Cardiovascular disease is the leading cause of morbidity and mortality in developed nations, and heart disease is predicted to remain the leading killer for the foreseeable future. Acute myocardial infarctions—nearly 1.1 million annually occurring in the U.S. alone—are the major cardiovascular disease subgroup. Current treatments for myocardial infarction are limited to interventions that serve to mitigate the initial insult, but clinical applications to protect or regenerate damaged myocardium are lacking. This dissertation examines three therapeutic systems to treat the infarcted heart. First, the decoration of a polymeric nanoparticle with N-acetylglucosamine for the uptake of anti-­apoptotic therapeutics to ameliorate cardiomyocyte cell death. Second, novel dendrimeric structure architecture to allow for regio­selected decoration of ligands to induce angiogenesis. Third, exosomes secreted from hypoxic cardiac progenitor cells as a naturally derived therapeuticfor angiogenesis and anti-fibrosis, and to provide bio-inspired clues for future therapies.

In this dissertation, an exosome capturing ciliary micropillar array microfluidics is introduced and applied to evaluate the response of resistant cancer cells under nanoparticle encapsulated chemotherapy. Cancer cells are able to develop different mechanisms to resist therapeutic treatment, frequently causing chemotherapy failure. Active drug expulsion is one of the usual resisting schemes to reduce intracellular drug accumulation to a non-effective level. Evidence has suggested a potential exosomal pathway is used by multi-drug resistant (MDR) cancer cells to expel drugs. Here I study the exosomes derived from MDR cancer cells treated by nanotherapeutics aiming to establish the correlation between nanotherapeutics and exosomal pathway for drug expulsion. The outcome would boost further understanding of cancer MDR, and in turn direct the development of pharmaceutical nanoparticles to overcome MDR cancer. To effectively isolate exosomes for drug expulsion evaluation, a ciliary micropillar structure is fabricated employing microelectromechanical systems (MEMS) and metal assisted chemical etching (MACE) techniques. The ciliary micropillar is fabricated in two major steps: deep silicon etch (DSE) for pillars followed by a MACE process to etch nanowires on the pillars. The concept of using MACE as an alternative to DSE is also explored to reduce fabrication cost. With optimized parameters, it shows a comparable result to DSE. COMSOL simulation revealed that ciliated micropillars exhibited a unique advantage as a unit structure for capturing small particles in fluid flow, according to particle filtration theory. A nanowire layer with high permittivity allows fluid streamlines to pass through, and increases interaction with particle carrying fluid to increase the probability of particle interception. Nanowires on the pillar can trap specific sized particles due to their characteristic dimension. Thanks to the weaker stability of porous silicon nanowires, trapped particles can then be released by dissolving these nanowires without damage to the particles themselves. A microfluidic chip is fabricated with an optimized circular micropillar arrangement for resistance reduction, and its particle filtration performance is demonstrated by processing model cell culture medium. The device is applied to study MDR cells’ response to micelle encapsulated paclitaxel treatment. Cell culture medium from sensitive and MDR variant of MDA-MB-231 cells treated with pure and capsulated drugs are processed through the device for exosome isolation. Drug volume in collected exosomes is determined after measurement. By measuring drug efflux through exosomes, it is determined that MDA-MB-231MDR cells do use an exosomal pathway to expel drugs, but other mechanisms are also at play. Nanoparticle encapsulation results in higher drug concentration in exosomes partly because the origin of exosomes and nanoparticle intake through endocytosis share some similar route. / text

Human immunodeficiency virus type 1 (HIV-1) is the cause of acquired immune deficiency syndrome (AIDS). HIV-1 is a worldwide epidemic that currently affects over 35 million people worldwide, and continues to spread at an appalling rate. A universal HIV-1 preventive vaccine is considered to be the optimal solution in achieving the ultimate goal of AIDS eradication. Regretfully, most endeavors thus far of developing a prophylactic vaccine have been largely disappointing. Highly Active Anti-Retroviral Therapy (HAART) has been shown to reduce the plasma HIV-1 RNA level to below the detection limit of clinical assays (50 copies/ml); it combines three or more antiretroviral drugs which belong to at least two different classes – targeting distinct steps in the viral life cycle, and inhibiting viral replication. However, unless the infection is eradicated, strict adherence to a lifelong treatment regimen is required. HAART is limited by its high cost, drug availability, complicated administration schedules, serious side effects, and the potential that the virus will ultimately develop drug resistance. A more plausible approach lies in therapeutic vaccines that provide immunity to partially control viral replication postinfection – delaying or minimizing ART, and offering “drug holidays”. The primary goal of a therapeutic vaccine is to effectively induce HIV-1 specific cytotoxic T lymphocyte (CTL) responses, which plays a critical role in control of viral proliferation. Dendritic cells (DCs)-based therapeutic vaccines have been showing the most promising results. However, the therapeutic efficacy of DCs based vaccines is limited. This is partially due to the fact that DCs induced CD8+ T cell responses are largely CD4+ T cell dependent, while HIV-1 infection usually renders the immune system very “helpless” from CD4+ T cells. In addition, infection, impaired function, and physical depletion of DCs are often reported during the early stage. Furthermore, DCs are often found to be inflammatory and immunosuppressive, which is mainly mediated by the interaction between HIV-1 Env gp120 and DC receptors. Thus, the search for a novel therapeutic vaccine strategy is warranted. Using T-APC (T cells-antigen-presenting cells) as a novel T cell-based vaccine has emerged as a potential candidate for a HIV-1 therapeutic vaccine, which aims at boosting HIV-specific CTL responses. Our previous work demonstrated that CD4+ and CD8+ T cells derived from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic OT II and OT I mice via co-culture with OVA-pulsed DCs (DCOVA) can be activated, acquiring pMHC I, pMHC II, and costimulatory molecules, thus act as CD4+ T helper-antigen-presenting cells (Th-APCs) and CD8+ cytotoxic T-antigen-presenting cells (Tc-APCs). We also elucidated that DC-derived exosomes (EXO), which are 50- to 90-nm diameter vesicles containing antigen-presenting, tetraspan, adhesion, and costimulatory molecules, can transfer the antigen-presenting activity of DCs to activated CD4+T cells through EXO uptake. EXOOVA-targeted activated CD4+T (aTexo) cells can (1) stimulate more efficient central memory CD8+ CTL responses and T cell memory than EXOOVA or DCOVA, (2) activate CD8+ CTL responses independent of CD4+Th cells, and (3) counteract CD4+25+regulatory T (Tr) cell-mediated immune suppression. These results formed the new concept of novel EXO-targeted CD4+ T cell vaccines. In this study, we tailored EXO-targeted T cells vaccine by using polyclonal activated CD8+ T cells instead of CD4+ T cells, as CD4+ T cells served as the primary target for HIV-1 infection. We showed that (1) OVA-specific exosome-targeted CD8+ T cell-based vaccine (OVA-Texo) can stimulate efficient OVA-specific CD8+ CTL and memory responses, inducing sufficient antitumor immunity against OVA-expressing tumor cells in mouse models. (2) This exosome-targeted CD8+ T cell-based vaccine strategy could be applied to HIV-1-Gag protein, provoking effective Gag-specific CD8+ CTL, T cell memory, and antitumor immunity against Gag-expressing tumor cells. (3) Engineering Gag-Texo with up-regulated 4-1BBL (APC derived costimulatory molecule) expression could improve the performance of Gag-Texo vaccine. (4) OVA-Texo is able to evoke a successful immune response in bystander chronic infection, converting CD8+ T cell exhaustion, restoring effector functions of exhausted CD8+ T cells. Moreover, combination of OVA-Texo vaccine with PD-L1 blockage in a dual treatment could result in a synergistic effect in rescuing CTLs exhaustion in chronic infection. Those desired features make EXO-targeted CD8+ T cells vaccine an appealing novel strategy in HIV-1 infection. The EXO-targeted CD8+ T cells vaccine may be applicable to therapeutic HIV treatment through the use of autologous T cells with uptake of EXOs derived from engineered DCs.

Eukaryotic gene expression requires hundreds of proteins and several RNA factors to facilitate nuclear RNA processing. These RNA processing events include RNA transcription, pre-mRNA splicing, pre-ribosomal RNA (pre-rRNA) processing and trafficking of RNA to its proper location in the cell. As we learn more about the molecular details of the factors governing these highly coordinated processes it is becoming increasingly clear that a subset of factors participate in multiple RNA processing pathways to ensure faithful gene expression. Our work completes the characterization of the Abelson pre-mRNA splicing mutants. We have discovered that the prp27-1 splicing mutant is a severe loss of function allele of RAT1, an essential 5’→3’ exoribonuclease. Several alleles of RAT1 have been previously isolated with each conferring an array of phenotypes thus making the elucidation of its essential in vivo function difficult. We set out to determine how mutations within a specific region determines the RNA processing pathway in which Rat1p has been implicated to function within. In our analysis of Rat1p function we discovered the prp27-1 allele exhibits novel 3’ end processing defects never reported in previous rat1 mutants. We performed mutational analysis to examine the coupling of 5’ and 3’ exonucleolytic activities in nuclear RNA processing events. Through our study we have discovered a means by which the cell coordinately regulates the nuclear RNA degradation complexes to ensure efficient processing of pre-RNAs for the faithful execution of eukaryotic gene expression. Additionally, we offer evidence in support of role for Rat1p in promoting mitotic events in vivo. / text

The ultimate goal of antitumor vaccines is to develop memory CD8+ cytotoxic T lymphocytes (CTLs), which are critical mediators of antitumor immunity. Previous work in our lab demonstrated that the ovalbumin (OVA)-specific CD4+ T cell-based (OVA-TEXO) vaccine generated using OVA-pulsed dendritic cell (DCOVA)-released exosomes (EXOOVA) stimulates CTL responses via interleukin (IL)-2 and costimulatory CD80 signaling. To assess the potential involvement of other costimulatory pathways and to define the key constituent of costimulation for memory CTL development, we first immunized wild-type (WT) C57BL/6 and gene-knockout mice with WT CD4+ OVA-TEXO cells or OVA-TEXO cells with various molecular deficiencies. We then assessed OVA-specific primary and recall CTL responses using PE-H-2Kb/OVA257–264 tetramer and FITC-anti-CD8 antibody staining by flow cytometry. We also examined antitumor immunity against the OVA-expressing B16 melanoma cell line BL6-10OVA. We demonstrate that CD4+ OVA-TEXO cells form immunological synapses with cognate CD8+ T cells in vitro. By assessment of the pattern of ex vivo interactions between OTI CD8+ T cells and OVA-TEXO or (Kb-/-)TEXO cells lacking peptide/major histocompatibitity complex (pMHC)-I expression, we provide the first visible evidence on the critical role of exosomal pMHC-I in targeting OVA-TEXO to cognate CD8+ T cells using two-photon microscopy. By assessing primary and recall CTL responses in mice immunized with OVA-TEXO cells or with OVA-TEXO cells lacking the costimulatory molecules CD40L, 4-1BBL or OX40L, we demonstrated that these costimulatory signals are dispensable for CTL priming by OVA-TEXO cells. Interestingly, CD40L, but not 4-1BBL or OX40L, plays a crucial role in the development of functional memory CTLs against BL6-10OVA tumors. Overall, this work suggests that a novel CD4+ T cell-based vaccine that is capable of stimulating long-term functional CTL memory via CD40L signaling may represent a novel, efficient approach to antitumor vaccination. Breast cancer is the most common cancer among women in the western world. Approximately 20-30% of invasive breast carcinomas are proto-oncogene human epidermal growth factor receptor (HER)-2 positive and associated with increased metastatic potential and poor prognosis. The survival benefit of anti-HER2 driven therapies demonstrated in clinical trials indicates that HER2 is one of the most promising molecules for targeted therapy to date. Above results prompt us to assess whether CD4+ T-cell-based vaccine can stimulate efficient HER2-specific CD8+ CTL responses and antitumor immunity in transgenic mice with HER2-specific self-immune tolerance. We prepared HER2-specific HER2-TEXO using ConA-stimulated CD4+ T cells with uptake of exosomes released from HER2-expressing AdVHER2-transfected DCs. We found that HER2-TEXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2/HER2-expressing B16 melanoma BL6-10HLA-A2/HER2 in 2/8 double transgenic HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Therefore, the novel T cell-based HER2-TEXO vaccine may provide a new therapeutic alternative for women with HER2+ breast cancer. In contrast to CD4+CD25+ regulatory T cells (Tregs), mechanisms of CD8+CD25+ Treg-mediated immunosuppression are not well understood. In this study, we purified polyclonal CD8+CD25+ Tregs from C57BL/6 mouse splenocytes and expanded them in culture medium containing CD3/CD28 microbeads. By using these amplified CD8+CD25+ Tregs, we demonstrated that CD8+CD25+ Tregs inhibit naive CD4+ T-cell proliferation and induce naive T-cell anergy by up-regulating T-cell anergy-associated early growth response 2 (EGR2), and by decreasing T-cell proliferation and IL-2-secretion upon stimulation. They also impact the expression of perforin on effector CTLs and directly induce perforin-mediated CTL apoptosis. CD8+CD25+ Tregs, when pulsed with OVA323-339 peptide, exert an enhanced inhibition. Interestingly, CD8+CD25+ Tregs, when pulsed with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, become capable of inhibiting MOG35-55-induced experimental autoimmune encephalomyelitis (EAE). Two-photon microscopic observations suggest that OVA323-339-pulsed (armed) CD8+CD25+ Tregs reduce the interactions between DCs and cognate CD4+ T cells ex vivo by increasing velocities of T cells in mouse lymph nodes. Therefore, redirecting antigen-specificity to nonspecific CD8+CD25+ Tregs can be achieved for enhanced immunosuppression through their arming with the antigen-specific pMHC-II complexes. This approach may have great impact on improvement of endogenous polyclonal Treg-mediated immunotherapy for autoimmune diseases. Taken together, our studies demonstrate that nonspecific polyclonal CD4+ T cells and CD8+CD25+ Tregs, when armed with HER2 and MOG antigen-specific pMHC-I and -II complexes, become capable of stimulating enhanced HER2-specific CTL responses and antitumor immunity in double transgenic HLA-A2/HER2 mice and inducing enhanced MOG-specific immunosuppression in MOG-induced EAE mice, respectively. Therefore, redirecting antigen specificity to nonspecific CD4+ T and CD8+CD25+ Tregs by pMHC complex arming may have great impact in development of novel T cell-based vaccines for treatment of cancer and autoimmune diseases.

<p>Due to its close proximity to the gastrointestinal tract, the human urinary tract is</p><p>subjected to constant barrage by gut-­associated bacteria. However, for the most part, this tract has resisted infection by various microbes. The impregnability of the urinary tract to microbial colonization is attributable to the ability of the bladder to promptly sense and mount robust responses to microbial challenge. A powerful mechanism for the elimination of invading bacteria was recently described in bladder epithelial cells, involving non-­lytic ejection of intracellular bacteria back into the extracellular milieu. In spite of the effectiveness of this defense strategy, much of the underlying mechanisms surrounding how this powerful cellular defense activity detects intracellular UPEC and shuttles them from their intracellular location to the plasma membrane of BECs to be exported remains largely a mystery.</p><p> Here, we describe uropathogenic E.coli (UPEC) expelled from infected bladder</p><p>epithelium cells (BECs) within membrane-­bound vesicles as a distinct cellular defense</p><p>response. Examination of the intracellular UPEC revealed that intracellular bacteria were</p><p>initially processed via autophagy, the conventional degradative pathway, then delivered</p><p>into multivesicular bodies (MVBs) and encapsulated in nascent intraluminal vesicle membrane. We further show the bacterial expulsion is triggered when intracellular UPEC follow the natural degradative trafficking pathway and reach lysosomes and attempt to neutralize its pH to avoid degradation. This pathogen-­mediated activity is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized on lysosomes, which spontaneously initiates lysosome exocytosis resulting in expulsion of exosome-­encased bacteria. These studies reveal a cellular default system for lysosome homeostasis and also, how it is coopted by the autonomous defense program to clear recalcitrant pathogens.</p> / Dissertation

O acidente vascular encefálico (AVE) é uma doença comum e de grande impacto para a saúde da população, uma vez que é considerado a principal causa de incapacidades neurológicas, principalmente motora e cognitiva. Considerando que os exossomos e o seu conteúdo podem ser marcadores prognóstico de demência em pacientes com Doença de Alzheimer, avaliamos o perfil exossomal circulante em pacientes pós AVE isquêmico com e sem comprometimento cognitivo. Ainda, é de conhecimento que os pacientes tratados com trombolíticos apresentam redução na dependência funcional a longo prazo, assim é de interesse avaliar os exossomos periféricos destes pacientes. Este trabalho visou investigar o perfil de exossomos circulantes em pacientes após diagnóstico de AVE isquêmico (AVEI) nas fases aguda e crônica. Foram determinados o conteúdo de proteína total e a atividade da acetilcolinesterase (AChE), marcadores de exossomos, além de correlacionar escores de comprometimento neurológico com os parâmetros bioquímicos. Ainda, considerando que o estresse oxidativo tem um papel central na fisiopatologia da isquemia cerebral, determinamos o conteúdo de espécies reativas e a atividade da enzima superóxido dismutase (SOD) em exossomos circulantes. Os fatores comprometimento cognitivo e o uso do trombolítico na fase aguda da doença não mostraram ter influência sobre os parâmetros estudados. Os resultados sugerem que uma diminuição de exossomos circulantes, já que houve uma redução na quantificação de proteínas totais na fase crônica, pode sugerir um prejuízo no sistema de remoção de materiais tóxicos levando ao acúmulo de materiais indesejados disso, o estado oxidativo exossomal, representado pelos níveis de espécies reativas e a atividade da SOD, foi alterado na fase crônica quando comparado com a fase aguda. Estes resultados sugerem que há uma alteração do perfil dos exossomos ao longo do tempo nos pacientes com AVE isquêmico. Nossos dados indicam que os níveis de espécies reativas na fase aguda podem predizer alterações no perfil dos exossomos na fase crônica de pacientes com comprometimento cognitivo, especificamente nas atividades das enzimas SOD e AChE e na concentração de proteínas totais. Estas associações não foram observadas em pacientes sem comprometimento cognitivo. Correlacionamos os parâmetros bioquímicos com as escalas de déficit neurológico, os escores do Mini-Exame do Estado Mental (MEEM) foram inversamente proporcionais aos níveis de AChE naqueles pacientes que não receberam o tratamento trombolítico na fase aguda. Assim, nossos dados demonstram que o prejuízo cognitivo (menores escores de MEEM) está associado a maiores níveis de atividade da AChE em exossomos circulantes. O papel dosexossomos na fisiopatologia da isquemia cerebral, assim como na predição de diagnóstico de comprometimento cognitivo precisa ser melhor investigado. / Stroke is a common disease and has a major impact on the public health, since it is considered the main cause of neurological disabilities, mainly motor and cognitive impairment. Considering that exosomes’ content may be prognostic markers of dementia in patients with Alzheimer's disease we evaluated the circulating exosomal profile in patients with ischemic stroke with and without cognitive impairment. Furthermore, it is known that thrombolytic therapy reduces long-term disability, so it is interesting to evaluate the peripheral exosomes of these patients. This work aimed to investigate the profile of circulating exosomes in patients after diagnosis of ischemic stroke in the acute and chronic phases. Total protein content and acetylcholinesterase activity (AChE), markers of exosomes were determined, also it was correlated the scores of cognitive function tests with biochemical parameters. Considering that oxidative stress plays a central role in the pathophysiology of cerebral ischemia, we determined the content of reactive species and the activity of the superoxide dismutase enzyme (SOD) in circulating exosomes. The factors cognitive impairment and the use of thrombolytic in the acute phase of the disease did not show influence on the studied parameters. The results suggest that a decrease in circulating exosomes, since there was a reduction in the quantification of total proteins in the chronic phase, may suggest a damage in the system of removal of toxic materials leading to the accumulation of unwanted materials. The exosomal oxidative state, represented by the reactive species levels and the SOD activity, was altered in the chronic phase when compared to the acute phase These results suggest that the profile of the exosomes is altered over time in patients with ischemic stroke. Our data indicate that reactive species levels in the acute phase may predict changes in the exosome profile in the chronic phase of patients with cognitive impairment, specifically in the activities of SOD and AChE enzymes and in the concentration of total proteins. These associations were not observed in patients without cognitive impairment. We correlated the biochemical parameters with the the scores of the assessment of cognitive function tests, the Mini-Mental State Examination (MMSE) scores were inversely proportional to the AChE levels in those patients who did not receive the thrombolytic treatment in the acute phase. Thus, our data demonstrate that cognitive impairment (lower MMSE scores) is associated with higher levels of AChE activity in circulating exosomes. The role of exosomes in the pathophysiology of cerebral ischemia, as well as in the prediction of diagnosis of cognitive impairment, needs to be better investigated.

Changes in the biomechanical properties of cells accompanying the development of various pathological conditions have been increasingly reported as biomarkers for various diseases and as a predictor of disease progression stages. For instance, cancer cells have been found to be less stiff compared to their healthy counterparts due to the proteomic and lipidomic dysregulations conferred by the underlying pathology. The separation and selective recovery of cells or extracellular vesicles secreted from such cells that have undergone these changes have been suggested to be of diagnostic and prognostic value. This dissertation first describes the implementation of a stiffness-based separation of phosphatidylcholine-based vesicles using a method first introduced based on the research in this work and was dubbed thermally-assisted acoustophoresis, or thermo-acoustophoresis. By tuning the temperature, we achieved the separation of vesicles of the same size, shape, and charge but with different stiffness values. It was observed that at a specific transition point, the acoustic contrast factor of vesicles changed sign from positive to negative. This change was mainly due to change in the compressibility of the vesicles, which is inversely proportional to stiffness. The acoustic contrast temperature (Tϕ), corresponding to the temperature at which the contrast factor switches sign, was determined to be unique to the composition of the vesicles. This unique temperature signature allowed us to develop this separation method of vesicles with distinct membrane stiffness with target outlet purities exceeding 95%. We have further explored the functionality of this method by experimenting with cholesterol-containing vesicles. In cells, the cholesterol content plays a crucial role in determining stiffness. Changes in the cholesterol content in cellular membranes can be an indication of pathological disorders. We evaluated the Tϕ of vesicles at different cholesterol molar ratios (Xchol) and developed a multi-stage lab-on-a-chip method to accomplish for the first time the separation of a three-vesicle mixture. Using Xchol = 0.1, 0.2, and 0.3 vesicles, we obtained efficiencies exceeding 93%. The simplicity, rapidity, and label-free nature of this approach holds promise as a diagnostic and separation tool for cells affected by diseases that affect the stiffness and extracellular vesicles such as exosomes and microvesicles.

O acidente vascular encefálico (AVE) é uma doença comum e de grande impacto para a saúde da população, uma vez que é considerado a principal causa de incapacidades neurológicas, principalmente motora e cognitiva. Considerando que os exossomos e o seu conteúdo podem ser marcadores prognóstico de demência em pacientes com Doença de Alzheimer, avaliamos o perfil exossomal circulante em pacientes pós AVE isquêmico com e sem comprometimento cognitivo. Ainda, é de conhecimento que os pacientes tratados com trombolíticos apresentam redução na dependência funcional a longo prazo, assim é de interesse avaliar os exossomos periféricos destes pacientes. Este trabalho visou investigar o perfil de exossomos circulantes em pacientes após diagnóstico de AVE isquêmico (AVEI) nas fases aguda e crônica. Foram determinados o conteúdo de proteína total e a atividade da acetilcolinesterase (AChE), marcadores de exossomos, além de correlacionar escores de comprometimento neurológico com os parâmetros bioquímicos. Ainda, considerando que o estresse oxidativo tem um papel central na fisiopatologia da isquemia cerebral, determinamos o conteúdo de espécies reativas e a atividade da enzima superóxido dismutase (SOD) em exossomos circulantes. Os fatores comprometimento cognitivo e o uso do trombolítico na fase aguda da doença não mostraram ter influência sobre os parâmetros estudados. Os resultados sugerem que uma diminuição de exossomos circulantes, já que houve uma redução na quantificação de proteínas totais na fase crônica, pode sugerir um prejuízo no sistema de remoção de materiais tóxicos levando ao acúmulo de materiais indesejados disso, o estado oxidativo exossomal, representado pelos níveis de espécies reativas e a atividade da SOD, foi alterado na fase crônica quando comparado com a fase aguda. Estes resultados sugerem que há uma alteração do perfil dos exossomos ao longo do tempo nos pacientes com AVE isquêmico. Nossos dados indicam que os níveis de espécies reativas na fase aguda podem predizer alterações no perfil dos exossomos na fase crônica de pacientes com comprometimento cognitivo, especificamente nas atividades das enzimas SOD e AChE e na concentração de proteínas totais. Estas associações não foram observadas em pacientes sem comprometimento cognitivo. Correlacionamos os parâmetros bioquímicos com as escalas de déficit neurológico, os escores do Mini-Exame do Estado Mental (MEEM) foram inversamente proporcionais aos níveis de AChE naqueles pacientes que não receberam o tratamento trombolítico na fase aguda. Assim, nossos dados demonstram que o prejuízo cognitivo (menores escores de MEEM) está associado a maiores níveis de atividade da AChE em exossomos circulantes. O papel dosexossomos na fisiopatologia da isquemia cerebral, assim como na predição de diagnóstico de comprometimento cognitivo precisa ser melhor investigado. / Stroke is a common disease and has a major impact on the public health, since it is considered the main cause of neurological disabilities, mainly motor and cognitive impairment. Considering that exosomes’ content may be prognostic markers of dementia in patients with Alzheimer's disease we evaluated the circulating exosomal profile in patients with ischemic stroke with and without cognitive impairment. Furthermore, it is known that thrombolytic therapy reduces long-term disability, so it is interesting to evaluate the peripheral exosomes of these patients. This work aimed to investigate the profile of circulating exosomes in patients after diagnosis of ischemic stroke in the acute and chronic phases. Total protein content and acetylcholinesterase activity (AChE), markers of exosomes were determined, also it was correlated the scores of cognitive function tests with biochemical parameters. Considering that oxidative stress plays a central role in the pathophysiology of cerebral ischemia, we determined the content of reactive species and the activity of the superoxide dismutase enzyme (SOD) in circulating exosomes. The factors cognitive impairment and the use of thrombolytic in the acute phase of the disease did not show influence on the studied parameters. The results suggest that a decrease in circulating exosomes, since there was a reduction in the quantification of total proteins in the chronic phase, may suggest a damage in the system of removal of toxic materials leading to the accumulation of unwanted materials. The exosomal oxidative state, represented by the reactive species levels and the SOD activity, was altered in the chronic phase when compared to the acute phase These results suggest that the profile of the exosomes is altered over time in patients with ischemic stroke. Our data indicate that reactive species levels in the acute phase may predict changes in the exosome profile in the chronic phase of patients with cognitive impairment, specifically in the activities of SOD and AChE enzymes and in the concentration of total proteins. These associations were not observed in patients without cognitive impairment. We correlated the biochemical parameters with the the scores of the assessment of cognitive function tests, the Mini-Mental State Examination (MMSE) scores were inversely proportional to the AChE levels in those patients who did not receive the thrombolytic treatment in the acute phase. Thus, our data demonstrate that cognitive impairment (lower MMSE scores) is associated with higher levels of AChE activity in circulating exosomes. The role of exosomes in the pathophysiology of cerebral ischemia, as well as in the prediction of diagnosis of cognitive impairment, needs to be better investigated.

Periodontal disease affects 47% of Americans over 30. Characterized by microbial dysbiosis and unregulated inflammation, severe periodontitis causes degradation of bone and soft tissue around teeth. Current treatments have limited regenerative outcomes and frequent reinfection by harmful bacteria. Human mesenchymal stem cells (hMSCs) have been shown to promote wound healing and tissue regeneration. Many therapeutic benefits of hMSCs are due to their secretome products, like exosomes. Our long-term goal is to develop periodontal therapies with hMSC exosomes. The objectives of this study were to determine the effect of hMSC-derived exosomes on cellular activity of hMSCs and investigate whether hMSC exosome treatment reduces pro-inflammatory cytokine production in LPS-activated RAW264.7 cells. The specific aims of this study were: 1) Determine the characteristics of hMSC-derived exosomes, 2) Determine the biological effect of exosomes on cellular activity of hMSCs, 3) Determine whether exosomes treatment can inhibit cytokine production in activated RAW264.7 cells, and 4) Determine the role of exosomal miRNA in pro-inflammatory cytokine production of RAW264.7 cells. To investigate, exosomes were first harvested from hMSCs culture media through ultracentrifugation. Exosomes were then observed under a transmission electron microscope (TEM) and assessed for surface markers using Western Blot. A transwell migration assay was used to evaluate the chemotactic effect of exosomes. To study the effect of exosomes on stem cell proliferation, exosomes were administered to hMSCs. The immunogenicity of MSC exosome was also evaluated. After 72 hours, cells were lysed and DNA was measured. To study anti-inflammatory effects of exosomes, LPS stimulated RAW264.7 cells were treated with exosomes. Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) levels of supernatant were measured by ELISA. To study exosomal miRNA, exosomal miRNAs were overexpressed in RAW264.7 cells and these cells were stimulated with LPS. IL-6 and TNFα were measured by ELISA. TEM images showed that exosomes are nano-sized vesicles (~100 nm). Western blot images showed that CD63 and CD81 are enriched in exosomes compared to total cell lysates. Exosome treatment increased cell proliferation and migration in hMSCs. At the doses that are chemotactic and mitogenic, MSC exosomes had minimal effect on the inflammatory cytokine IL-6 production. Treatment with exosomes significantly decreased IL-6 and TNFα production in RAW264.7 cells activated by LPS. Transfecting RAW264.7 cells with exosomal miR-760 significantly decreased IL-6 production, but had minimal effect on TNFα. Our results indicate that exosomes have a pleiotropic activity, which includes stimulating stem cell migration and proliferation, and mitigating the inflammatory response. Therefore, hMSC exosome delivery is promising for the treatment of periodontal diseases.