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/patologia , Transtornos Neurocognitivos/tratamento farmacológico , Transtornos Neurocognitivos/metabolismo , Transtornos Neurocognitivos/patologia , Assistência Perioperatória/métodos , Piridinas/farmacologia , Pirróis/farmacologia , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
Microglial activation, increased proinflammatory cytokine production, and a reduction in synaptic density are key pathological features associated with HIV-associated neurocognitive disorders (HAND). Even with combination antiretroviral therapy (cART), more than 50% of HIV-positive individuals experience some type of cognitive impairment. Although viral replication is inhibited by cART, HIV proteins such as Tat are still produced within the nervous system that are neurotoxic, involved in synapse elimination, and provoke enduring neuroinflammation. As complement deposition on synapses followed by microglial engulfment has been shown during normal development and disease to be a mechanism for pruning synapses, we have tested whether complement is required for the loss of synapses that occurs after a cortical Tat injection mouse model of HAND. In Tat-injected animals evaluated 7 or 28 days after injection, levels of early complement pathway components, C1q and C3, are significantly elevated and associated with microgliosis and a loss of synapses. However, C1qa knockout mice have the same level of Tat-induced synapse loss as wild-type (WT) mice, showing that the C1q-initiated classical complement cascade is not driving synapse removal during HIV1 Tat-induced neuroinflammation.
Assuntos
Disfunção Cognitiva/patologia , Complemento C1q/metabolismo , Infecções por HIV/complicações , Sinapses/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia , Animais , Medula Óssea/metabolismo , Transplante de Medula Óssea , Proteínas de Ligação ao Cálcio/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/virologia , Complemento C1q/genética , Complemento C3/farmacologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Gliose/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Sinapses/metabolismo , Sinapses/patologiaRESUMO
BACKGROUND: The mixed lineage kinase type 3 inhibitor URMC-099 facilitates amyloid-beta (Aß) clearance and degradation in cultured murine microglia. One putative mechanism is an effect of URMC-099 on Aß uptake and degradation. As URMC-099 promotes endolysosomal protein trafficking and reduces Aß microglial pro-inflammatory activities, we assessed whether these responses affect Aß pathobiogenesis. To this end, URMC-099's therapeutic potential, in Aß precursor protein/presenilin-1 (APP/PS1) double-transgenic mice, was investigated in this model of Alzheimer's disease (AD). METHODS: Four-month-old APP/PS1 mice were administered intraperitoneal URMC-099 injections at 10 mg/kg daily for 3 weeks. Brain tissues were examined by biochemical, molecular and immunohistochemical tests. RESULTS: URMC-099 inhibited mitogen-activated protein kinase 3/4-mediated activation and attenuated ß-amyloidosis. Microglial nitric oxide synthase-2 and arginase-1 were co-localized with lysosomal-associated membrane protein 1 (Lamp1) and Aß. Importatly, URMC-099 restored synaptic integrity and hippocampal neurogenesis in APP/PS1 mice. CONCLUSIONS: URMC-099 facilitates Aß clearance in the brain of APP/PS1 mice. The multifaceted immune modulatory and neuroprotective roles of URMC-099 make it an attractive candidate for ameliorating the course of AD. This is buttressed by removal of pathologic Aß species and restoration of the brain's microenvironment during disease.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Modelos Animais de Doenças , Piridinas/uso terapêutico , Pirróis/uso terapêutico , Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/genética , Animais , Células Cultivadas , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Presenilina-1/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/farmacologia , Pirróis/farmacologiaRESUMO
Gray matter degeneration contributes to progressive disability in multiple sclerosis (MS) and can occur out of proportion to measures of white matter disease. Although white matter pathology, including demyelination and axon injury, can lead to secondary gray matter changes, we hypothesized that neurons can undergo direct excitatory injury within the gray matter independent of these. We tested this using a model of experimental autoimmune encephalomyelitis (EAE) with hippocampal degeneration in C57BL/6 mice, in which immunofluorescent staining showed a 28% loss of PSD95-positive excitatory postsynaptic puncta in hippocampal area CA1 compared with sham-immunized controls, despite preservation of myelin and VGLUT1-positive excitatory axon terminals. Loss of postsynaptic structures was accompanied by appearance of PSD95-positive debris that colocalized with the processes of activated microglia at 25 d after immunization, and clearance of debris was followed by persistently reduced synaptic density at 55 d. In vitro, addition of activated BV2 microglial cells to hippocampal cultures increased neuronal vulnerability to excitotoxic dendritic damage following a burst of synaptic activity in a manner dependent on platelet-activating factor receptor (PAFR) signaling. In vivo treatment with PAFR antagonist BN52021 prevented PSD95-positive synapse loss in hippocampi of mice with EAE but did not affect development of EAE or local microglial activation. These results demonstrate that postsynaptic structures can be a primary target of injury within the gray matter in autoimmune neuroinflammatory disease, and suggest that this may occur via PAFR-mediated modulation of activity-dependent synaptic physiology downstream of microglial activation. SIGNIFICANCE STATEMENT: Unraveling gray matter degeneration is critical for developing treatments for progressive disability and cognitive impairment in multiple sclerosis (MS). In a mouse model of MS, we show that neurons can undergo injury at their synaptic connections within the gray matter, independent of the white matter pathology, demyelination, and axon injury that have been the focus of most current and emerging treatments. Damage to excitatory synapses in the hippocampus occurs in association with activated microglia, which can promote excitotoxic injury via activation of receptors for platelet-activating factor, a proinflammatory signaling molecule elevated in the brain in MS. Platelet-activating factor receptor blockade protected synapses in the mouse model, identifying a potential target for neuroprotective treatments in MS.
Assuntos
Pareamento Cromossômico/fisiologia , Encefalomielite Autoimune Experimental/patologia , Hipocampo/patologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Modelos Animais de Doenças , Proteína 4 Homóloga a Disks-Large , Feminino , Fibrinolíticos/farmacologia , Ginkgolídeos/farmacologia , Substância Cinzenta/metabolismo , Substância Cinzenta/patologia , Guanilato Quinases/metabolismo , Lactonas/farmacologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microglia/metabolismo , Microglia/patologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/imunologia , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
Leucine-rich repeat kinase 2 (LRRK2) is the single most common genetic cause of both familial and sporadic Parkinson's disease (PD), both of which share pathogenetic and neurologic similarities with human immunodeficiency virus 1 (HIV-1)-associated neurocognitive disorders (HAND). Pathologic LRRK2 activity may also contribute to neuroinflammation, because microglia lacking LRRK2 exposed to proinflammatory stimuli have attenuated responses. Because microglial activation is a hallmark of HIV-1 neuropathology, we have investigated the role of LRRK2 activation using in vitro and in vivo models of HAND. We hypothesize that LRRK2 is a key modulator of microglial inflammatory responses, which play a pathogenic role in both HAND and PD, and that these responses may cause or exacerbate neuronal damage in these diseases. The HIV-1 Tat protein is a potent neurotoxin produced during HAND that induces activation of primary microglia in culture and long-lasting neuroinflammation and neurotoxicity when injected into the CNS of mice. We found that LRRK2 inhibition attenuates Tat-induced pS935-LRRK2 expression, proinflammatory cytokine and chemokine expression, and phosphorylated p38 and Jun N-terminal kinase signaling in primary microglia. In our murine model, cortical Tat injection in LRRK2 knock-out (KO) mice results in significantly diminished neuronal damage, as assessed by microtubule-associated protein 2 (MAP2), class III ß-tubulin TUJ1, synapsin-1, VGluT, and cleaved caspase-3 immunostaining. Furthermore, Tat-injected LRRK2 KO animals have decreased infiltration of peripheral neutrophils, and the morphology of microglia from these mice were similar to that of vehicle-injected controls. We conclude that pathologic activation of LRRK2 regulates a significant component of the neuroinflammation associated with HAND.
Assuntos
Transtornos Cognitivos/metabolismo , Infecções por HIV/metabolismo , Inflamação/metabolismo , Degeneração Neural/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Complexo AIDS Demência/complicações , Complexo AIDS Demência/metabolismo , Animais , Biomarcadores/metabolismo , Córtex Cerebral/efeitos dos fármacos , Transtornos Cognitivos/complicações , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Produtos do Gene tat/administração & dosagem , Produtos do Gene tat/toxicidade , Infecções por HIV/complicações , Inflamação/induzido quimicamente , Mediadores da Inflamação/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Camundongos Knockout , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microinjeções , Fármacos Neuroprotetores/metabolismo , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Amyloid-ß (Aß)-stimulated microglial inflammatory responses engage mitogen-activated protein kinase (MAPK) pathways in Alzheimer's disease (AD). Mixed-lineage kinases (MLKs) regulate upstream MAPK signaling that include p38 MAPK and c-Jun amino-terminal kinase (JNK). However, whether MLK-MAPK pathways affect Aß-mediated neuroinflammation is unknown. To this end, we investigated if URMC-099, a brain-penetrant small-molecule MLK type 3 inhibitor, can modulate Aß trafficking and processing required for generating AD-associated microglial inflammatory responses. METHODS: Aß1-42 (Aß42) and/or URMC-099-treated murine microglia were investigated for phosphorylated mitogen-activated protein kinase kinase (MKK)3, MKK4 (p-MKK3, p-MKK4), p38 (p-p38), and JNK (p-JNK). These pathways were studied in tandem with the expression of the pro-inflammatory cytokines interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α. Gene expression of the anti-inflammatory cytokines, IL-4 and IL-13, was evaluated by real-time quantitative polymerase chain reaction. Aß uptake and expression of scavenger receptors were measured. Protein trafficking was assessed by measures of endolysosomal markers using confocal microscopy. RESULTS: Aß42-mediated microglial activation pathways were shown by phosphorylation of MKK3, MKK4, p38, and JNK and by expression of IL-1ß, IL-6, and TNF-α. URMC-099 modulated microglial inflammatory responses with induction of IL-4 and IL-13. Phagocytosis of Aß42 was facilitated by URMC-099 with up-regulation of scavenger receptors. Co-localization of Aß and endolysosomal markers associated with enhanced Aß42 degradation was observed. CONCLUSIONS: URMC-099 reduced microglial inflammatory responses and facilitated phagolysosomal trafficking with associated Aß degradation. These data demonstrate a new immunomodulatory role for URMC-099 to inhibit MLK and to induce microglial anti-inflammatory responses. Thus, URMC-099 may be developed further as a novel disease-modifying AD therapy.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Citocinas/metabolismo , Microglia/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pirróis/farmacologia , Peptídeos beta-Amiloides/farmacologia , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Células Cultivadas , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Camundongos , Microscopia Confocal , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estatísticas não Paramétricas , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
During studies to extend the half-life of crystalline nanoformulated antiretroviral therapy (nanoART) the mixed lineage kinase-3 inhibitor URMC-099, developed as an adjunctive neuroprotective agent was shown to facilitate antiviral responses. Long-acting ritonavir-boosted atazanavir (nanoATV/r) nanoformulations co-administered with URMC-099 reduced viral load and the numbers of HIV-1 infected CD4+ T-cells in lymphoid tissues more than either drug alone in infected humanized NOD/SCID/IL2Rγc-/- mice. The drug effects were associated with sustained ART depots. Proteomics analyses demonstrated that the antiretroviral responses were linked to affected phagolysosomal storage pathways leading to sequestration of nanoATV/r in Rab-associated recycling and late endosomes; sites associated with viral maturation. URMC-099 administered with nanoATV induced a dose-dependent reduction in HIV-1p24 and reverse transcriptase activity. This drug combination offers a unique chemical marriage for cell-based viral clearance. From the Clinical Editor: Although successful in combating HIV-1 infection, the next improvement in antiretroviral therapy (nanoART) would be to devise long acting therapy, such as intra-cellular depots. In this report, the authors described the use of nanoformulated antiretroviral therapy given together with the mixed lineage kinase-3 inhibitor URMC-099, and showed that this combination not only prolonged drug half-life, but also had better efficacy. The findings are hoped to be translated into the clinical setting in the future.
Assuntos
Sulfato de Atazanavir/administração & dosagem , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Nanocápsulas/química , Piridinas/administração & dosagem , Pirróis/administração & dosagem , Animais , Antirretrovirais/administração & dosagem , Terapia Antirretroviral de Alta Atividade/métodos , Quimioterapia Combinada/métodos , Infecções por HIV/diagnóstico , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Camundongos , Camundongos SCID , Nanocápsulas/administração & dosagem , Nanocápsulas/ultraestrutura , Inibidores de Proteínas Quinases/administração & dosagem , Resultado do Tratamento , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
UNLABELLED: Limitations of antiretroviral therapy (ART) include poor patient adherence, drug toxicities, viral resistance, and failure to penetrate viral reservoirs. Recent developments in nanoformulated ART (nanoART) could overcome such limitations. To this end, we now report a novel effect of nanoART that facilitates drug depots within intracellular compartments at or adjacent to the sites of the viral replication cycle. Poloxamer 407-coated nanocrystals containing the protease inhibitor atazanavir (ATV) were prepared by high-pressure homogenization. These drug particles readily accumulated in human monocyte-derived macrophages (MDM). NanoATV concentrations were â¼1,000 times higher in cells than those that could be achieved by the native drug. ATV particles in late and recycling endosome compartments were seen following pulldown by immunoaffinity chromatography with Rab-specific antibodies conjugated to magnetic beads. Confocal microscopy provided cross validation by immunofluorescent staining of the compartments. Mathematical modeling validated drug-endosomal interactions. Measures of reverse transcriptase activity and HIV-1 p24 levels in culture media and cells showed that such endosomal drug concentrations enhanced antiviral responses up to 1,000-fold. We conclude that late and recycling endosomes can serve as depots for nanoATV. The colocalization of nanoATV at endosomal sites of viral assembly and its slow release sped antiretroviral activities. Long-acting nanoART can serve as a drug carrier in both cells and subcellular compartments and, as such, can facilitate viral clearance. IMPORTANCE: The need for long-acting ART is significant and highlighted by limitations in drug access, toxicity, adherence, and reservoir penetrance. We propose that targeting nanoformulated drugs to infected tissues, cells, and subcellular sites of viral replication may improve clinical outcomes. Endosomes are sites for human immunodeficiency virus assembly, and increasing ART concentrations in such sites enhances viral clearance. The current work uncovers a new mechanism by which nanoART can enhance viral clearance over native drug formulations.
Assuntos
Antirretrovirais/farmacocinética , Endossomos/metabolismo , HIV-1/efeitos dos fármacos , Macrófagos/metabolismo , Nanopartículas , Oligopeptídeos/farmacocinética , Poloxâmero/farmacocinética , Piridinas/farmacocinética , Antirretrovirais/farmacologia , Sulfato de Atazanavir , Transporte Biológico , Células Cultivadas , Proteína do Núcleo p24 do HIV/análise , HIV-1/crescimento & desenvolvimento , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Modelos Teóricos , Oligopeptídeos/farmacologia , Poloxâmero/farmacologia , Piridinas/farmacologia , Cultura de VírusRESUMO
Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) is a significant source of disability in the HIV-infected population. Even with stringent adherence to anti-retroviral therapy, >50% of patients living with HIV-1 will develop HAND (Heaton et al., 2010). Because suppression of viral replication alone is not enough to stop HAND progression, there is a need for an adjunctive neuroprotective therapy in this population. To this end, we have developed a small-molecule brain-penetrant inhibitor with activity against mixed-lineage kinase 3 (MLK3), named URMC-099. MLK3 activation is associated with many of the pathologic hallmarks of HAND (Bodner et al., 2002, 2004; Sui et al., 2006) and therefore represents a prime target for adjunctive therapy based on small-molecule kinase inhibition. Here we demonstrate the anti-inflammatory and neuroprotective effects of URMC-099 in multiple murine and rodent models of HAND. In vitro, URMC-099 treatment reduced inflammatory cytokine production by HIV-1 Tat-exposed microglia and prevented destruction and phagocytosis of cultured neuronal axons by these cells. In vivo, URMC-099 treatment reduced inflammatory cytokine production, protected neuronal architecture, and altered the morphologic and ultrastructural response of microglia to HIV-1 Tat exposure. In conclusion, these data provide compelling in vitro and in vivo evidence to investigate the utility of URMC-099 in other models of HAND with the goal of advancement to an adjunctive therapeutic agent.
Assuntos
Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Inflamação/prevenção & controle , MAP Quinase Quinase Quinases/antagonistas & inibidores , Fármacos Neuroprotetores/uso terapêutico , Animais , Transplante de Medula Óssea , Receptor 1 de Quimiocina CX3C , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/virologia , Células Cultivadas , Citocinas , Modelos Animais de Doenças , Embrião de Mamíferos , Produtos do Gene tat/imunologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Hipocampo/patologia , Humanos , Inflamação/genética , Inflamação/patologia , Inflamação/virologia , Camundongos , Camundongos Transgênicos , Microscopia Imunoeletrônica , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Piridinas/uso terapêutico , Pirróis/farmacologia , Pirróis/uso terapêutico , Ratos , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Estatísticas não Paramétricas , Fatores de Tempo , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência Humana , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
BACKGROUND: Seizures are relatively common among children with HIV in low- and middle-income countries and are associated with significant morbidity and mortality. Early treatment with antiretroviral therapy (ART) may reduce this risk by decreasing rates of central nervous system infections and HIV encephalopathy. METHODS: We conducted a prospective, unmatched case-control study. We enrolled children with new-onset seizure from University Teaching Hospital in Lusaka, Zambia and 2 regional hospitals in rural Zambia. Controls were children with HIV and no history of seizures. Recruitment took place from 2016 to 2019. Early treatment was defined as initiation of ART before 12 months of age, at a CD4 percentage >15% in children aged 12-60 months or a CD4 count >350 cells/mm 3 for children aged 60 months or older. Logistic regression models were used to evaluate the association between potential risk factors and seizures. RESULTS: We identified 73 children with new-onset seizure and compared them with 254 control children with HIV but no seizures. Early treatment with ART was associated with a significant reduction in the odds of seizures [odds ratio (OR) 0.04, 95% confidence interval: 0.02 to 0.09; P < 0.001]. Having an undetectable viral load at the time of enrollment was strongly protective against seizures (OR 0.03, P < 0.001), whereas history of World Health Organization Stage 4 disease (OR 2.2, P = 0.05) or CD4 count <200 cells/mm 3 (OR 3.6, P < 0.001) increased risk of seizures. CONCLUSIONS: Early initiation of ART and successful viral suppression would likely reduce much of the excess seizure burden in children with HIV.
Assuntos
Fármacos Anti-HIV , Infecções por HIV , Criança , Humanos , Lactente , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Zâmbia/epidemiologia , Estudos de Casos e Controles , Fatores de Risco , Convulsões/tratamento farmacológico , Convulsões/prevenção & controle , Convulsões/complicações , Contagem de Linfócito CD4 , Fármacos Anti-HIV/uso terapêuticoRESUMO
OBJECTIVE: To determine the long-term outcomes, including mortality and recurrent seizures, among children living with HIV (CLWH) who present with new onset seizure. METHODS: Zambian CLWH and new onset seizure were enrolled prospectively to determine the risk of and risk factors for recurrent seizures. Demographic data, clinical profiles, index seizure etiology, and 30-day mortality outcomes were previously reported. After discharge, children were followed quarterly to identify recurrent seizures and death. Given the high risk of early death, risk factors for recurrent seizure were evaluated using a model that adjusted for mortality. RESULTS: Among 73 children enrolled, 28 died (38%), 22 within 30-days of the index seizure. Median follow-up was 533 days (IQR 18-957) with 5% (4/73) lost to follow-up. Seizure recurrence was 19% among the entire cohort. Among children surviving at least 30-days after the index seizure, 27% had a recurrent seizure. Median time from index seizure to recurrent seizure was 161 days (IQR 86-269). Central nervous system opportunistic infection (CNS OI), as the cause for the index seizure was protective against recurrent seizures and higher functional status was a risk factor for seizure recurrence. SIGNIFICANCE: Among CLWH presenting with new onset seizure, mortality risks remain elevated beyond the acute illness period. Recurrent seizures are common and are more likely in children with higher level of functioning even after adjusting for the outcome of death. Newer antiseizure medications appropriate for co-usage with antiretroviral therapies are needed for the care of these children. CNS OI may represent a potentially reversible provocation for the index seizure, while seizures in high functioning CLWH without a CNS OI may be the result of a prior brain injury or susceptibility to seizures unrelated to HIV and thus represent an ongoing predisposition to seizures. PLAIN LANGUAGE SUMMARY: This study followed CLWH who experienced a new onset seizure to find out how many go on to have more seizures and identify any patient characteristics associated with having more seizures. The study found that mortality rates continue to be high beyond the acute clinical presentation with new onset seizure. Children with a CNS OI causing the new onset seizure had a lower risk of later seizures, possibly because the trigger for the seizure can be treated. In contrast, high functioning children without a CNS OI were at higher risk of future seizures.
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Epilepsia Generalizada , Infecções por HIV , Criança , Humanos , Anticonvulsivantes/uso terapêutico , Estudos de Coortes , Convulsões/tratamento farmacológico , Epilepsia Generalizada/tratamento farmacológico , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Dano Encefálico Crônico/induzido quimicamente , Dano Encefálico Crônico/complicações , Dano Encefálico Crônico/tratamento farmacológicoRESUMO
CEP-1347 is a potent inhibitor of mixed lineage kinase (MLK), which was investigated for ameliorating HIV-associated neurocognitive disorders. CEP-1347 and atazanavir pharmacokinetics were determined when CEP-1347 50 mg twice daily was administered to HIV-infected patients (n = 20) receiving combination antiretroviral therapy including atazanavir and ritonavir (ATV/RTV, 300/100 mg) once daily continuously. Co-administration of CEP-1347 and ATV/RTV resulted with significant changes in pharmacokinetics of ATV but not RTV. Specifically, an increase in ATV accumulation ratio of 15 % (p = 0.007) and a prolongation of T(½) from 12.7 to 15.9 h (p = 0.002) were observed. The results suggested that co-administration of CEP-1347 with ATV/RTV in HIV-infected patients might result in limited impact on ATV but not on RTV pharmacokinetics.
Assuntos
Fármacos Anti-HIV/farmacocinética , Carbazóis/farmacocinética , Infecções por HIV/tratamento farmacológico , Nootrópicos/farmacocinética , Oligopeptídeos/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Piridinas/farmacocinética , Ritonavir/farmacocinética , Adulto , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/sangue , Sulfato de Atazanavir , Contagem de Linfócito CD4 , Carbazóis/administração & dosagem , Carbazóis/sangue , Esquema de Medicação , Interações Medicamentosas , Quimioterapia Combinada , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Meia-Vida , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Masculino , Pessoa de Meia-Idade , Nootrópicos/administração & dosagem , Nootrópicos/sangue , Oligopeptídeos/administração & dosagem , Oligopeptídeos/sangue , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/sangue , Piridinas/administração & dosagem , Piridinas/sangue , Ritonavir/administração & dosagem , Ritonavir/sangueRESUMO
Various oligomeric species of amyloid-beta have been proposed to play different immunogenic roles in the cellular pathology of Alzheimer's Disease. However, investigating the role of a homogenous single oligomeric species has been difficult due to highly dynamic oligomerization and fibril formation kinetics that convert between many species. Here we report the design and construction of a quantum dot mimetic for larger spherical oligomeric amyloid species as an "endogenously" fluorescent proxy for this cytotoxic species to investigate its role in inducing inflammatory and stress response states in neuronal and glial cell types.
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Background: Links between acute lung injury (ALI), infectious disease, and neurological outcomes have been frequently discussed over the past few years, especially due to the COVID-19 pandemic. Yet, much of the cross-communication between organs, particularly the lung and the brain, has been understudied. Here, we have focused on the role of neutrophils in driving changes to the brain endothelium with ensuing microglial activation and neuronal loss in a model of ALI. Methods: We have applied a three-dose paradigm of 10µg/40µl intranasal lipopolysaccharide (LPS) to induce neutrophilia accompanied by proteinaceous exudate in bronchoalveolar lavage fluid (BALF) in adult C57BL/6 mice. Brain endothelial markers, microglial activation, and neuronal cytoarchitecture were evaluated 24hr after the last intranasal dose of LPS or saline. C57BL/6-Ly6g(tm2621(Cre-tdTomato)Arte (Catchup mice) were used to measure neutrophil and blood-brain barrier permeability following LPS exposure with intravital 2-photon imaging. Results: Three doses of intranasal LPS induced robust neutrophilia accompanied by proteinaceous exudate in BALF. ALI triggered central nervous system pathology as highlighted by robust activation of the cerebrovascular endothelium (VCAM1, CD31), accumulation of plasma protein (fibrinogen), microglial activation (IBA1, CD68), and decreased expression of proteins associated with postsynaptic terminals (PSD-95) in the hippocampal stratum lacunosum moleculare, a relay station between the entorhinal cortex and CA1 of the hippocampus. 2-photon imaging of Catchup mice revealed neutrophil homing to the cerebral endothelium in the blood-brain barrier and neutrophil extravasation from cerebral vasculature 24hr after the last intranasal treatment. Conclusions: Overall, these data demonstrate ensuing brain pathology resulting from ALI, highlighting a key role for neutrophils in driving brain endothelial changes and subsequent neuroinflammation. This paradigm may have a considerable translational impact on understanding how infectious disease with ALI can lead to neurodegeneration, particularly in the elderly.
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Background: Links between acute lung injury (ALI), infectious disease, and neurological outcomes have been frequently discussed over the past few years, especially due to the COVID-19 pandemic. Yet, much of the cross-communication between organs, particularly the lung and the brain, has been understudied. Here, we have focused on the role of neutrophils in driving changes to the brain endothelium with ensuing microglial activation and neuronal loss in a model of ALI. Methods: We have applied a three-dose paradigm of 10µg/40µl intranasal lipopolysaccharide (LPS) to induce neutrophilia accompanied by proteinaceous exudate in bronchoalveolar lavage fluid (BALF) in adult C57BL/6 mice. Brain endothelial markers, microglial activation, and neuronal cytoarchitecture were evaluated 24hr after the last intranasal dose of LPS or saline. C57BL/6-Ly6g(tm2621(Cre-tdTomato)Arte (Catchup mice) were used to measure neutrophil and blood-brain barrier permeability following LPS exposure with intravital 2-photon imaging. Results: Three doses of intranasal LPS induced robust neutrophilia accompanied by proteinaceous exudate in BALF. ALI triggered central nervous system pathology as highlighted by robust activation of the cerebrovascular endothelium (VCAM1, CD31), accumulation of plasma protein (fibrinogen), microglial activation (IBA1, CD68), and decreased expression of proteins associated with postsynaptic terminals (PSD-95) in the hippocampal stratum lacunosum moleculare, a relay station between the entorhinal cortex and CA1 of the hippocampus. 2-photon imaging of Catchup mice revealed neutrophil homing to the cerebral endothelium in the blood-brain barrier and neutrophil extravasation from cerebral vasculature 24hr after the last intranasal treatment. Conclusions: Overall, these data demonstrate ensuing brain pathology resulting from ALI, highlighting a key role for neutrophils in driving brain endothelial changes and subsequent neuroinflammation. This paradigm may have a considerable translational impact on understanding how infectious disease with ALI can lead to neurodegeneration, particularly in the elderly.
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Despite limited evidence for infection of SARS-CoV-2 in the central nervous system, cognitive impairment is a common complication reported in "recovered" COVID-19 patients. Identification of the origins of these neurological impairments is essential to inform therapeutic designs against them. However, such studies are limited, in part, by the current status of high-fidelity probes to visually investigate the effects of SARS-CoV-2 on the system of blood vessels and nerve cells in the brain, called the neurovascular unit. Here, we report that nanocrystal quantum dot micelles decorated with spike protein (COVID-QDs) are able to interrogate neurological damage due to SARS-CoV-2. In a transwell co-culture model of the neurovascular unit, exposure of brain endothelial cells to COVID-QDs elicited an inflammatory response in neurons and astrocytes without direct interaction with the COVID-QDs. These results provide compelling evidence of an inflammatory response without direct exposure to SARS-CoV-2-like nanoparticles. Additionally, we found that pretreatment with a neuro-protective molecule prevented endothelial cell damage resulting in substantial neurological protection. These results will accelerate studies into the mechanisms by which SARS-CoV-2 mediates neurologic dysfunction.